Toxicologic Pathology for Non-Pathologists [1st ed. 2019] 978-1-4939-9776-3, 978-1-4939-9777-0

This extensive volume began as a short course primarily geared toward toxicologists who want to expand their understandi

525 81 34MB

English Pages X, 919 [918] Year 2019

Report DMCA / Copyright

DOWNLOAD FILE

Polecaj historie

Toxicologic Pathology for Non-Pathologists [1st ed. 2019]
 978-1-4939-9776-3, 978-1-4939-9777-0

Table of contents :
Front Matter ....Pages i-x
Introduction to Toxicologic Pathology (George A. Parker)....Pages 1-44
The Pathology Report, Peer Review, and Pathology Working Group (Ted A. Birkebak, Peter C. Mann)....Pages 45-77
Routine and Special Techniques in Toxicologic Pathology (Pamela Blackshear, Erica Carroll, Sasmita Mishra, Matthew Renninger, Arun Tatiparthi)....Pages 79-112
Pathology of the Liver and Gallbladder (Robert R. Maronpot, David E. Malarkey)....Pages 113-136
Pathology of the Gastrointestinal Tract and Exocrine Pancreas (Mark J. Hoenerhoff, Arun Kumar R. Pandiri)....Pages 137-199
Pathology of the Urinary System (Kendall S. Frazier)....Pages 201-250
Pathology of the Nervous System (Juliana S. Lee, Sarah D. Cramer, Mark T. Butt)....Pages 251-277
Pathology of the Cardiovascular System (Joshua H. Decker, Radhakrishna Sura, Paul W. Snyder)....Pages 279-309
Pathology of the Respiratory System (Jack R. Harkema, James G. Wagner)....Pages 311-354
Pathology of the Lymphoid System (Tracey L. Papenfuss, Marlon C. Rebelatto, Brad Bolon)....Pages 355-395
Pathology of the Male and Female Reproductive System and Mammary Gland (Justin D. Vidal)....Pages 397-482
Pathology of the Integumentary System (Kelly L. Diegel)....Pages 483-535
Pathology of the Endocrine System (Brent E. Walling, Thomas J. Rosol)....Pages 537-569
Pathology of Bone, Skeletal Muscle, and Tooth (Stacey L. Fossey, D. Greg Hall, Andrew W. Suttie, Martin Guillot, Aurore Varela)....Pages 571-618
Pathology of the Eye (Leandro B. C. Teixeira)....Pages 619-659
Pathology of the Ear (Kenneth A. Schafer)....Pages 661-688
Principles of Toxicologic Clinical Pathology (Adam Aulbach, Laura Cregar)....Pages 689-743
Carcinogenicity (Paul Howroyd)....Pages 745-778
Pathology of Juvenile Animals (Catherine A. Picut, Amera K. Remick)....Pages 779-849
Non-mammalian Laboratory Species: Fish, Frogs, and Beyond (Shannon M. Wallace, Jeffrey C. Wolf)....Pages 851-873
Back Matter ....Pages 875-919

Citation preview

Thomas J. Steinbach Daniel J. Patrick Mary Ellen Cosenza Editors

Toxicologic Pathology for NonPathologists

T o x i c o l o g i c P at h o l o g y f o r N o n - P at h o l o g i s t s

Toxicologic Pathology for Non-Pathologists Edited by

Thomas J. Steinbach North Carolina Laboratory, Experimental Pathology Laboratories, Inc., Durham, NC, USA

Daniel J. Patrick Charles River Laboratories, Inc., Mattawan, MI, USA

Mary Ellen Cosenza MEC Regulatory and Toxicology Consulting, LLC , Moorpark, CA, USA

Editors Thomas J. Steinbach North Carolina Laboratory Experimental Pathology Laboratories, Inc. Durham, NC, USA

Daniel J. Patrick Charles River Laboratories, Inc. Mattawan, MI, USA

Mary Ellen Cosenza MEC Regulatory and Toxicology Consulting, LLC Moorpark, CA, USA

ISBN 978-1-4939-9776-3    ISBN 978-1-4939-9777-0 (eBook) https://doi.org/10.1007/978-1-4939-9777-0 © Springer Science+Business Media, LLC, part of Springer Nature 2019 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Humana imprint is published by the registered company Springer Science+Business Media, LLC part of Springer Nature. The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.

Preface This book is based on the successful American College of Toxicology (ACT) and Society of Toxicologic Pathology (STP) “Pathology for Non-Pathologists” short course that is held every other year in the United States. This course is primarily geared toward toxicologists who want to expand their understanding of toxicologic pathology in order to be better study directors; however, it has also proven to be of great interest to other drug development scientists and regulatory reviewers. In 2003, a small group of ACT members felt that a practical pathology course for non-­ pathologists/toxicologists would be useful to aid experienced toxicologists and regulatory officials. Because of the breadth of topics to present, multiple days would be needed to properly cover the topics of interest. A decision was made to hold the first course separate from the ACT annual meeting. During the first year, these members selected appropriate topics, recruited knowledgeable instructors, and identified companies that could provide financial support and meeting space. The inaugural 2004 course committee included one of the editors of this book, Mary Ellen Cosenza, as well as Leigh Ann Burns-Naas, Debbie Hoivik, Laura Dill Morton, Jerry Hardisty, Winston Evering, Isaac Hayward, Paul Howroyd, Stuart Levin, Douglas Wolf, and Farrel Fort. The Society of Toxicologic Pathology agreed to formally partner with the ACT on the initial and subsequent short course efforts, and this partnership has steadily strengthend the collaboration between these two organizations. The first committee members felt there was a need to start the short course with an overview of general pathology concepts that included fundamental vocabulary and the basics of pathophysiological processes (e.g., degenerative, regenerative, hyperplasia, hypertrophy, neoplasia, etc.; see this book’s appendix on SEND terminology and definitions). These concepts cover findings typically seen in toxicology studies. The course would then cover organ system pathology. Some of the other important topics included addressing biomarkers, correlation of clinical pathology endpoints (chemistry and hematology) with microscopic changes, and well-known pathology findings for classes of toxic substances. The first course was held at Baxter Healthcare Corporation in Deerfield, Illinois. Other sponsors that year included GlaxoSmithKline, National Institute of Environmental Health Sciences, and Pfizer. Due to growing attendance over the years, the course moved to larger locations and is now held at a hotel conference center. The course has benefitted from outstanding course speakers and dedicated course organizers who are often members of both the ACT and STP. Dan Patrick has helped organize the course and secure presenters since 2010 and met Tom Steinbach when he agreed at the last minute to be a substitute presenter when one of the scheduled speakers couldn’t attend the course in 2012; their camaraderie began at that point and continues. Tom has been a course organizer since 2014. Repeatedly, Tom and Dan have been asked about the possibility of sharing previous course notes, course slides, and recommendations on textbooks from individuals who couldn’t attend the course. These frequent inquiries made it clear that there was a need to reproduce some of the important education from the course in an easy-­ to-­understand reference book. About 2 years ago, Tom and Dan set out to develop such a

v

vi

Preface

book. The overall goal would be to help non-pathologists understand, contextualize, and communicate the pathology data and interpretations from the study pathologist in a practical and usable format. They also wanted to include a highly respected non-pathologist to help ensure that the product would fulfill these goals, and they were fortunate that their first choice, Mary Ellen Cosenza, accepted. The editors reached out to some of the highly regarded speakers from past courses as well as respected and well-known colleagues with expertise in specific organ systems or other specific aspects of toxicologic pathology. They were extremely happy in the outstanding group of pathologists who agreed to take this project on and volunteer many hours of their busy lives to write these chapters. So, that is a brief summary on how this book before you came to be. We are incredibly indebted and grateful to the many authors who contributed their time and expertise in this final product that we are very proud of. David Sabio of EPL Inc. is also commended for producing many of the high quality medical illustrations and images. We sincerely hope that the original intent of helping non-pathologists understand, converse in, and apply a basic understanding of pathology in their day-to-day careers is fulfilled. Durham, NC, USA Mattawan, MI, USA Moorpark, CA, USA

Thomas J. Steinbach Daniel J. Patrick Mary Ellen Cosenza

Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .   v Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .   ix 1 Introduction to Toxicologic Pathology�����������������������������������������������������������������  1 George A. Parker 2 The Pathology Report, Peer Review, and Pathology Working Group ������������������� 45 Ted A. Birkebak and Peter C. Mann 3 Routine and Special Techniques in Toxicologic Pathology ����������������������������������� 79 Pamela Blackshear, Erica Carroll, Sasmita Mishra, Matthew Renninger, and Arun Tatiparthi 4 Pathology of the Liver and Gallbladder �������������������������������������������������������������� 113 Robert R. Maronpot and David E. Malarkey 5 Pathology of the Gastrointestinal Tract and Exocrine Pancreas���������������������������� 137 Mark J. Hoenerhoff and Arun Kumar R. Pandiri 6 Pathology of the Urinary System������������������������������������������������������������������������ 201 Kendall S. Frazier 7 Pathology of the Nervous System������������������������������������������������������������������������ 251 Juliana S. Lee, Sarah D. Cramer, and Mark T. Butt 8 Pathology of the Cardiovascular System�������������������������������������������������������������� 279 Joshua H. Decker, Radhakrishna Sura, and Paul W. Snyder 9 Pathology of the Respiratory System ������������������������������������������������������������������ 311 Jack R. Harkema and James G. Wagner 10 Pathology of the Lymphoid System�������������������������������������������������������������������� 355 Tracey L. Papenfuss, Marlon C. Rebelatto, and Brad Bolon 11 Pathology of the Male and Female Reproductive System and Mammary Gland������������������������������������������������������������������������������������������ 397 Justin D. Vidal 12 Pathology of the Integumentary System�������������������������������������������������������������� 483 Kelly L. Diegel 13 Pathology of the Endocrine System�������������������������������������������������������������������� 537 Brent E. Walling and Thomas J. Rosol 14 Pathology of Bone, Skeletal Muscle, and Tooth�������������������������������������������������� 571 Stacey L. Fossey, D. Greg Hall, Andrew W. Suttie, Martin Guillot, and Aurore Varela 15 Pathology of the Eye������������������������������������������������������������������������������������������ 619 Leandro B. C. Teixeira 16 Pathology of the Ear������������������������������������������������������������������������������������������ 661 Kenneth A. Schafer

vii

viii

Contents

17 Principles of Toxicologic Clinical Pathology�������������������������������������������������������� 689 Adam Aulbach and Laura Cregar 18 Carcinogenicity�������������������������������������������������������������������������������������������������� 745 Paul Howroyd 19 Pathology of Juvenile Animals���������������������������������������������������������������������������� 779 Catherine A. Picut and Amera K. Remick 20 Non-mammalian Laboratory Species: Fish, Frogs, and Beyond���������������������������� 851 Shannon M. Wallace and Jeffrey C. Wolf Appendix: Fundamental Pathology Terminology Based on the Standard for the Exchange of Nonclinical Data (SEND)�����������������������������875 Index �������������������������������������������������������������������������������������������������������������������������893

Contributors Adam Aulbach  •  Charles River Laboratories, Inc., Mattawan, MI, USA Ted A. Birkebak  •  Experimental Pathology Laboratories, Inc., Redwood City, CA, USA Pamela Blackshear  •  Early Development, Covance Laboratories, Greenfield, IN, USA Brad Bolon  •  GEMpath, Inc., Longmont, CO, USA Mark T. Butt  •  Tox Path Specialists, LLC, Frederick, MD, USA Erica Carroll  •  Early Development, Covance Laboratories, Greenfield, IN, USA Sarah D. Cramer  •  Tox Path Specialists, LLC, Frederick, MD, USA Laura Cregar  •  Charles River Laboratories, Inc., Mattawan, MI, USA Joshua H. Decker  •  Charles River Laboratories, Inc., Mattawan, MI, USA Kelly L. Diegel  •  GlaxoSmithKline, Collegeville, PA, USA Stacey L. Fossey  •  Abbvie, Inc., North Chicago, IL, USA Kendall S. Frazier  •  Pathology, GlaxoSmithKline, Collegeville, PA, USA Martin Guillot  •  Charles River Laboratories, Inc., Senneville, QC, Canada D. Greg Hall  •  Lilly Research Laboratories, Indianapolis, IN, USA Jack R. Harkema  •  Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, MI, USA Mark J. Hoenerhoff  •  In Vivo Animal Core, Unit for Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA Paul Howroyd  •  Charles River Laboratories Edinburgh Ltd, Tranent, UK Juliana S. Lee  •  Alizée Pathology, Inc., Thurmont, MD, USA David E. Malarkey  •  National Toxicology Program, National Institute of Environmental Sciences, Research Triangle Park, NC, USA Peter C. Mann  •  Experimental Pathology Laboratories, Inc., Seattle, WA, USA Robert R. Maronpot  •  Maronpot Consulting LLC, Raleigh, NC, USA Sasmita Mishra  •  Early Development, Covance Laboratories, Greenfield, IN, USA Arun Kumar R. Pandiri  •  Molecular Pathology Group, Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle, NC, USA Tracey L. Papenfuss  •  Charles River Laboratories, Inc., Ashland, OH, USA George A. Parker  •  Global Pathology, Charles River Laboratories, Durham, NC, USA Catherine A. Picut  •  Charles River Laboratories, Inc., Durham, NC, USA Marlon C. Rebelatto  •  Precision Medicine, Astrazeneca, Gaithersburg, MD, USA Amera K. Remick  •  Charles River Laboratories, Inc., Durham, NC, USA Matthew Renninger  •  Early Development, Covance Laboratories, Greenfield, IN, USA Thomas J. Rosol  •  Department of Biomedical Sciences, College of Osteopathic Medicine, Ohio University, Athens, OH, USA Kenneth A. Schafer  •  Greenfield Pathology Services, Inc., Greenfield, IN, USA Paul W. Snyder  •  Experimental Pathology Laboratories, Inc., West Lafayette, IN, USA Radhakrishna Sura  •  AbbVie Inc., North Chicago, IL, USA Andrew W. Suttie  •  Covance Laboratories, Inc., Chantilly, VA, USA

ix

x

Contributors

Arun Tatiparthi  •  Early Development, Covance Laboratories, Greenfield, IN, USA Leandro B. C. Teixeira  •  Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA Aurore Varela  •  Charles River Laboratories, Inc., Senneville, QC, Canada Justin D. Vidal  •  Charles River Laboratories, Inc., Mattawan, MI, USA James G. Wagner  •  Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, MI, USA Shannon M. Wallace  •  Experimental Pathology Laboratories, Inc., Sterling, VA, USA Brent E. Walling  •  Charles River Laboratories, Inc., Ashland, OH, USA Jeffrey C. Wolf  •  Experimental Pathology Laboratories, Inc., Sterling, VA, USA

Chapter 1 Introduction to Toxicologic Pathology George A. Parker Abstract Toxicologic pathology involves microscopic examination of organ and tissue specimens from laboratory animals that have been exposed to candidate drugs, devices, or various chemical or biological agents. The goals are to identify organ system toxicity, dose levels that produce toxicity, and biomarkers of toxicity. A variety of investigative techniques are employed in the detection of histomorphologic alterations, most commonly light microscopic examination of histologic tissue sections and preparation of reports containing subjectively based diagnostic terms and interpretations that convey the identity and anticipated significance of observations. Contemporary clinical pathology evaluations are performed to help identify changes in bodily fluids which may precede and/or accompany histological alterations and further characterize these changes and their adversity. Clinical pathology evaluations also help identify potential clinical biomarkers of xenobiotic-associated tissue damage. Key words Pathology, Histopathology, Toxicologic pathology, Toxicology, Safety assessment

1  Overview of Pathology and Pathologists The pathology evaluation of toxicology studies is a common source of toxicity data that can substantially alter the development or marketing of drugs and chemicals, to the degree that a decision for continued development may be based largely on the pathology data. The histopathology data result from subjective analysis of histological sections performed by pathologists who may be in a separate division, company, or geographic location from the personnel who conduct the in-life study. Study directors and other responsible individuals may come from a background that included little or no exposure to pathology. The combination of (a) the subjective nature of the analysis, (b) lack of familiarity with pathology, and (c) the potentially significant impact of the pathology analysis often results in concerns among those who are responsible for the overall study conduct or product development. Toxicology stakeholders may benefit from an understanding of the challenges within the science and practice of pathology. A major goal of this book is to provide insight into the basic nature of pathology and Thomas J. Steinbach et al. (eds.), Toxicologic Pathology for Non-Pathologists, https://doi.org/10.1007/978-1-4939-9777-0_1, © Springer Science+Business Media, LLC, part of Springer Nature 2019

1

2

George A. Parker

the pathology analysis that is performed in the conduct of nonclinical toxicology studies for non-pathologists. Histopathology consists of a form of subjective image analysis that is performed on thin sections of tissue that are stained by various dyes to allow visualization of the tissue components. Without these stains, the tissue sections have few interpretable microscopic features. When viewing tissue sections by light microscopy, it is important to remember that the histologic presentation is not precisely the same as the in vivo tissue structure and that all differences in color as seen microscopically are merely artifacts introduced by the histotechnology procedures. The tissue specimen, as seen microscopically, typically originated from a laboratory animal that was exsanguinated at the time of necropsy; thus it lacks the blood perfusion that characterizes the living tissue. The specimen was preserved by immersion in a fixative, commonly neutral buffered formalin, dehydrated through graded alcohol solutions, cleared with xylene, and infiltrated with and embedded in paraffin. Thin paraffin sections collected from the paraffin block were deparaffinized by immersion in xylene or a xylene substitute, rehydrated through graded alcohols up to water, stained to allow visualization of the tissue, and protected by application of a thin glass coverslip using a mountant. These technical processes may introduce tissue artifacts that must be distinguished from histopathological changes. There is some truth to the smug assertion that histopathology is the study of tissue artifacts. The key to success in this endeavor is the training and experience in the observer, i.e., the pathologist (Seaton 2014). Untrained microscopists, regardless of their level of effort, enthusiasm, and general scientific ability, are rarely able to function above a rudimentary level in histopathological evaluation. Anyone with normal visual acuity is able to see structures in histological sections, but interpreting the significance and meaning of those microscopic observations relative to toxicity and overall effect on the health status of the animal requires a considerable amount of background training and experience. The importance of this perspective in the interpretation of microscopic observations has been emphasized on numerous occasions, e.g., a highly publicized situation in which multiple long-term safety assessment studies in rats were initially thought to result in test substance-related lymphoma involving the lungs. It was eventually determined that the proliferative lymphoid tissue in the lungs of affected rats represented a known component of the response to Mycoplasma pulmonis infection (Schoeb and McConnell 2011a, b; Schoeb et al. 2009). Though infectious and parasitic diseases are not as common in modern laboratory animal colonies as in previous times, the possible involvement of these diseases in toxicology studies must be considered. Exacerbation of cryptic or indolent infectious diseases is particularly likely in studies of test materials that are intentionally or incidentally immunomodulatory. These disease interferences are a real-life problem, not

Introduction to Toxicologic Pathology

3

merely an academic exercise (Hutto 2010). In some instances the laboratory animals used in toxicology studies may have a substantial prevalence of potentially significant infectious diseases. Polyomavirus infection was reported in 12 of 57 i­mmunosuppressed cynomolgus monkeys (van Gorder et al. 1999), and 95% of rhesus macaques in one colony were seropositive for rhesus cytomegalovirus (Andrade et al. 2003). The cited rhesus macaque colony had been closed to incoming animals for more than 70 years, indicating the latent rhesus cytomegalovirus infection was endemic. An exhaustive presentation of possible infectious or parasitic disease interferences in all of the common laboratory animal species is beyond the scope of this chapter. Table 1 presents a list of possible disease interferences in nonhuman primates that may illustrate the potential magnitude of this issue (Haley 2012; Parker and Snyder 2017). Toxicologic pathologists are most commonly graduates of veterinary medical colleges who undertake additional training in pathology. The veterinary pathology training programs encompass all aspects of pathology in domestic, laboratory, and zoo/wildlife animals. The training programs typically prepare candidates to complete board certification examinations as administered by the American College of Veterinary Pathologists, the European College of Veterinary Pathology, the Japanese College of Veterinary Pathologists, and the Royal College of Pathologists in the UK. Residency programs in veterinary pathology typically involve three to four years of full-time training following completion of veterinary college. Individuals who successfully complete the requirements to become board-certified in veterinary pathology indicate that status by abbreviations such as DACVP, DECVP, DJCVP, or FRC-Path. In addition to board certification in pathology, many pathology trainees also complete a master’s or Ph.D. degree in pathology or a related biomedical area. Many pathology training programs consist of combination residency and graduate programs that result in Ph.D. or master’s degrees in addition to preparation for board certification examinations. Non-veterinarian toxicologic pathologists are uncommon in North America, but are encountered with greater frequency in Europe/UK and Japan. In the formative days of toxicologic pathology, a number of physicians with an interest in comparative medicine became involved in toxicologic pathology and made remarkable contributions to the field. Many physician researchers remain highly involved in the field of toxicologic pathology, but modern organizations related to toxicologic pathology are largely populated by veterinarians with specialty training in pathology. It should be noted that Japan also has a board certification procedure, administered by the Japanese Society of Toxicologic Pathology (JSTP) that is open to individuals who are not veterinarians. Some of the certifying organizations, e.g., ACVP, have separate board certification procedures for anatomic and clinical pathology.

4

George A. Parker

Table 1 Selected pathogens of nonhuman primates used in toxicology studies Protozoan and metazoan parasites

Viral agents

Bacterial agents

Fungal agents

Adenovirus

Campylobacter

Aspergillus spp. Acanthamoeba spp.

Cercopithecine herpesvirus I (B virus)

E. coli, enteropathogenic Candida albicans

Balantidium coli

Cynomolgus polyomavirus

Helicobacter pylori

Blastocystis spp.

Cytomegalovirus

Helicobacter heilmannii-type

Cryptosporidium spp.

Hepatitis A virus

Lawsonia

Cyclospora spp.

Lymphocryptovirus

Moraxella catarrhalis

Demodex spp.

Measles virus

Mycobacterium tuberculosis

Endolimax nana

Polyomavirus

Rhodococcus equi

Entamoeba coli

Rhesus rhabdovirus

Salmonella spp.

Enterocytozoon bieneusi

Simian immunodeficiency virus

Shigella spp.

Giardia duodenalis

Simian parvovirus

Yersinia spp.

Oesophagostomum spp.

Dermatophyte spp.

Simian type D retrovirus

Plasmodium spp.

Simian varicella virus

Pneumonyssus semicola

Simian virus 40

Sarcocystis spp. Schistosoma spp. Strongyloides fuelleborni Toxoplasma gondii Trichomonas spp. Trichuristrichiura

Modified from Haley (2012) and Parker (2016)

Following completion of basic training in veterinary pathology, achievement of board certification status, and possibly completion of an additional academic degree, novice pathologists enter employment in private industry, academia, or government service. Those entering private industry may be employed by pharmaceutical firms, contract research organizations (CROs), or various facets of the chemical industry, including agricultural chemical firms. The early career pathologist, though fully trained and accredited, typically requires a period of training and close supervision while attaining additional familiarity in toxicologic pathology. The early career

Introduction to Toxicologic Pathology

5

pathologist must learn there are additional expectations involved other than simply observing and interpreting pathological changes. In most industrial situations the toxicologic pathologist is expected to function as part of a team, which may be different from previous experiences in academia or traditional diagnostic veterinary pathology. Integration into the investigative team requires the pathologist to become familiar with regulatory requirements, procedures, and policies related to the in-life phase of toxicology studies. Learning to provide these complex services in compliance with good laboratory practice (GLP) regulatory requirements is a major step in the development of the early career pathologist. In addition, most corporations have an internal culture that must be navigated to achieve the goal of providing accurate, timely, and cost-effective pathology services in support of the firm’s mission. Pathology as it relates to nonclinical safety assessment toxicology is broadly divided into anatomic and clinical pathology. The anatomic pathology area typically includes necropsy, histotechnology, and histopathology and often includes organ weight analysis, electron microscopy, and other special morphology-based analyses. Clinical pathology includes the traditional clinical pathology analyses, a multitude of special assays, and various biomarker assays. Close coordination between clinical pathology and anatomic pathology reporting is required in order to avoid conflicts between these two major forms of pathology interpretation. Ideally, the pathology reporting includes correlation between clinical observations, necropsy observations, clinical pathology data, organ weights, and histopathological findings.

2  Necropsy The necropsy, or postmortem examination, of animals from toxicology studies may be performed by pathologists, but more commonly is performed by non-pathologist personnel who are specifically trained in these procedures. Pathologists may or may not be in attendance for the necropsy examinations, but pathologists are typically available if questions or issues arise. The necropsy examinations are performed in accordance with study protocols as well as guidelines delineated in the facility’s standard operating procedures (SOPs). Facility SOPs typically provide very specific guidelines regarding specimen collection and fixation as well as the terminology used in recording necropsy observations. Standardization of necropsy terminology is necessary for tabulating necropsy observations relative to experimental treatments. Phosphate-buffered formalin is commonly used as a fixative, which prevents postmortem degeneration (autolysis) of tissue specimens. Non-buffered formalin should be avoided, as the slightly acidic non-buffered solution enhances the formation of

6

George A. Parker

acid hematin from hemoglobin contained in erythrocytes. Presence of brown acid hematin in histological sections is considered a ­technical defect though, in reality, its presence rarely precludes the possibility of competent histopathological interpretations. All involved in necropsy planning or conduct should be familiar with the technical details of formalin fixation, as failure to understand the terminology can result in serious failures in tissue preservation. Pure formaldehyde is a gas, which is dissolved in water to form formaldehyde solution. This formaldehyde solution is sometimes referenced as “pure formaldehyde,” though it consists of only 37–39% formaldehyde. Formaldehyde solution is diluted with water to form a 10% solution known as formalin. Various buffers are added to yield solutions such as neutral buffered 10% formalin, but it must be recognized that this final use solution is typically only 3.5–3.7% formaldehyde. Formaldehyde solution may contain up to 15% methanol as a stabilizing agent, which may interfere with ultrastructural studies (Bolon et al. 2013). As a result of these variables, it is difficult to know the exact formaldehyde content of the initial formaldehyde solution or the resultant neutral buffered formalin. In situations where a more exact concentration of formaldehyde is needed, or exclusion of methanol is necessary, it is customary to prepare fresh solutions of paraformaldehyde (polyoxymethylene) for use in tissue fixation. Formaldehyde solution and formalin are clear solutions that cannot be visually differentiated from water, saline, or other clear solutions. In bygone days it was customary to sniff the formalin container as it was opened to verify the presence of pungent formaldehyde. This practice must be discouraged, due to the potential health effects of formaldehyde exposure. A reliable, centrally controlled procedure (e.g., addition of a small amount of eosin) for identification of formaldehyde-containing fixatives must be instituted in order to ensure that (a) fixative solutions actually contain formaldehyde and (2) fixatives are not used for some other purpose where the formaldehyde content may have detrimental effects.

3  Histotechnology Procedures These procedures for preparation of histological sections are part of the field of histotechnology, though in our common workday terminology they are often referenced as “histology procedures.” Knowledgeable scientists in toxicology and pathology know that histology is actually defined as the biomedical specialty that deals with the study of tissue structure, as exemplified in multiple textbooks (Bloom et al. 1994; Young et al. 2006), rather than the technical procedures that result in histological sections. Conversion of fixed tissue specimens into histological sections suitable for light microscopic examination involves sequen-

Introduction to Toxicologic Pathology

7

tial steps known as gross trimming, processing, embedding in a matrix such as paraffin wax, microtomy, staining, and coverslipping. While these activities are the province of the histotechnology staff, it is useful for toxicologists and pathologists to have a general knowledge of the processes involved in preparing histological sections (Fig. 1a–g). 1. In the gross trimming step, the fixed tissue specimens from necropsy are trimmed to a dimension that will fit into process/ embed cassettes (see additional steps below) and are sufficiently thin to allow ready penetration of fluids. It is common practice to trim tissue specimens to approximately 3 mm thickness. The surface of the tissue specimen typically occupies no more than 50% of the surface area of the cassette in order that sufficient embedding medium remains to securely hold the tissue specimens for microtomy. 2. The term “processing,” which has special meaning in histotechnology, starts with passage of trimmed tissue specimens through sequential alcohol solutions of increasing concentration, with the goal of removing water from the tissue. Following ­dehydration, the tissue specimens are immersed in a clearing agent such as xylene or xylene substitutes to remove fat from the specimen, and the specimen is finally infiltrated with paraffin or other molten embedding medium. Tissue processing can be accomplished by manually moving tissues through glass jars containing the appropriate fluids, but in modern histotechnology laboratories, these procedures are performed by robotic tissue processors that typically have a single reaction chamber containing tissue specimens. The various processing fluids are sequentially introduced into the sealed reaction chamber, which is subjected to cycles of vacuum and pressure to aid penetration of fluids into the tissue specimens and heated to maintain the molten status of the final embedding media. All these activities are controlled by computer programs that can be varied to optimize the histological processing of various types of specimens. 3. Embedding of tissue specimens into paraffin blocks is necessary to hold the tissue specimens in a solid matrix that will allow preparation of the thin slices (“histological sections”) that are typically 3–6 μm in thickness. This is accomplished with the aid of an embedding center, which maintains a vat of molten paraffin that is dispensed into a cassette mold held on a small hot plate to maintain the paraffin in molten status while the tissue specimen is properly aligned in the future block. Once the specimen is properly aligned, the cassette mold is moved to a chilled plate that quickly cools the molten paraffin to a solid state.

8

George A. Parker

Fig. 1 (a) Formalin-fixed tissue specimens are gross trimmed to a size that will allow the specimens to fit into a process-embed cassette. Tissue specimens are approximately 3 mm in thickness, which would theoretically allow preparation of 600 serial sections of 5 μM thickness. In reality, technical issues make it impossible to collect and preserve each individual section. A major loss of tissue occurs as the paraffin block is faced to allow preparation of full-face sections. Paraffin blocks should be faced as few times as possible to avoid depletion of the tissue specimens. If special stains are anticipated, the full listing of stains should be determined in advance and the paraffin blocks should be faced only once, ideally at the same time the initial sections for routine staining are prepared. (b) Trimmed tissue specimens are subjected to histological processing in an automated tissue processor. “Processing” has specific meaning in a histology laboratory, where it refers to sequential passage of tissue specimens through graded alcohols to remove water, a clearing agent such as xylene to remove lipids, and infiltration with a molten embedding medium such as paraffin. Modern tissue

Introduction to Toxicologic Pathology

9

Fig. 1 (continued) processors have a processing chamber that holds the specimens in process-embed cassettes. Reagents are pumped from the various reservoirs, here shown at the bottom of the instrument. The processing chamber is sealed and typically has alternating cycles of pressure and vacuum to aid in replacement of air with the processing fluids. The processing chamber must also be heated to maintain embedding media in a molten state. The tissue processor instrument has computerized control that allows the use of multiple processing cycles for different types or sizes of tissue specimens. (c) Following processing the paraffin-infiltrated tissue specimens are embedded in a solid block of paraffin, using the labeled process-embed cassette as a mold that determines the final size of the paraffin block. The embedding center has a heated plate that maintains the paraffin in a molten state, which allows manipulation of tissue specimens to proper alignment in the final block (upper left image). Once proper alignment of the specimens is achieved, the process-embed cassette is placed on the embedding mold (upper right image), and the embedding mold is filled with liquid paraffin (lower left image) and finally moved to a refrigerated plate on the embedding center (lower right image). This results in rapid cooling of the block containing the tissue specimens. (d) Microtomy is aided by cooling the paraffin blocks on wet ice. (e) The paraffin block containing tissue specimens is clamped in a microtome, which moves the block up and down across the face of a knife blade. The microtome causes the block to advance at specified intervals, resulting in thin ribbons of paraffin containing tissue sections. (f) The paraffin ribbon containing tissue sections is floated on a warm water bath to allow dissipation of any wrinkles that may have been introduced during microtomy. One or more sections are picked up onto glass slides for staining or other uses. If multiple ribbons are placed on the water bath simultaneously, great care must be taken in maintaining the identity of the individual ribbons. (g) The autostainer on the right side of the image consists of multiple reservoirs filled with reagents used for staining tissue sections. The instrument is computer-controlled; thus it can move racks of slides through any designated series of fluid reservoirs, as opposed to older instruments that were limited to a linear pathway of progress. The initial steps in the staining process are essentially a reversal of the tissue processing steps, with passage through xylene or xylene substitutes to remove paraffin and graded alcohols to return the tissue specimens to an aqueous state. Autostainers result in uniform staining of tissue sections only if proper attention is given to rotation and replenishment of the various fluids used in the instrument. The coverslipping robot on the left side of the image dispenses mountant medium and places a very thin glass coverslip on the surface of the slide. Once the mountant has dried, the stained tissue section is protected and ready for microscopic examination. More modern versions of autostainers and coverslipping robots are connected, with the autostainers mechanically passing racks of stained slide through to the coverslipping robot

10

George A. Parker

4. Microtomy, often considered the centerpiece activity of histotechnology, is accomplished by clamping the solid paraffin block containing embedded tissue specimens into a microtome. The microtome moves the paraffin block up and down across the face of an extremely sharp knife edge and advances the block toward the knife by whatever incremental distance represents the desired thickness of the final histological section. Sequential sections from the block adhere side-to-side, forming a “ribbon” that is floated on a warm water bath that allows the ribbon to flatten, thus removing folds and wrinkles from the histological section. Labeled glass microslides are inserted into the water beneath the floating ribbon, and one or more sections are lifted onto the microslide for subsequent staining. 5. The staining process requires removal of the paraffin by immersion of the microslides in xylene or xylene substitute and rehydration of the tissue specimens by sequential immersion in decreasing concentrations of alcohol, ending with water, i.e., a reversal of the steps employed in tissue processing. Once in an aqueous phase, the tissue specimens are subjected to various staining procedures that allow visualization of tissue components. Staining with hematoxylin and eosin (H&E) is most commonly used in nonclinical toxicology studies, though numerous histochemical and immunohistochemical stains are available for specific purposes. Routine staining in modern facilities typically is accomplished by computer-driven autostainers that can be programmed to perform a variety of staining tasks. 6. The stained tissue section lying on a glass microslide is very fragile and must be protected by applying a thin glass coverslip that essentially encases the tissue specimen in a small glass box. The coverslip is adhered to the microslide by use of various mounting media that are fluid when held in a container, but quickly solidify when placed in a thin film between the coverslip and the underlying microslide. All these materials are engineered to allow the greatest possible light transmission and the least possible refractive index, resulting in a stained tissue specimen that visually appears to be floating in air. Coverslip application in modern laboratories is accomplished by coverslipping robots, some of which are linked to the antecedent autostaining robot. The great majority of the histological sections used in toxicology studies are prepared from paraffin-embedded specimens, but there are occasions where other embedding materials are required. For example, the use of xylene or xylene substitutes in histological processing removes neutral lipids from the specimens; therefore, frozen sections have traditionally been used for demonstration of neutral lipid accumulations. The requirement for frozen sections for lipid staining can be circumvented by use of osmium post-­

Introduction to Toxicologic Pathology

11

Fig. 2 This H&E-stained histological section was prepared from a specimen of rat liver that was treated with osmium tetroxide following fixation, prior to histological processing. Osmium complexes with lipids in the tissue, rendering the lipids insoluble in the solvents used in routine histological processing. This eliminates the need for frozen sections for demonstration of fatty change in tissues. The dense black staining indicates osmium-complexed lipids in the tissue. Note the distinct pattern of distribution relative to the lobular microarchitecture of the liver. Osmium staining for neutral lipids, 2× objective magnification

fixation of formalin-fixed specimens, followed by routine hematoxylin and eosin staining. Osmium complexes with neutral lipids, rendering them insoluble in standard histologic solvents, resulting in dense black deposits of osmicated lipids in the H&E-stained sections (Fig. 2). Modern histotechnology procedures most commonly are centered on the use of process/embed cassettes, which are perforated plastic containers that hold tissues as they are subjected to the various steps of histological processing and eventually serve as molds and labels for paraffin blocks. In modern laboratories the cassettes are commonly labeled by computer-driven robots. When circumstances dictate, the plastic process/embed cassettes may be labeled with pencils, but not the commonly used felt-tip pens that are in abundant supply in most laboratories. The ink used in many felt-tip pens is soluble in one or more of the solvents used in histotechnology. Even the pens that are purchased specifically for use in histotechnology should be viewed with suspicion until the ink is tested and found to be insoluble in the laboratory’s current histology solvents.

4  Routine Microscopy Standard toxicology study protocols typically refer to “routine light microscopy” as the process used to perform the histopathological evaluation. A more precise term for this form of microscopy

12

George A. Parker

Fig. 3 (a) This lung specimen from a rhesus macaque has multiple white to gray discolored areas that represent inflammation associated with the presence of lung mites (Pneumonyssus semicola) (Andrade et al. 2003; Leonovich 2010). (b) Incident light microscopy of material expressed from the lung lesions shown in Fig. 8a revealed the causative lung mites

is “transmitted visible light microscopy,” which indicates a light source in the visible spectrum is placed beneath the specimen and the light beam passes through the specimen to the eyes of the observer. In “reflected visible light microscopy,” the light beam is incident upon the surface of the (typically opaque) specimen and then reflected back to the eyes of the observer. This latter form of visible light microscopy is commonly applied in metallurgical studies and the dissecting microscopes that may be used for subgross examination of specimens from toxicology studies (Fig. 3a, b).

5  Specialized Microscopy Other alterations in the light path or characteristics of the light beam result in different forms of microscopy that are variably applicable to toxicology studies. Substitution of the visible light beam by ultraviolet (UV) light, coupled with application of fluorochromes to the specimen, results in fluorescence microscopy, which is widely used in localization of specific molecules to cells or regions of histological specimens. Fluorescence microscopy is also utilized in Fluoro-Jade staining (Schmued and Hopkins 2000), which accentuates the autofluorescence seen in degenerating neurons (Fig. 4). In polarization microscopy, polarized lenses are placed in the incident light beam above and below the specimen. Rotation of the lenses to a point where they are at 90° to each other effectively blocks the light path to the observer. If the specimen contains crystalline material, it may rotate the light beam to a plane where it passes the upper polarized lens, thus allowing light to reach the eyes of the observer and revealing the presence of crystals in the tissue specimen (Fig. 5a, b). Some types of crystalline mate-

Introduction to Toxicologic Pathology

13

Fig. 4 Fluorescence microscopy performed on a brain sections stained with Fluoro-Jade B reveals intense fluorescence in degenerating neurons in a model of experimentally induced neurotoxicity. Fluoro-Jade B stain, UV fluorescence microscopy, 40× objective magnification

Fig. 5 (a) The H&E-stained section of the kidney from a dog has faintly discernible light brown material within cortical tubules. The dog was submitted to necropsy following consumption of automobile antifreeze. H&E stain, 10× objective magnification. (b) Examination via partially polarized light reveals bright, multicolored birefringent material within cortical tubules. Partially polarized light was used to permit visualization of the background renal structure. With fully polarized microscopy, the crystalline material would appear in a black background. The intratubular material is typical of the oxalate crystal accumulation seen in renal tubules following ingestion of ethylene glycol, which is used in automobile antifreeze. H&E stain with polarization microscopy, 10× objective magnification

rial have specific polarization characteristics, such as the red color and “Maltese cross” configurations seen with porphyrin crystals in tissues (Greijdanus-van der Putten et al. 2005) (Fig. 6a, b). In phase-contrast microscopy, the microscope converts phase shifts, which are invisible to the human eye, into variations in brightness that are visible. This allows visualization of biological structures

14

George A. Parker

Fig. 6 (a) This H&E-stained histological section of the liver is from a dog treated with a drug candidate. The biliary tract has mixed inflammatory cell infiltration and a prominent accumulation of unidentified amorphous brown material. H&E stain, 40× objective magnification. (b) Examination of the same microscopic field via partially polarized light reveals the brown material to have a distinct red color. Some of the pigment accumulations have intersecting dark bands that are generally consistent with “Maltese crosses.” The image was captured using partially polarized light in order that the background hepatic architecture would be visible. With completely polarized light, the red color was more intense and the “Maltese cross” configurations were more distinctive. The overall histologic presentation is consistent with hepatic accumulation of porphyrin pigment. H&E stain with polarization microscopy, 40× objective magnification

that are not visible by other means and may be performed on living organisms contained with liquid media (Fig. 7a, b). In dark-field microscopy, the direct path of the visible incident light is blocked from reaching the observer’s eye; thus only light that is scattered by the specimen reaches the eye of the observer. The result appearance is brightly illuminated specimens “floating” in a dark background (Fig. 7c). A combination of phase-contrast and dark-field microscopy may be employed (Fig. 7d).

6  Histochemistry Histochemical stains other than routine hematoxylin and eosin, commonly known as “special stains,” involve the use of various chemical reactions to demonstrate tissue components, microbes, etc., in histological sections. Typical histochemical stains involve a series of chemical treatments that may range from very simple to highly complex and often involve subjective evaluations of staining progress by histologic technicians/technologists. Due to this subjective input, performance of histochemical stains involves a mixture of art and science. As such, this work is typically assigned to the more experienced histology lab personnel. Some examples of special stains include PAS (periodic acid-Schiff) for polysaccha-

Introduction to Toxicologic Pathology

15

Fig. 7 (a) The H&E-stained section of the colon from a rhesus macaque has numerous intraluminal organisms that are consistent with Balantidium coli. Very little of the internal structure of the organisms is visible in histological sections, which are essentially two-dimensional. H&E stain, 20× objective magnification. (b) Phase-­ contrast microscopy performed on a wet mount of colon contents from a rhesus macaque reveals surface cilia and internal structures in a Balantidium coli organism. Wet mount, phase-contrast microscopy, 100× objective magnification. (c) Dark-field microscopy performed on a wet mount of colon contents from a rhesus macaque reveals a number of ovoid organisms that are consistent with Balantidium coli. Wet mount, dark-field microscopy, 40× objective magnification. (d) Combined phase-contrast and dark-field microscopy on a wet mount of colon contents from a rhesus macaque reveals additional structural details of the Balantidium coli organisms. Phase-contrast and dark-field microscopy, 100× objective magnification

rides, von Kossa for calcium, Perls’ Prussian blue for iron, and Sudan black B for lipids (Fig. 8a, b). Specific requirements of the histopathology evaluation may necessitate the use of special tissue fixatives. For example, it is common practice to use Davidson’s or modified Davidson’s fixatives for preservation of eye and testis specimens (Latendresse et al. 2002). It is critically important that those involved in the design of toxicology studies should determine any specific tissue collection or preservation requirements well in advance of the planned necropsy, as procurement of the necessary reagents may involve delays.

16

George A. Parker

Fig. 8 (a) The histological section of the lung is from a rat that received a test article in corn oil vehicle via oral gavage. Note the focally extensive infiltration of inflammatory cells near distal airways. H&E stain, 5× objective magnification. (b) A frozen section of the lung, from the same animal shown in Fig. 1a, stained with Sudan black B reveals the presence of corn oil in association with the inflammatory cell infiltrates. Reflux and aspiration of minor quantities of test article/vehicle is seen with some frequency in gavage studies and should be considered a possible basis for unexplained pulmonary findings in gavage studies (Crabbs et al. 2013)

Failure to meet these fixation requirements may have a disastrous effect on meeting the specific goals of the study. For further information on this complex topic, readers are referred to histotechnology texts (Carson 1997; Sheehan and Hrapchak 1980) or reference publications on histochemistry (Thompson 1966).

7  Immunohistochemistry Traditional histopathology involves microscopic evaluation of the histomorphology of tissues, which largely ignores the major contribution of physiological and biochemical abnormalities in many pathological processes. The development of i­ mmunohistochemistry (IHC) was a major advance in the field of histopathology, as it allowed detection of many of the molecules that define cell types and cellular structures as well as signaling and effector molecules that contribute to many pathological processes (Figs. 9a–d and 10a–c). Immunohistochemistry depends on the non-covalent bonding of primary antibodies that recognize the three-­dimensional presentation of antigenic epitopes. The primary antibodies may be directly labeled with a fluorochrome that is visualized via fluorescence microscopy or an enzyme such as horseradish peroxidase that converts a chromogen to a product that is visible by routine visible light microscopy. More commonly, a “sandwich technique” is employed whereby the primary antibody is recognized by a fluorochrome- or enzyme-labeled secondary antibody, followed by

Introduction to Toxicologic Pathology

17

Fig. 9 (a) The image is from a Swiss roll preparation of the small intestine from a rat collected at postnatal day 28. Note the plaque-like array of deeply stained lymphoid follicles that constitute a mucosal lymphoid aggregate (“Peyer’s patch”). Examination of the routinely stained histological section suggests intact mucosal immune system structures, but provides little information regarding the functional subcategories of immune cell populations. H&E stain, 5× objective magnification. (b) An immunohistochemical stain directed at CD45RA reveals a dense population of brown-stained CD45RA+ B cells in lymphoid follicles of the Peyer’s patch. CD45RA IHC stain with 3, 3′-diaminobenzidine chromogen and hematoxylin counterstain, 5× objective magnification. (c) An immunohistochemical stain directed at CD3 reveals a dense population of brown-stained CD3+ lymphocytes in the spaces between lymphoid follicles in the Peyer’s patch. These aggregates of CD3+ T cells are equivalent to the paracortex zone of lymph nodes. Note the regularly spaced population of surveillance T cells in the superficial intestinal mucosa, as well as the substantial population of T cells scattered throughout the follicles. This intrafollicular T-cell population is critically important in development of B-cell-­ mediated immune responses. CD3 IHC stain with 3, 3′-diaminobenzidine chromogen and hematoxylin counterstain, 5× objective magnification. (d) An immunohistochemical stain directed at Ki67 proliferation marker reveals marked proliferative activity in the intestinal crypts and within germinal centers at the base of lymphoid follicles within the Peyer’s patch. Presence of active germinal centers indicates intact responsiveness to antigenic stimulation and initiation of a humoral immune response. Ki67 IHC stain with 3, 3′-diaminobenzidine chromogen and hematoxylin counterstain, 5× objective magnification

fluorescence microscopy or enzyme-chromogen interaction in chromogenic IHC (Fig. 11). The common use of IHC staining as a second-tier investigative technique in pathology evaluation warrants specific consideration of tissue preservation requirements for IHC. Aldehyde-mediated cross-linking of amino acids starts to occur soon after formalin immersion, and with prolonged formalin immersion, the protein cross-linking may block access of the antibodies used in IHC

18

George A. Parker

Fig. 10 (a) The histological section is from a rhesus macaque that was subjected to 11 Gy ionizing radiation 53 days prior to necropsy. The original pleural layer (∗) consists of a dense layer of pink-stained fibrous connective tissue, while the superficial surface has a thick layer (∗∗) of immature fibrous connective tissue. H&E stain, 10× objective magnification. (b) A Masson’s trichrome stain performed on the same tissue specimen shown in Fig. 3a shows a thin layer of mature collagenous tissue (∗) in the original pleural surface, with a smaller amount of collagenous tissue in the immature superficial layer (∗∗). Masson’s trichrome stain, 10× objective magnification. (c) An immunohistochemical stain directed at alpha-smooth muscle actin (αSMA) shows little αSMA staining in the original mature pleural layer (∗), but there is abundant αSMA staining in the superficial layer of immature fibrous connective tissue (∗∗). Presence of abundant αSMA immunoreactivity suggests the involvement of myofibroblasts in the pathogenesis of the radiation-induced pleural fibrosis. Alpha-smooth muscle actin IHC staining, 10× objective magnification

s­ taining. This technical defect may be overcome by judicious use of antigen retrieval processes, but those processes are imperfect and introduce an additional variable into the experiment. If there is any expectation that IHC staining will be required during the pathology evaluation, immersion of tissue specimens in aldehyde-based fixatives should be limited to approximately 48 hours, after which the tissue specimens should be transferred to 70% ethanol.

8  In Situ Hybridization In situ hybridization (ISH) is somewhat similar to IHC, but base pairing between nucleic acids replaces antigen-antibody binding in the initial recognition step. An additional strength of ISH over IHC

Introduction to Toxicologic Pathology

19

Fig. 11 In the “sandwich technique” of chromogenic immunohistochemical staining, the target molecule on the tissue is detected by a primary antibody to that molecule. A secondary antibody directed at the primary antibody is tagged with an enzyme such as horseradish peroxidase. After sequential steps that allow binding of the primary and tagged secondary antibodies, the section is flooded with a chromogen such as 3, 3′-diaminobenzidine (DAB) that reacts with the enzyme to form a colored precipitate. The end result is a colored deposit at the site of primary antibody binding. In fluorescent immunohistochemical staining, either the primary or secondary antibody is tagged with a fluorochrome which is directly visualized by fluorescence microscopy. Different fluorochromes emit different wavelengths upon UV stimulation; thus it is possible to demonstrate multiple molecular targets simultaneously. (Artwork compliments of Cynthia L. Swanson, M.S.)

is the possibility of repeated temperature-based nucleic acid dissociation-association cycles, which results in amplification of faint signals. In situ hybridization may be used to visualize production of messenger RNA; thus it is particularly valuable in detection of early cellular responses or responses where production of signaling or effector molecules is very low or the existence of those molecules is highly transient due to the action of reversal or control pathways (Fig. 12a, b).

9  Ancillary Morphological Assay Procedures 9.1  Tissue Microarray

Tissue microarray (TMA) is a histological technique whereby small cores of tissues are oriented in an array in a single paraffin block, thus allowing multiple tissues to be efficiently analyzed by various

20

George A. Parker

Fig. 12 (a) Chromagenic in situ hybridization (ISH) allows visualization of nucleic acid target sequences in tissues, as opposed to the peptide targets that are typically revealed by immunohistochemistry. Messenger RNA (mRNA), a common target for ISH, is very labile in fixed tissue specimens; therefore, it is desirable to demonstrate the presence of the mRNA product of an invariably present “housekeeping” gene in order to confirm that mRNA species were adequately preserved in the specimen. This ISH-stained section of the small intestine from a rhesus macaque reveals an abundance of the PPIB mRNA target. PPIB in situ hybridization, 20× objective magnification. (b) Chromogenic in situ hybridization (ISH) staining directed at Lgr5 mRNA expression reveals a population of positively stained cells in the deep aspect of small intestinal crypts of a rhesus macaque, thus confirming the presence of an Lgr5-positive population of intestinal stem cells. Lgr5 in situ hybridization staining, 20× objective magnification

histochemical stains, immunohistochemistry, or in situ hybridization. Tissue microarrays may represent a broad selection of tissues, or selected tissues that represent one or more organ systems. It is common practice to prepare TMAs based on multiple neoplasms, e.g., multiple lung carcinoma specimens from individual subjects. Most often TMAs contain duplicate or triplicate samples from individual tissue specimens, thus allowing confirmation of any observations in individual specimens. The number of tissue cores included in each TMA is determined by the bore size of the punch used to create the tissue cylinders. Standard TMA blocks commonly have 30, 60, or 90 individual tissue cylinder cross-sections (Fig. 13a, b). Tissue microarrays are routinely employed in basic biomedical research and are applied with some frequency in toxicology investigations (Morgan et al. 2002; Irwin et al. 2004; Luebke et al. 2006). MALDI analysis (see below) is also possible on TMA sections (Mascini et al. 2015; Powers et al. 2014). 9.2  Tissue Cross-Reactivity

Tissue cross-reactivity (TCR) studies are based on immunohistochemistry staining of multiple tissues, typically using a primary antibody that is prepared to indicate the presence of a bound test material such as a biopharmaceutical molecule. The underlying principle is the expectation that binding of a test material to an unexpected tissue site may be a harbinger of an untoward reaction in a tissue other than the pharmaceutically relevant tissues. The initial procedure for TCR studies was outlined in the US FDA publication entitled “Points to Consider in the Manufacture and

Introduction to Toxicologic Pathology

21

Fig. 13 (a) Histological sections of the small and large intestine of a cynomolgus macaque demonstrate the relative size of the tissue cores that result from use of 1.04-, 1.5-, and 2-mm microarray tissue punches. Though smaller cores allow a greater number of tissue samples on a microslide, the small cores may not be completely representative of the entire tissue. Selection of core diameter requires judgments related to tissue structure and the overall goals of the analytical procedure. (b) This H&E-stained tissue microarray contains numerous tissue specimens; thus it is a very efficient tool for use in many tissue-based analytical procedures. It is standard practice to include multiple sections of a tissue microarray block on a single slide, thus allowing duplication of analytical procedures with a minimum of reagent and labor investment

Testing of Monoclonal Antibody Products for Human Use,” first published in 1983 and revised in 1987, 1994, and 1997. Earlier versions of TCR tests included tissues from humans, rats, and nonhuman primates, but more recent versions are focused largely on human tissues. More details on the TCR assay are available in multiple publications (Bussiere et al. 2011; Hall et al. 2008; Leach et al. 2010). 9.3  Laser-Capture Microdissection

Laser-capture microdissection (LCM) is a general term for a number of histological techniques that allow selective capture of individual cells or tissues from within a broader expanse of the histological section. These procedures involve the use of very fine (e.g., 1 μm diameter) laser beams to cut histological tissue sections as they are viewed via light microscopy. The areas of interest may be translocated to a capture container, or may be retained on the slide as extraneous areas are deleted. One procedure involves application of a thin film of a plastic-type material to the surface of a histological section, followed by directing a very fine laser beam to the area of film that overlies the cell or tissue of interest. Interaction of the laser beam with the film and underlying tissue causes the cell or tissue to become adhered to the film. The selected subpopulation of cells or tissues is subsequently detached from the film into a separate container. After collection, the cells or tissues of interest are used for various investigational purposes, often involving molecular biology assays. The following references present the salient features of LCM (Emmert-Buck et al. 1996; Espina et al.

22

George A. Parker

2006) as well as examples of its application in toxicology (Cullen et al. 2010; Dunnick et al. 2016). 9.4  Morphometric Analysis

In some situations the standard subjective analysis of histological sections is not sufficiently precise to accomplish established goals. A greater level of precision may be possible with carefully selected counts or measurements, though these procedures tend to be labor-intensive and costly (Fig. 14a–d). Historically, morphometric analysis involved preparation of photomicrographic prints with superimposed measurement lines or grids to introduce the quantitative element to the analysis. More recently, morphometric analysis has been performed on digital images or scanned (“virtual”) slides, using software that greatly facilitates the capture of morphometric data. In performing morphometric analysis, the analyst must remain cognizant of the underlying requirement for technically adequate histological preparations that are representative of the pathologic entity of interest. No degree of analytical precision will yield meaningful data if the substrate specimens fail to meet these basic requirements. Those proposing to undertake the additional expense of morphometric analysis must also decide whether the additional level of precision would contribute significantly to the overall decision-making process. As a commonly encountered example, in routine circumstances, it would make little sense to employ histological morphometric analysis to precisely characterize the degree of xenobiotic-associated hepatocellular hypertrophy when the degree of that liver alteration is revealed with adequate precision by the organ weights collected at necropsy. A specific form of morphometric analysis consisting of linear measurements of various brain structures is incorporated into developmental neurotoxicity study guidelines (US Environmental Protection Agency OPPTS 870.6300) and the neurotoxicity arm of extended one-generation developmental toxicity studies (EOGRTS) (Organisation of Economic Cooperation and Development Test Guideline 443). These linear measurements are a response to the recognition that chemically mediated alteration in brain size may occur in the absence of detectable histological alterations. See Chapter, “Pathology of Juvenile Animals,” for a presentation of linear measurements used in the analysis of developmental neurotoxicity studies. Linear measurements of the height of thyroid follicular epithelial cells, and areal measurements of the colloid content of thyroid follicles, may provide a more quantitative indication of the effects of thyrotoxicants (Fig. 15a, b). Semi-automated counting of ovarian follicles, particularly when stained by selective immunohistochemical staining procedures, may offer advantages over manual counting when definitive estimates of ovarian follicle populations are needed (Picut et al. 2008) (Fig. 16a, b).

Introduction to Toxicologic Pathology

23

Fig. 14 (a) A section of normal lung tissue from a rhesus macaque has a small amount of blue-stained collagenous tissue within alveolar walls. Masson’s trichrome stain, 20× objective magnification. (b) In this type of morphometric analysis, the blue color of the Masson’s trichrome-stained collagenous tissue in the lung is selected by the analytical program and “painted” a primary color for subsequent analysis. In this rudimentary example, the number of green-colored pixels would be expressed per the total area of the lung image. In a real-life example, it would be necessary to compensate for the amount of lung tissue versus the air-filled spaces to arrive at a meaningful estimate of the amount of collagenous tissue in the lung. (c) This section of lung tissue is from a rhesus macaque collected 119 days after receiving 10 Gy of whole-body ionizing radiation with 5% bone marrow shielding. Note the larger amount of blue-stained collagenous tissue within alveolar walls, as compared to the naïve control animal in the previous images. Masson’s trichrome stain, 20× objective magnification. (d) The blue-stained collagenous tissue is “painted” green by the analytical program, and the amount of collagen in the lung is estimated by counting the green pixels, which equates to relative area of collagen deposition

9.5  Stereological Analysis

Stereological analysis in a broad sense consists of techniques for statistically based analysis of three-dimensional objects or spaces. As applied to toxicologic pathology, stereological analysis most commonly applies to counting or measuring objects or structures in entire organs. Rather than relying on exhaustive step-sectioning of the entire organ, histological sections collected at intervals are

24

George A. Parker

Fig. 15 (a) This H&E-stained section of the thyroid gland was collected from a rat at postnatal day (PND) 61. Note the variation in size of the pink-stained colloid accumulations within follicles. H&E stain, 10× objective magnification. (b) In a basic color segmentation-type analysis, the light pink follicular colloid is “painted” a distinctive color such as green, which can then be subjected to various forms of morphometric analysis. Common forms of analysis would include overall colloid content relative to the total tissue or mean size of colloid accumulations. H&E stain with color segmentation morphometric analysis, 10× objective magnification

Fig. 16 (a) This section of rat ovary was subjected to immunohistochemical staining for proliferating cell nuclear antigen (PCNA), which selectively stains the small round primordial and primary follicles as well as several other structures in the section (Picut et al. 2008). (b) The morphometric analytical program detected the population of primordial and primary follicles via a combination of color segmentation, size of the structures, and the “roundness” of the structures. A variety of structures were selectively stained by the PCNA IHC staining, but the morphometric analytical process displayed excellent fidelity in selecting only primordial and primary follicles

Introduction to Toxicologic Pathology

25

used to estimate the population of the entire organ, as based on the Cavalieri principle (Gundersen and Jensen 1987). In its simplest form, the Cavalieri principle states that the three-dimensional ­volume of a structure can be estimated by preparing cross-sections of the structure, performing multiple two-dimensional area ­measurements of those cross-sections, and multiplying by the linear interval between the two-dimensional sections. Preparation of histological sections for stereological analysis is based on a process known as systematic uniform random sampling (SURS) (Gundersen and Jensen 1987). Various probes are then used to interrogate the specimens in order to arrive at statistically relevant counts or measurements (Boyce et al. 2010a, b). Many journals in the neuroscience area require unbiased stereological analysis if structure-based counts or measurements are end-points. Similar requirements are proposed for studies of respiratory tissues (Knudsen and Ochs 2011). Though not typically mandated by regulatory requirements, stereological analysis should be considered in situations where definitive counts or measurements are required. For example, studies involving potential therapies for Parkinson’s disease commonly require definitive enumeration of tyrosine hydroxylase-­ positive “dopaminergic” neurons in the substantia nigra region of the brain (Fig. 17). Stereological analysis is required in situations involving neuronal population counts, as studies have shown that simple visual examination of histological sections may be inadequate for detection of toxicologically relevant alterations in neuronal numbers (de Groot et al. 2005). It is critical for those involved in study design to understand that stereological analysis must be planned in advance of specimen collection and should not be attempted retrospectively after routine studies have been completed. While the immediate application of stereological analysis in toxicologic pathology is based on analysis of histological sections, it should be noted that other forms of planar, two-dimensional tissue presentations (e.g., computerized tomography) could also be subjected to stereological analysis techniques. 9.6  Matrix-Assisted Laser Desorption Ionization (MALDI)Mass Spectrometry Imaging (MSI)

MALDI-MSI represents an additional morphology-based investigational technique in the progression from routine light microscopy, special histochemical stains, immunohistochemistry (IHC), and in situ hybridization (ISH). MALDI coupled with mass spectrometry imaging (MALDI-MSI) utilizes frozen or formalin-fixed, paraffin-embedded (FFPE) specimens coated with a matrix, which is then probed with a two-dimensional laser to collect a mass spectrum of the area of interest. The associated computer program assembles the spectral data upon an optical image, thus allowing determinations of the molecular content of the areas of interest (Maronpot et al. 2017). MALDI-MSI coupled with gene expression analysis has the potential to render exquisite insight into the basic nature of pathological processes (Brown et al. 2016). MALDI

26

George A. Parker

Fig. 17 The image illustrates the procedure for counting tyrosine hydroxylase-­ positive neurons using physical dissector stereological analysis. Scanned images of adjacent tissue sections are aligned digitally by the computer program, and the cells within a counting frame in one histological section (the reference section) but not the other (the lookup section) are counted, using the presence of the nucleolus as a marker. This insures that large cells are counted only once. Cells with nuclei touching the green lines are included in the count and cells with nuclei touching the red lines are excluded. The blue dotted line defines the sampling region. The blue “A” indicates a cell which has been included in the count for this dissector pair. (Image compliments of Dr. Danielle Brown and Cynthia L. Swanson, M.S., Charles River Laboratories, Durham, NC)

coupled with Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR MS) may be used to identify the chemical constituents of crystalline deposits in tissues (Lenz et al. 2018). When coupled with histopathological analysis, molecules unique to distinct cell populations and pathologies can be identified (Fig. 18). When used to localize test articles in tissues, MALDI has the advantage of identifying the exact molecular signature of test article molecules (Carter et al. 2015), while autoradiography reveals both labeled test article molecules and any metabolite molecules containing the labeling isotope. As with special histochemical stains, immunohistochemical staining, and in situ hybridization, MALDI studies can be performed retrospectively after routine histopathological analysis has been completed. 9.7  Magnetic Resonance Imaging (MRI)

Magnetic resonance imaging (MRI) uses a powerful magnet and radio-frequency energy to detect atomic nuclei, such as hydrogen protons, within tissues or bodies (Maronpot et al. 2017) (Fig. 19). MRI is widely used as a clinical investigative tool and may have applicability in the in-life phase of some toxicology studies, particularly in situations where repeated non-destructive tissue examination is required (Hockings et al. 2002; Wu and Wu 2009; Ramot et al. 2017; Shaltiel-Karyo et al. 2017). In addition to these in-life

Introduction to Toxicologic Pathology

27

Fig. 18 MALDI-FTICR-MS images of the N-glycans unique to distinct cell populations and pathologies in lung tissue from a rhesus macaque collected 180 days after receiving 10 Gy of whole-body ionizing radiation with 5% bone marrow shielding. Hex3HexNac5 shown in blue (a), Hex5HexNac4 displayed in green (b), and Hex5HexNAc4dHex1 in red (c). Hex = hexose, dHex = fucose, and HexNAc = N-acetylhexosamine, for N-glycan sugar composition. A merged ion image of the three N-glycans is shown (d) to highlight their differential distribution in regions of specific cell populations and pathologies. This is more readily observed by comparing the merged ion image with its corresponding H&E section (e); specific pathologies highlighted by the glycans are shown at a higher magnification in the boxes to the right. Hex5HexNAc4dHex1 shown in red is detected across the parenchyma with increases observed in regions of hyperplasia. Hex3HexNac5 shown in blue is highly localized to regions of mucus accumulation. Hex5HexNac4 shown in green is detected across the parenchyma with an increased detection in signal observed in regions of alveolar macrophage accumulation. (Image compliments of Dr. Claire L. Carter and Dr. Maureen A. Kane (University of Maryland School of Pharmacy Mass Spectrometry Center). Methodology for MALDI-FTICR-MS images is described in Carter et al. (2018))

applications, there is some interest in the use of MRI as an aid in routine histology specimen preparation, particularly in neurotoxicology (Hanig et al. 2014; Johnson et al. 2011). The basis for this interest is the known focal or multifocal nature of many forms of neurotoxicity and the potential that standard prospective histological sectioning may fail to reveal significant tissue alterations. 9.8  Computed Tomography (CT)

Computer-aided tomography (CT) uses a rotating X-ray source to produce slices through a living body or a specific organ (Maronpot et al. 2017). Computer assistance allows the slices to be presented as a three-dimensional view of the body or organ. Micro-CT imag-

28

George A. Parker

Fig. 19 Magnetic resonance imaging (MRI) has considerable potential for in vivo and ex vivo detection of lesions in toxicology studies. (a) High-performance compact MRI imaging instrument. (b) Two-dimensional views of the same liver specimens using different MRI image acquisition settings. (c) MRI rendering and segmentation of a whole liver showing multiple focal lesions in an Mdr -/- knockout mouse. (d) H&E-stained section of one of the focal lesions shown in (c). (Image compliments of Dr. Abraham Nyska) (Ramot et al. 2017)

ing systems allow resolutions as low as 3.25–9 μm, but movement of the subject during image capture is a complicating factor. There are numerous publications that demonstrate the use of CT imaging in toxicologic pathology (Maronpot et al. 2017). 9.9  Confocal Microscopy

In biosciences applications, confocal microscopy is commonly used to identify fluorochrome-labeled target molecules in cells or tissues (Fig. 20). Confocal microscopy systems involve an excitation light source, intermediate optics that typically include a high-quality microscope, and detection devices such as CCD cameras or photomultiplier tubes (PMTs) (Zucker 2006). Computer programs allow specimens to be visually rotated so that cells or structures may be examined in three dimensions. Confocal microscopy may have applications in aspects of toxicologic pathology that are difficult to investigate using two-dimensional techniques, e.g., alterations in synaptogenesis (McLeod et al. 2017).

Introduction to Toxicologic Pathology

29

Fig. 20 Confocal microscopy on cultured malignant mouse B-1 cells treated with fluorescein-labeled antisense IL-10 (asIL-10) and counterstained with rhodamine-­labeled annexin V to label surface expression of phosphatidylserine (apoptosis marker). Antisense IL-10 was administered to the malignant cells by incorporating asIL-10 into amorphous liposomes (“cochleates”), which repeatedly fuse with the surface membrane of cells and deliver small doses of asIL-10 to the malignant cells. B-1 cells depend on IL-10 as a growth and survival factor; therefore, binding of IL-10 mRNA by asIL-10 results in prompt apoptosis of the neoplastic cells (Parker et al. 2000)

9.10  Radiography/ Densitometry

Histopathological evaluation is commonly inadequate in the detection of physiological alterations such as those seen in the cardiovascular system or nervous system and, on occasion, may be inadequate for detection of microanatomic abnormalities. The latter issue is commonly encountered with alterations in bone or tooth density, which are rendered largely invisible to histopathological detection as the hard tissue is decalcified in routine histological processing. Radiographic or densitometric analysis may be employed in detecting these forms of hard tissue alteration (Boyce et al. 2014; Doyle et al. 2017; Ominsky et al. 2017; Varela et al. 2017). See Chapter, “Pathology of Bone, Skeletal Muscle, and Tooth,” for additional information regarding radiography and densitometry in evaluation of skeletal alterations.

9.11  Non-decalcified Tissue Sections

While the most common histology preparations are based on cutting thin sections of tissue held in some form of matrix, such as paraffin or resins, it is also possible to embed un-decalcified hard tissues in a matrix and grind the surface away to result in a relatively thin tissue section that can be visualized by light microscopy

30

George A. Parker

(Hillmann et al. 1991). Examination of un-decalcified bone specimens is obviously critical in the investigation of medicinal products aimed at therapy of altered bone metabolism and calcification (Boyce et al. 1990, 1995). This approach is particularly valuable in the investigation of interactions between medical devices and surrounding hard or soft tissues (Hunt and Callaghan 2008; ­ Pinheiro et al. 2014).

10  Ultrastructural Assay Procedures 10.1  Transmission Electron Microscopy

Light microscopic magnification of stained tissue specimens allows detection of changes that are invisible to the naked eye, but there is an upper limit on magnification that is related to the resolving power of light microscopy. Resolving power, defined in practice as the ability to determine that two closely approximated small structures are indeed two structures rather than a single structure, is determined by the physical properties of the microscope and the incident light. The resolving power of a light microscope is determined by the numerical aperture of the objective, the wavelength of the incident light, and the number of phase changes in the light path. The wavelength of visible light ranges from roughly 400 to 700 nanometers, which equates to a resolving power of approximately 0.2 μm under optimal conditions. Detection and interpretation of histological changes in toxicologic pathology is most commonly based on relatively low magnifications that are easily achievable with routine light microscopy but, on occasion, it is necessary to visualize structures that are beyond the resolving power of light microscopy. Transmission electron microscopy (TEM) is commonly employed in these situations. Rather than using a visible light source, TEM utilizes an electron beam with very short wavelength to visualize ultra-thin tissue sections stained with metallic stains, which allows resolution at extremely high magnification. Optimal TEM examination requires special tissue fixatives such as glutaraldehyde and special fixation procedures such as organ or whole-body perfusion, but in some situations it is possible to derive the required ultrastructural information from TEM performed on routine formalin-fixed specimens. Use of TEM is commonly required for confirmation in safety assessment studies where test article-related phospholipid accumulation (“phospholipidosis”) is suspected (Fig. 21).

10.2  Scanning Electron Microscopy

Scanning electron microscopy (SEM) involves many of the same principles that are utilized in TEM, but specimens are coated rather than sectioned. As a result, the external surface of structures is visualized at high magnification. SEM is particularly valuable in detecting alterations in intestinal mucosa or ciliated respiratory

Introduction to Toxicologic Pathology

31

Fig. 21 Alveolar macrophages in the lung of a cynomolgus macaque contain the lamellar structures (∗) that characterize phospholipidosis. Transmission electron microscopy, 3000× magnification. (Image compliments of Dr. Vivian Chen and Alfred Inman, Charles River Laboratories)

passages, where three-dimensional alterations in villi or cilia may not be readily visible in two-dimensional sections. Special techniques in toxicologic pathology are presented in greater depth in Chapter “Routine and Special Techniques in Toxicologic Pathology” .

11  Histopathology Terminology The terminology applied to histopathological alterations has historically been a source of problems in communications between pathologists and toxicology study stakeholders. The majority of the problems have not been with detection of histological alterations, but rather with the terminology used to record and describe the alterations and interpretations of the biological significance of the observed histological alterations. Problems related to terminology are presented here, and problems related to interpretation are presented below under “Adversity.” Most readers have heard assertions that showing a histological alteration to three pathologists will result in at least four diagnoses. At first glance, such comments suggest uncertainty regarding the existence and/or biological nature of the histological alteration. However, in most cases the problems arise from the use of language to present visual observations. Languages have synonymous terms, i.e., an object of substantial size may be described as big, large,

32

George A. Parker

huge, massive, etc. All the terms may be correct and applicable but, taken individually or out of context, may appear to be incorrect or conflicting. This problem in communication of pathology evaluations has long been recognized. In medical diagnostic pathology, the Systematized Nomenclature of Pathology (“SNOP”), first published by the College of American Pathologists in 1965 (Sommers 1967), has long served as the basis for diagnostic pathology terminology. Various publications have attempted to introduce similar harmonization into the diagnostic terminology used in veterinary pathology, and it became apparent that specialized terminology was needed to address pathology observations in laboratory animals used in nonclinical safety assessment studies. Attempts to address this issue included the Standardized System of Nomenclature and Diagnostic Criteria (SSNDC) Guides (“STP Guides”) that were published by the Society of Toxicologic Pathology (STP) in conjunction with the American Registry of Pathology (ARP) and the Armed Forces Institute of Pathology (AFIP). More recently, a multinational effort entitled International Harmonization of Nomenclature and Diagnostic Criteria (INHAND) has been mounted to harmonize toxicologic pathology terminology. As with the predecessor STP Guides, the INHAND publications are based on organs or organ systems and are abundantly illustrated. The INHAND publications on rodent pathology are essentially complete as of this writing, and publications on large animal (dog, pig, and nonhuman primate) pathology are underway. Additional and updated INHAND publications are available on the websites of various societies of toxicologic pathology, e.g., the North American-based Society of Toxicologic Pathology (STP). Updates to the list of INHAND documents are available on the website of the Society of Toxicologic Pathology (https:// www.toxpath.org).

12  Severity Grading In addition to conveying information regarding the presence and identity of histological alterations, the pathologist is also expected to give some indication of the extent or toxicologic importance of the alterations. That indication has historically taken the form of severity grades such as minimal, mild, moderate, marked, or severe. In diagnostic pathology, in contrast to toxicologic pathology, these appellations were initially related to clinical significance, with minimal or mild alterations expected to have little impact on overall well-being of the patient, and marked or severe alterations having serious, potentially life-threatening impact. These historically based severity grades have been translated into toxicologic pathology and, in the age of computerization, have been given numerical

Introduction to Toxicologic Pathology

33

counterparts; however, the assignment by the study pathologist is typically independent of perceived functional impact, adversity, clinical relevance, etc. The application of severity grades to histopathological alterations has served an obvious purpose in communication between pathologists and clinicians and may be similarly useful in communication between pathologists and toxicology study stakeholders. However, there are differences between clinical practice and toxicology research that complicate the application of traditional severity grades. Toxicology studies typically involve multiple laboratory animals that are assembled into groups based on experimental treatments, as opposed to the single subject that is the basis for communications between pathologists and clinicians. In the clinical setting the severity grade may influence the type or immediacy of therapy, but in toxicology research a difference in severity may be the basis for a major conclusion such as a positive or negative result of the study. Given the potential for severity grades to have a serious impact on study interpretation, particular attention must be given to diagnostic criteria and uniformity in applying severity grades in toxicology studies. Precise communication is necessary if the applied severity grades can be accurately interpreted. For example, does a severity grade of “mild” relate only to comparisons within the immediate toxicology study, all studies in the pathologist’s experience, a comparison of similar studies in the departmental or corporate historical control ranges, or a discipline-wide comparison as supported by open literature publications? All these definitions may be applicable under certain circumstances. In circumstances where the precise definition of severity grades is an important facet of decision-­ making, the definitions should be clearly presented in the pathology report. The severity grades represent somewhat arbitrary ordinal categories of observations that actually exist as a continuum. While severity grades of mild versus moderate (grades 2 versus 3) may appear substantially different, the underlying histological alterations may be of severity 2.9 versus 3.1. The mathematical grading system implies a greater level of precision than is possible with human ability to visualize subtle differences. For these reasons, differences in subjectively graded tissue alterations of only one severity score are commonly disregarded. This applies only to purely subjective grading processes, as morphometric analysis can be expected to provide more precise data. In the field of traditional diagnostic pathology, severity grades are generally related to expected clinical impact. However, this approach is unsatisfactory in the field of toxicologic pathology, as many xenobiotic-related histological alterations may have no clinical manifestation in the animals yet remain toxicologically important. Attempts have been made to harmonize the general application

34

George A. Parker

of severity grades, with various levels of success. Unfortunately, it probably is not possible to propose a uniform severity grading scheme that is applicable to all histological observations in all tissues. A histological alteration graded as mild in one tissue may be interpreted as inconsequential, while another alteration graded as mild in another tissue may be cause for great concern. For example, mild lymphoid depletion in the thymic cortex of rats is ­commonly encountered as a stress response that is expected to resolve following cessation of the stress-inducing factor. By contrast, mild reduction in the cellularity of a retinal cell layer would be viewed with considerable concern. This element of contextdependency is a major obstacle to the development of universally applicable severity grades. When the severity of critical test articlerelated changes potentially impacts the NOAEL/LOAEL, specific histopathologic grading criteria should be clearly described within the pathology report (Schafer et al. 2018). Conveying the overall severity and/or importance of histopathologic observations may be problematic in communication between pathologists and toxicology stakeholders. Toxicology stakeholders must be aware that histopathological evaluation is based on the examination of a limited number of 4–6-micron-thick histological sections typically presented on 1 × 3 inch glass microslides. The microslide may contain an entire cross-section of the lung lobes of a rat or mouse, but contains only representative areas of the lung from a dog, nonhuman primate, or pig. The histological alteration may involve a large percentage of the lung tissue presented on the microslide of these large animals, but if only a single lesion is present in the lung, then the overall toxicologic significance and clinical impact on the animal may be slight. Comments on the relative importance of the histopathology observations are a critical component of toxicologic pathology reports, as the pathologist is uniquely positioned to perform the required correlation between clinical records, necropsy observations, clinical pathology data, and histopathological observations. As indicated throughout this chapter, thorough and accurate communication between pathologists and toxicology study stakeholders is necessary. When a novel test article-related tissue alteration is encountered, the severity of the alteration is of interest but may not be critically important to the overall pathology interpretation of the study. However, when there is an apparent treatment-related increase in severity of a spontaneous or background tissue alteration, then precise analysis of the relative severities may be important in making a final interpretation that a test article-related tissue alteration is truly present. In these situations, it may be desirable to employ more sophisticated analysis methods (Holland and Holland 2011), as follows:

Introduction to Toxicologic Pathology

35

Ordering Method The pathologist physically sorts the slides from control and treated animals into order from the most to the least (or the converse) expression of the histological alteration. Simple visualization of the resultant slide order is an aid in determining whether the suspected treatment-related effect is more prominent in the treated animals. If a more detailed evaluation is desired, the ­Wilcoxon-Mann-­Whitney test is an excellent objective method (Holland and Holland 2011). Score Method The observation of interest is divided into ordinal classes such as minimal, mild, etc. as presented above and subjectively analyzed for association between treatment and tissue alteration. This method is most commonly employed by toxicologic pathologists (Holland 2001), but it is less sensitive than the ordering method. Studies indicate that histological alterations must be approximately 25% more severe to be detected by the score method versus the ordering method. Affected Method The pathologist determines the normal range and then examines those tissues from control and treated animals while “blinded” to treatment group and identifies tissues that are outside the normal range. The normal range can be taken from personal experience, historical control data, expert opinion, or any other source. If concurrent control values are used to establish the normal range, then the affected method becomes identical to the outside-control method presented below. A Fisher’s exact test is the most powerful way of analyzing data presented in this format (Sprent and Smeeton 2007). This method is popular, but insensitive. Tissue alterations must be approximately 50% more severe for detection by this method versus the ordering method. Pair-Contrast Method Specimens from individual animals in treated groups are paired against individual animals from the control group. The identity of the animals is coded, or blocked from the pathologist’s view, and the pathologist then chooses which of each pair of slides bears the histological alteration of interest. After breaking the code or “un-­ blinding” the slides, the pathologist determines whether animals from the treated group are more commonly affected by the histological change of interest. If the treated group has the alteration of interest in 8, 9, or 10 of the pairs, then the confidence limit is at least 94% that the alteration is treatment-related. To achieve the arbitrary 95% confidence level, then 9 or 10 of the pairs must show the treated group is affected. The sign test is the standard test for data of this type (Siegel and Castellan 1988). This method is easy

36

George A. Parker

to perform but is very insensitive, requiring a change that is 75% greater than that required for detection by the ordering method. Outside-Control Method The most extreme example of the histological alteration in the control group animals is identified, and then the treated group animals are compared to that extreme example from the control group. This method is insensitive, requiring approximately 40% increase in the histological change as compared to the ordering method. There is no routinely applied statistical test for this method (Holland and Holland 2011). Use of the most severely affected control animal as the representative of the control group is highly inappropriate, as it greatly reduces the likelihood of detecting a treatment-related alteration in the treated group. The outside-­ control method has one strength: it is only slightly affected by the presence of non-responders in the treated group. The data gathered during the ordering method are easily transferred into the outside-control method; thus the outside-control method may be used to reinforce a major weakness of the ordering method, which is significant influence of non-responders in the treated group. With the exception of the score method, these methods are not in routine use. However, each method has an advantage which can be leveraged in certain situations to increase confidence in the pathology data.

13  Adversity The major purposes of nonclinical safety (IPCS 2004) assessment studies are the identification of organ systems of toxicity, dose levels that result in toxicity, and biomarkers that provide in-life evidence of organ system toxicity. In achieving these goals, it is necessary to distinguish between potentially adverse and inconsequential test article-related tissue alterations. Despite multiple attempts, a wholly satisfactory definition of “adverse” has not been forthcoming. A definition that is widely, but not universally, accepted defines adversity as a “...change in the morphology, physiology, growth, development, reproduction, or life span of an organism, system, or (sub)population that results in an impairment of functional capacity, an impairment of the capacity to compensate for additional stress, or an increase in susceptibility to other influences” (IPCS 2004). Some of these factors are readily observable in the context of a short-term toxicology study, while other factors require estimation or speculation on the part of observers. For example, within the confines of a typical 28-day toxicology study, it may not be possible to determine effects on life span other than obvious effects on survival during the study. Determination of

Introduction to Toxicologic Pathology

37

impairments of the capacity to compensate for additional stress or increase in susceptibility to other influences raises questions as to the nature of the additional stressors or influences as well as the period of time those additional factors influence the test system. For example, it is unlikely that effects on development or reproductive capacity would be revealed in typical short-term toxicology studies. These definitions are not merely academic, as they serve as the practical basis for determining the no observed effect level (NOEL) and no observed adverse effect level (NOAEL) that are used for setting doses for subsequent animal studies and human exposures. In recognition of the importance of adversity determinations, professional societies have convened panels to provide guidelines to toxicologic pathologists. Ten recommendations of the Scientific and Regulatory Policy Committee of the Society of Toxicologic Pathology (Kerlin et al. 2016) are summarized below: 1. Adversity is a term indicating harm to the test animal. 2. The decision about whether or not test article-related effects (or a group of related effects) in a nonclinical study are considered adverse or non-adverse should be unambiguously stated and justified in sub-reports and/or the study report. 3. Adversity as identified in a nonclinical study report should be applied only to the test species and under conditions of the study. 4. Toxic effects on cells, tissues, organs, or systems within the test animal should be assessed on their own merits. 5. Communication of what is considered adverse and assignment of the NOAEL in the overall study report should be consistent with, and supported by, the information provided in the study sub-reports. 6. Communication of adverse findings and the NOAEL should include direct interaction between staff within different contributing scientific disciplines. 7. The NOAEL for a test article should be communicated in an overview document based upon data from multiple studies. 8. In order to place them in appropriate context, the use of NOAELs in data tables should be referenced to explanatory text. 9. Nonclinical scientists, including toxicologists, pathologists, and other contributing subject matter experts who interpret data from nonclinical studies, should be active participants in assessing and communicating human risk. 10. All available data from all nonclinical studies must be evaluated together to define any potential toxicities and to predict human risk.

38

George A. Parker

Concepts and guidelines in adversity interpretations are presented in greater detail in Chapter “The Pathology Report, Peer Review, and Pathology Working Group”.

14  Philosophical Considerations in Toxicologic Pathology Histopathological observations and interpretations are based in part on the training and experience of the observer. While it is true that an untrained observer with normal visual acuity should be able to make competent histopathological observations with the light microscope, experience indicates that trained and experienced observers are able to make more meaningful and accurate observations. The process of observing histomorphologic alterations in tissues is relatively straightforward once the observer becomes sufficiently familiar with normal tissue structure. However, interpreting the meaning and significance of those structural alterations requires knowledge of the underlying physiologic and biochemical functions as well as possible effects of the structural alterations. The existence of histopathologic alterations is an uncommon source of conflict in toxicologic pathology, but conflicts in interpretation commonly arise. In addition to conflicts arising from terminology and other communication issues, there may be variations in interpretation based on the training, background, and experience of the various observers. An illustrative parody may be useful here. If a group of educated observers were asked to identify the animal in Fig. 22, all would identify the animal as a horse. Those with zoological inclinations might respond with the genus and species name (Equus caballus). Some might note the visible external genitalia, thus identifying the animal as a male. Observers with a particularly keen eye might notice the horse’s back is a bit shorter than expected, and those familiar with equine anatomy would know that Arabian and Morgan horses commonly have only five lumbar vertebrae, rather than the more typical six lumbar vertebrae. If it was possible to observe the animal’s movement, the high-stepping gait that is characteristic of Morgan horses would be obvious to knowledgeable observers, but meaningless to casual observers. Thus there could be multiple correct indications of the identity of the animal, and differences in the precision of those indications, based primarily on the background of the observer and the context in which the observations are made. Some questions regarding performance of the animal presented above would be answered uniformly, while answers to other questions would vary. If asked if the animal could fly, there would be uniformly negative responses. The animal has no wings, and the ponderous body could not glide even if it possessed the gliding apparatus of a gliding gecko. There would probably be disparate

Introduction to Toxicologic Pathology

39

Fig. 22 Morgan horse stallion. Identification and interpretation of the image is dependent on the background and experiences of the observer as well as the context in which the observations are made. https://www.morganhorse.com/ about_morgan/ideal-morgan/

answers if observers were asked whether the animal could swim. The huge body mass relative to the small feet suggests the animal would have difficulties in swimming, but observers who have seen old Western movies would recall cowboys being towed across ­rivers by their horses. Those with training in equine anatomy would know the massive lungs of the horse provide substantial buoyancy, as does the partially gas-filled intestinal tract; thus the horse essentially floats for whatever time is necessary to translocate across a body of water. This same principle applies to interpretations in toxicologic pathology: opinions are influenced to some degree by training and experience. Formal training and certification of pathologists provides an indication of the basic qualifications of the pathologist, but the more subtle influences on interpretation can be determined only by thorough communication between pathologists and toxicology study stakeholders on a study-by-study basis.

15  Challenges in Toxicologic Pathology Toxicologic pathology is an exceptionally demanding professional endeavor, and those entering the specialty rarely make the choice without substantial thought. Veterinary pathologists have a number of career options related to biomedical research, teaching, or diagnosis of diseases in companion animals, animals of economic interest, or zoo animals/wildlife. Pathologists typically have important roles in each of those areas, resulting in rewarding interpersonal relationships with positive feedback regarding the contribution

40

George A. Parker

of pathologists. The end result is careers that are very pleasant and personally rewarding. Many aspects of toxicologic pathology make this area less appealing to novice veterinary pathologists as they consider a specialty area. 1. Bench-level toxicologic pathology involves extensive involvement in microscopy, which is very tedious work and can lead to physical impairments such as repetitive stress injury. The process of detecting minor histological alterations in tissue sections involves hundreds or thousands of subjective decisions each day. The pathologist is expected to apply consistent criteria for making these decisions throughout the days, weeks, or months the study is underway and to apply those criteria consistently to subsequent studies. There is also an expectation that these decisions will be harmonious with those made by other pathologists. This latter expectation presents major challenges, particularly in the area of diagnostic terminology (see above). 2. In most areas of veterinary pathology, the pathologist arrives at a diagnosis, presents and discusses that diagnosis with various stakeholders, and then prepares a final report on the case. These case reports are rarely revisited. By contrast, second and third opinions are very common in toxicologic pathology. Regulations require retention of microslides and pathology records, and the study pathologist must anticipate review of each histopathology observation and essentially every word in the pathology report. Different stakeholders may have different goals during their review; thus the reviews may become somewhat contentious. Experience gained during four decades of toxicologic pathology practice by the author suggests the most significant challenges faced by toxicologic pathologists are related to interpretation of toxicologic significance rather than the technical process of detecting histological alterations. Professional societies have attempted to harmonize these interpretive processes by the publication of thoughtful and carefully prepared guidance documents, but much remains to be accomplished in this area. 3. The demanding work of the toxicologic pathologist is performed in a background of highly stringent regulatory requirements known as the good laboratory practice (GLP) regulations. Since their introduction in the late 1970s, the GLP regulations have had an unquestionably beneficial effect that allows confidence in results obtained in nonclinical safety assessment. However, the GLP-mandated requirements for record maintenance add another layer of complexity to the work of the toxicologic pathologist. 4. The sheer size of toxicology studies may be daunting to the novice pathologist, particularly if there has been no previous

Introduction to Toxicologic Pathology

41

introduction to computer-assisted data retrieval programs. Routine veterinary diagnostic pathology primarily involves a light microscope, a word processor for generating reports, and a database for logging case material. By contrast, a toxicology study may involve thousands of laboratory animals with 60–70 histological specimens examined from each animal and numerous treatment groups that include various dose levels of one or more test materials, plus one or more components of the vehicle material. One or more pre-study necropsies are commonly performed to determine a baseline for various analyses, multiple interim necropsies may be included, and one or more recovery necropsies are commonly included in study protocols. Analysis of the histopathology data is quite different from arriving at a diagnosis based on a single biopsy specimen from a companion animal. Analysis of toxicologic pathology data requires a different mindset by the toxicologic pathologist, as well as competence in computer logistics and some level of comfort with statistical principles. Reasonable readers would question why anyone would deliberately enter a field as demanding as toxicologic pathology. While there are numerous specific reasons for this career choice, a common thread is a desire to serve humanity by helping to provide safe and effective products for the benefit of humans and animals. References Andrade MR, Yee J, Barry P, Spinner A, Roberts JA, Cabello PH, Leite JP, Lerche NW (2003) Prevalence of antibodies to selected viruses in a long-term closed breeding colony of rhesus macaques (Macaca mulatta) in Brazil. Am J Primatol 59(3):123–128. https://doi. org/10.1002/ajp.10069 Bloom W, Fawcett DW, Raviola E (1994) A textbook of histology. Chapman & Hall, New York Bolon B, Garman RH, Pardo ID, Jensen K, Sills RC, Roulois A, Radovsky A, Bradley A, Andrews-Jones L, Butt M, Gumprecht L (2013) STP position paper: recommended practices for sampling and processing the nervous system (brain, spinal cord, nerve, and eye) during nonclinical general toxicity studies. Toxicol Pathol 41(7):1028–1048. https://doi.org/10.1177/0192623312474865 Boyce RW, Franks AF, Jankowsky ML, Orcutt CM, Piacquadio AM, White JM, Bevan JA (1990) Sequential histomorphometric changes in cancellous bone from ovariohysterectomized dogs. J Bone Miner Res 5(9):947–953 Boyce RW, Paddock CL, Gleason JR, Sletsema WK, Eriksen EF (1995) The effects of risedronate on canine cancellous bone remodeling: three-­dimensional kinetic reconstruction of the remodeling site. J Bone Miner Res 10(2): 211–221

Boyce JT, Boyce RW, Gundersen HJ (2010a) Choice of morphometric methods and consequences in the regulatory environment. Toxicol Pathol 38(7):1128–1133 Boyce RW, Dorph-Petersen KA, Lyck L, Gundersen HJ (2010b) Design-based stereology: introduction to basic concepts and practical approaches for estimation of cell number. Toxicol Pathol 38(7):1011–1025 Boyce RW, Varela A, Chouinard L, Bussiere JL, Chellman GJ, Ominsky MS, Pyrah IT (2014) Infant cynomolgus monkeys exposed to denosumab in utero exhibit an osteoclast-poor osteopetrotic-­like skeletal phenotype at birth and in the early postnatal period. Bone 64:314–325. https://doi.org/10.1016/j.bone.2014.04.002 Brown HR, Castellino S, Groseclose MR, Elangbam CS, Mellon-Kusibab K, Yoon LW, Gates LD, Krull DL, Cariello NF, Arrington-Brown L, Tillman T, Fowler S, Shah V, Bailey D, Miller RT (2016) Drug-induced liver fibrosis: testing Nevirapine in a viral-like liver setting using histopathology, MALDI IMS, and gene expression. Toxicol Pathol 44(1):112–131. https:// doi.org/10.1177/0192623315617033 Bussiere JL, Leach MW, Price KD, Mounho BJ, Lightfoot-Dunn R (2011) Survey results on the use of the tissue cross-reactivity immunohisto-

42

George A. Parker

chemistry assay. Regul Toxicol Pharmacol 59(3):493–502 Carson FL (1997) Histotechnology- a self-­ instructional text. ASCP Press, Chicago Carter CL, Jones JW, Barrow K, Kieta K, Taylor-­ Howell C, Kearney S, Smith CP, Gibbs A, Farese AM, MacVittie TJ, Kane MA (2015) A MALDIMSI approach to the characterization of radiation-induced lung injury and medical countermeasure development. Health Phys 109(5):466–478. https://doi.org/10.1097/ HP.0000000000000353 Carter CL, Parker GA, Hankey KG, Farese AM, MacVittie TJ, Kane MA (2018) MALDI-MSI spatially maps N-glycan alterations to histologically distinct pulmonary pathologies following lethal-doses of irradiation. Scientific Reports in prep Crabbs TA, Miller RA, Malarkey DE (2013) Poster: centriacinar lung lesions in control rats associated with oral gavage administration. Paper presented at the 2013 ESTP, Ghent, Belgium Cullen JM, Falls JG, Brown HR, Yoon LW, Cariello NF, Faiola B, Kimbrough CL, Jordan HL, Miller RT (2010) Time course gene expression using laser capture microscopy-extracted bile ducts, but not hepatic parenchyma, reveals acute alpha-naphthylisothiocyanate toxicity. Toxicol Pathol 38(5):715–729 de Groot DM, Hartgring S, van de Horst L, Moerkens M, Otto M, Bos-Kuijpers MH, Kaufmann WS, Lammers JH, O’Callaghan JP, Waalkens-Berendsen ID, Pakkenberg B, Gundersen HG (2005) 2D and 3D assessment of neuropathology in rat brain after prenatal exposure to methylazoxymethanol, a model for developmental neurotoxicty. Reprod Toxicol 20(3):417–432 Doyle N, Varela A, Haile S, Guldberg R, Kostenuik PJ, Ominsky MS, Smith SY, Hattersley G (2017) Abaloparatide, a novel PTH receptor agonist, increased bone mass and strength in ovariectomized cynomolgus monkeys by increasing bone formation without increasing bone resorption. Osteoporos Int. https://doi. org/10.1007/s00198-017-4323-6 Dunnick JK, Merrick BA, Brix A, Morgan DL, Gerrish K, Wang Y, Flake G, Foley J, Shockley KR (2016) Molecular changes in the nasal cavity after N, N-dimethyl-p-toluidine exposure. Toxicol Pathol 44(6):835–847. https://doi. org/10.1177/0192623316637708 Emmert-Buck MR, Bonner RF, Smith PD, Chuaqui RF, Zhuang Z, Goldstein SR, Weiss RA, Liotta LA (1996) Laser capture microdissection. Science 274(5289):998–1001 Espina V, Wulfkuhle JD, Calvert VS, VanMeter A, Zhou W, Coukos G, Geho DH, Petricoin EF 3rd, Liotta LA (2006) Laser-capture microdissection. Nat Protoc 1(2):586–603

Greijdanus-van der Putten SW, van Esch E, Kamerman J, Ballering LA, van den Dobbelsteen DJ, TdR EP (2005) Drug-induced protoporphyria in beagle dogs. Toxicol Pathol 33(6):720–725 Gundersen HJ, Jensen EB (1987) The efficiency of systematic sampling in stereology and its prediction. J Microsc 147(Pt 3):229–263 Haley PJ (2012) Small molecule immunomodulatory drugs: challenges and approaches for balancing efficacy with toxicity. Toxicol Pathol 40(2):261–266 Hall WC, Price-Schiavi SA, Wicks J, Rojko JL (2008) Tissue cross-reactivity studies for monoclonal antibodies: predictive value and use for selection of relevant animal species for toxicity testing. In: Cavagnaro JA (ed) Preclinical safety evaluation of biopharmaceuticals: a science-­ based approach to facilitating clinical trials. Wiley, Hoboken, pp 207–240 Hanig J, Paule MG, Ramu J, Schmued L, Konak T, Chigurupati S, Slikker W Jr, Sarkar S, Liachenko S (2014) The use of MRI to assist the section selections for classical pathology assessment of neurotoxicity. Regul Toxicol Pharmacol 70(3):641–647. https://doi.org/10.1016/j. yrtph.2014.09.010 Hillmann G, Hillman B, Donath K (1991) Enzyme, lectin and immunohistochemistry of plastic embedded undecalcified bone and other hard tissues for light microscopic investigations. Biotech Histochem 66(4):185–193 Hockings PD, Roberts T, Campbell SP, Reid DG, Greenhill RW, Polley SR, Nelson P, Bertram TA, Kramer K (2002) Longitudinal magnetic resonance imaging quantitation of rat liver regeneration after partial hepatectomy. Toxicol Pathol 30(5):606–610 Holland (2001) A survey of discriminant methods used in toxicological histopathology. Toxicol Pathol 29(2):269–273 Holland T, Holland C (2011) Analysis of unbiased histopathology data from rodent toxicity studies (or, are these groups different enough to ascribe it to treatment?). Toxicol Pathol 39(4):569–575 Hunt JA, Callaghan JT (2008) Polymer-­ hydroxyapatite composite versus polymer interference screws in anterior cruciate ligament reconstruction in a large animal model. Knee Surg Sports Traumatol Arthrosc 16(7):655– 660. https://doi.org/10.1007/ s00167-008-0528-8 Hutto DL (2010) Opportunistic infections in non-­ human primates exposed to immunomodulatory biotherapeutics: considerations and case examples. J Immunotoxicol 7(2):120–127. https:// doi.org/10.3109/15476910903258252 IPCS (2004) International programme on chemical safety (IPCS) harmonization project: risk assessment terminology. Part 1: IPCS/OECD

Introduction to Toxicologic Pathology key generic terms used in chemical hazard/risk assessment. Part 2: IPCS glossary of key exposure assessment terminology. World Health Organization Irwin RD, Boorman GA, Cunningham ML, Heinloth AN, Malarkey DE, Paules RS (2004) Application of toxicogenomics to toxicology: basic concepts in the analysis of microarray data. Toxicol Pathol 32(Suppl 1):72–83 Johnson AG, Badea A, Jiang Y (2011) Quantitative neuromorphometry using magnetic resonance histology. Toxicol Pathol 39(1):85–91 Kerlin R, Bolon B, Burkhardt J, Francke S, Greaves P, Meador V, Popp J (2016) Scientific and Regulatory Policy Committee: recommended (“Best”) practices for determining, communicating, and using adverse effect data from nonclinical studies. Toxicol Pathol 44(2):147–162. https://doi.org/10.1177/0192623315623265 Knudsen L, Ochs M (2011) Microscopy-based quantitative analysis of lung structure: application in diagnosis. Expert Opin Med Diag 5(4):319–331 Latendresse JR, Warbrittion AR, Jonassen H, Creasy DM (2002) Fixation of testes and eyes using a modified Davidson’s fluid: comparison with Bouin’s fluid and conventional Davidson’s fluid. Toxicol Pathol 30(4):524–533 Leach MW, Halpern WG, Johnson CW, Rojko JL, Maclachlan TK, Chan CM, Galbreath EJ, Ndifor AM, Blanset DL, Polack E, Cavagnaro JA (2010) Use of tissue cross-reactivity studies in the development of antibody-based biopharmaceuticals: history, experience, methodology, and future directions. Toxicol Pathol 38(7):1138–1166 Lenz B, Brink A, Siam M, De Paepe A, Bassett S, Eichinger-Chapelon A, Maliver P, Neff R, Niederhauser U, Steinhuber B, Zurbach R, Singer T, Funk C, Schuler F, Albassam M, Schadt S (2018) Application of imaging techniques to cases of drug-induced crystal nephropathy in preclinical studies. Toxicol Sci 163(2):409–419. https://doi.org/10.1093/ toxsci/kfx044 Leonovich SA (2010) The lung mite Pneumonyssus semicola Banks (Halarachidae) in lungs of the rhesus monkey Macaca mulatta. Acarina 18:89–90 Luebke RW, Holsapple MP, Ladics GS, Luster MI, Selgrade M, Smialowicz RJ, Woolhiser MR, Germolec DR (2006) Immunotoxicogenomics: the potential of genomics technology in the immunotoxicity risk assessment process. Toxicol Sci 94(1):22–27 Maronpot RR, Nyska A, Troth SP, Gabrielson K, Sysa-Shah P, Kalchenko V, Kuznetsov Y, Harmelin A, Schiffenbauer YS, Bonnel D, Stauber J, Ramot Y (2017) Regulatory forum

43

opinion piece∗: imaging applications in toxicologic pathology-recommendations for use in regulated nonclinical toxicity studies. Toxicol Pathol 45(4):444–471. https://doi. org/10.1177/0192623317710014 Mascini NE, Eijkel GB, ter Brugge P, Jonkers J, Wesseling J, Heeren RM (2015) The use of mass spectrometry imaging to predict treatment response of patient-derived xenograft models of triple-negative breast cancer. J Proteome Res 14(2):1069–1075. https://doi.org/10.1021/ pr501067z McLeod F, Marzo A, Podpolny M, Galli S, Salinas P (2017) Evaluation of synapse density in Hippocampal Rodent brain slices. (128):e56153. https://doi.org/10.3791/56153 Morgan KT, Ni H, Brown HR, Yoon L, Qualls CW Jr, Crosby LM, Reynolds R, Gaskill B, Anderson SP, Kepler TB, Brainard T, Liv N, Easton M, Merrill C, Creech D, Sprenger D, Conner G, Johnson PR, Fox T, Sartor M, Richard E, Kuruvilla S, Casey W, Benavides G (2002) Application of cDNA microarray technology to in vitro toxicology and the selection of genes for a real-time RT-PCR-based screen for oxidative stress in Hep-G2 cells. Toxicol Pathol 30(4):435–451 Ominsky MS, Boyd SK, Varela A, Jolette J, Felx M, Doyle N, Mellal N, Smith SY, Locher K, Buntich S, Pyrah I, Boyce RW (2017) Romosozumab improves bone mass and strength while maintaining bone quality in ovariectomized cynomolgus monkeys. J Bone Miner Res 32(4):788–801. https://doi.org/10.1002/jbmr.3036 Parker GA (2016) Pathology evaluation for detection of immunomodulation. In: Parker GA (ed) Immunopathology in toxicology and drug development, vol 1. Humana Press (Springer Nature), Cham, pp 371–432 Parker GA, Snyder PW (2017) Pathology evaluation for detection of immunomodulation. In: Parker GA (ed) Immunopathology in toxicology and drug development, Molecular and integrative toxicology, vol 1, 1st edn. Springer International Publishing AG/Human Press, Cham, pp 371–442 Parker GA, Peng B, He M, Gould-Fogerite S, Chou CC, Raveche ES (2000) In vivo and in vitro antiproliferative effects of antisense interleukin 10 oligonucleotides. Methods Enzymol 314:411–429 Picut CA, Swanson CL, Scully KL, Roseman VC, Parker RF, Remick AK (2008) Ovarian follicle counts using proliferating cell nuclear antigen (PCNA) and semi-automated image analysis in rats. Toxicol Pathol 36(5):674–679 Pinheiro FA, Mourao CF, Diniz VS, Silva PC, Meirelles L, Santos Junior E, Schanaider A (2014) In-vivo bone response to titanium screw

44

George A. Parker

implants anodized in sodium sulfate. Acta Cir Bras 29(6):376–382 Powers TW, Neely BA, Shao Y, Tang H, Troyer DA, Mehta AS, Haab BB, Drake RR (2014) MALDI imaging mass spectrometry profiling of N-glycans in formalin-fixed paraffin embedded clinical tissue blocks and tissue microarrays. PLoS One 9(9):e106255. https://doi. org/10.1371/journal.pone.0106255 Ramot Y, Schiffenbauer YS, Maronpot R, Nyska A (2017) Compact magnetic resonance imaging systems-novel cost-effective tools for preclinical drug safety and efficacy evaluation. Toxicol Sci 157(1):3–7. https://doi.org/10.1093/toxsci/ kfx024 Schafer KA, Eighmy J, Fikes JD, Halpern WG, Hukkanen RR, Long GG, Meseck EK, Patrick DJ, Thibodeau MS, Wood CE, Francke S (2018) Use of severity grades to characterize histopathologic changes. Toxicol Pathol 46(3):256–265. https://doi.org/10.1177/0192623318761348 Schmued LC, Hopkins KJ (2000) Fluoro-Jade: novel fluorochromes for detecting toxicant-­ induced neuronal degeneration. Toxicol Pathol 28(1):91–99 Schoeb TR, McConnell EE (2011a) Mycoplasma pulmonis and lymphoma in a methanol bioassay. Vet Pathol 48(4):903–905. https://doi. org/10.1177/0300985811404713 Schoeb TR, McConnell EE (2011b) Commentary: further comments on mycoplasma pulmonis and lymphoma in bioassays of rats. Vet Pathol 48(2):420–426. https://doi.org/10.1177/ 0300985810377183 Schoeb TR, McConnell EE, Juliana MM, Davis JK, Davidson MK, Lindsey JR (2009) Mycoplasma pulmonis and lymphoma in bioassays in rats. Vet Pathol 46(5):952–959. https://doi.org/ 10.1354/vp.08-VP-0240-S-COM Seaton M (2014) The study pathologist’s role in GLP studies: a regulator’s perspective. Toxicol Pathol 42(1):285. https://doi.org/10.1177/ 0192623313506878 Shaltiel-Karyo R, Tsarfati Y, Rubinski A, Zawoznik E, Weinstock I, Nemas M, Schiffenbauer YS, Ramot Y, Nyska A, Yacoby-Zeevi O (2017)

Magnetic resonance imaging as a noninvasive method for longitudinal monitoring of infusion site reactions following administration of a novel apomorphine formulation. Toxicol Pathol 45(4):472–480. https://doi.org/10. 1177/0192623317706111 Sheehan DC, Hrapchak BB (1980) Theory and practice of histotechnology. Battelle Press, Columbus Siegel S, Castellan HJ (1988) Nonparametric statistics for the behavioural sciences. McGraw-Hill, New York Sommers SC (1967) Systematized nomenclature of pathology. Pathol Microbiol (Basel) 30(5): 826–827 Sprent P, Smeeton NC (2007) Applied nonparametric statistical methods, 4th edn. Chapeman & Hall/CRC, Boca Raton Thompson SW (1966) Selected histochemical and histopathological methods. Charles C Thomas Publisher, Springfield van Gorder MA, Della Pelle P, Henson JW, Sachs DH, Cosimi AB, Colvin RB (1999) Cynomolgus polyoma virus infection: a new member of the polyoma virus family causes interstitial nephritis, ureteritis, and enteritis in immunosuppressed cynomolgus monkeys. Am J Pathol 154(4): 1273–1284 Varela A, Chouinard L, Lesage E, Guldberg R, Smith SY, Kostenuik PJ, Hattersley G (2017) One year of abaloparatide, a selective peptide activator of the PTH1 receptor, increased bone mass and strength in ovariectomized rats. Bone 95:143–150. https://doi.org/10.1016/j. bone.2016.11.027 Wu Y, Wu EX (2009) MR study of postnatal development of myocardial structure and left ventricular function. J Magn Reson Imaging 30(1): 47–53 Young B, Lowe JS, Stevens A, Heath JW (2006) Wheater’s functional histology-a text and colour atlas. Churchill Livingston (Elsevier), Philadelphia Zucker RM (2006) Quality assessment of confocal microscopy slide based systems: performance. Cytometry A 69(7):659–676. https://doi. org/10.1002/cyto.a.20314

Chapter 2 The Pathology Report, Peer Review, and Pathology Working Group Ted A. Birkebak and Peter C. Mann Abstract The pathology report is the mechanism by which the important results of the pathology assessment are communicated to the interested parties, which range from study directors, sponsors, regulators, and investors. In this chapter, we review the structure of a pathology report and what should be expected to be in each section. The sections of a pathology report generally include a summary, description of methods, and results. The results section consists of a discussion on organ weights, macroscopic findings, and microscopic findings and should identify test article-related findings and address their significance. There is a review of terminology that is often used in pathology reports and which can be confusing to a non-­ pathologist. Interpretation of adversity is also discussed. The chapter expands on pathology reporting by discussing quality assessment of the pathology report through peer review and pathology working groups. The types of peer reviews and the methodology for performing them are presented as is a discussion on conducting a pathology working group. Key words Pathology reports, Summary, Methods, Organ weights, Macroscopic, Microscopic, Adversity, Peer review, Pathology working group

1  Overview and Format The goals of the pathology evaluation are to identify potential test article-related effects, name and categorize findings in a reproducible fashion that enables correlations between test article exposure and biological effects, and then interpret and present those findings in a report that accurately conveys that information. The pathology report should enable the reader, whether a pathologist, toxicologist, or regulatory reviewer, to understand what findings are related to test article administration, whether they are adverse in the test species, their potential reversibility, and if they are likely to be relevant to humans. There are two formats for pathology reporting in toxicology studies: a narrative that is part of an integrated study report that is

Thomas J. Steinbach et al. (eds.), Toxicologic Pathology for Non-Pathologists, https://doi.org/10.1007/978-1-4939-9777-0_2, © Springer Science+Business Media, LLC, part of Springer Nature 2019

45

46

Ted A. Birkebak and Peter C. Mann

signed by all contributors and a stand-alone phase report (also known as a pathology contributing scientist report) that is solely authored and signed by the study pathologist. The potential advantage of an integrated report is that it is thought to facilitate a more comprehensive interpretation of all the study data since all contributing scientists are cognizant of data from other disciplines and contribute to the interpretation of the data. The potential advantage of the stand-alone phase report is that there is better individual accountability for interpretation of the pathology data. Due to the regulatory preference (Vishwanathan 2005) for a sole point of control for the pathology evaluation and also to industry standard practices, one is more likely to see a stand-alone phase report, which is the focus of this chapter. Regardless of format, the report should contain a narrative that presents the pathologist’s interpretations and conclusions and individual and summary data tables that contain mortality, organ weight, macroscopic finding, and microscopic finding data. In some instances, it can include clinical pathology data, ultrastructural findings, or immunohistochemistry findings. As no-effect and no-adverse-effect levels are often determined based on the data in the pathology report, its contents are usually key to the overall study conclusions. The goal of this chapter is to familiarize the reader with the contents and layout of the stand-alone phase report and to provide guidance on interpretation of some aspects of the pathology data that may not be easily apparent to a non-pathologist. For each report component, the following text will address how the data and narrative are usually presented and will try to highlight items that should be considered when reviewing the report.

2  Report Contents The contents of a GLP-compliant pathology phase report consist of a Table of Contents, usually with hyperlinks to report components, a Summary or Executive Summary, a Methods section, a Results and Discussion section (may be separate sections), Individual and Summary Data Tables, a Conclusion section (can be optional), and a Signature Page. If the phase report is generated by a principal investigator, quality assurance documentation may be included in the phase report. Of these, the sections that can vary most and convey the most important content are the Summary or Executive Summary, Methods, Results and Discussion, Conclusion, and data tables. Ideally, text in these sections will be written so that key portions can be copied and placed directly into the main study report and into documents for submission to regulatory agencies; most toxicology laboratories have report guidelines in place that generate these sections with this in mind.

The Pathology Report, Peer Review, and Pathology Working Group

47

3  Summary The Summary, or Executive Summary, section is the first and most concise look at the study results; the overall goal of the summary is to clearly present test article-related findings by organ system and dose level. It is a high level overview with limited detail and typically consists of an introduction, a concise summary of findings, and a conclusion. The introductory paragraph should contain a short description of the methods, often taken directly from the protocol with appropriate modifications to address amendments and deviations, and should include the study objective, duration of the study, dose route, and species. This is followed by a summary of results that describes test article-related mortality, macroscopic findings, organ weight changes and microscopic findings by dose level and organ. When applicable, the summary includes short statements concerning the adversity or reversibility of these test article-related findings. Incidental findings or findings of uncertain relationship to treatment should not be discussed in the summary. Ideally, statements within the results summary are concise and definitive; there should be no speculative statements here as text from this section often finds its way into regulatory submission documents. A conclusion section may be present in the executive

Box 1 Example of an executive summary of the pathology report

48

Ted A. Birkebak and Peter C. Mann

summary; it varies with organizational preferences and is often most useful for studies with extensive or complex findings. If present, the conclusion is usually not a repeat of previous results but rather a statement summarizing the general pathologic or toxicologic processes (e.g., administration of the test article was associated with single cell necrosis of epithelial cells in the kidney, liver, and intestinal tract), the dose levels that were affected, the adversity of the effect(s), and an indication of the potential for reversibility. Example report summary text follows below in Box 1.

4  Methods The methods section content varies between laboratories but in most cases includes general study design information and information specific to the anatomic pathology data collection. In a report generated by a principal investigator, archiving information can also be present. The variation between labs usually results from differences in the extent to which they just reference the protocol rather than repeat the protocol text in this section. General study design information included in this section consists of the test species, test article, vehicle, study duration, dose-­ route, dose levels, and animal assignments; this information is present in both text and tabular format (often the Table of Experimental Design is copied from the Methods section of the protocol). Methodology specific to the anatomic pathology evaluation should indicate or reference which tissues were examined grossly, weighed, and collected at necropsy; a description of how the tissues were fixed, processed, and stained; a list of protocol tissues examined microscopically as part of a complete review1 and identification of animals and dose levels that had a complete review; and a list of potential target tissues that were examined microscopically 1

 With a few exceptions, a complete review is an examination of all protocollisted tissue samples collected at necropsy. A few samples on the protocol list, such as nasal cavity, spinal cord, or bone marrow smears, may be collected for conditional review and are not always part of a complete review. Complete reviews are typically conducted on all (main study and recovery) high-dose and control group animals in rodent studies and on all animals in all groups in nonrodent studies. Recommendations on the tissue sampling and examination and references to relevant regulatory guidance are presented in the following best practice article: Bregman, C.  L., Adler, R.  R., Morton, D.  G., Regan, K.  S., & Yano, B.  L. (2003). Recommended Tissue List for Histopathologic Examination in Repeat-Dose Toxicity and Carcinogenicity Studies: A Proposal of the Society of Toxicologic Pathology (STP). Toxicologic Pathology, 31(2): 252–253.

The Pathology Report, Peer Review, and Pathology Working Group

49

and identification of animals and dose levels in which they were examined. This is a protocol-derived text with the tense changed to the past tense and documentation of any changes in actual animals and tissues examined due to examination of unscheduled mortalities, identification and examination of target tissues in lower dose groups, and documentation of unexamined protocol tissues (more on that below). Finally, the method for collection, analysis, reporting, and archiving, if appropriate, of the data should be described; this will usually include identification of a pathology database. If clinical pathology endpoints are included in the evaluation, similar information concerning sample collection, handling, and analysis should be included. Special techniques, such as immunohistochemistry, ultrastructural evaluation, or in situ hybridization, would also be described in this section if performed and not reported as part of a separate phase report. Two examples of report methods text that illustrate this variation follow below in Boxes 2 and 3.

Box 2 Example of methods section describing procedures for pathology

50

Ted A. Birkebak and Peter C. Mann

Box 3 Alternate method for describing pathology procedures 4.1  Methods: Reviewing

One methodology-related item that is not always easily apparent to the non-pathologist is that there are often a few protocol-listed tissues that are not microscopically examined in some animals. These missing tissues may be due to insufficient tissue collection at necropsy; inability to identify, loss of, or damage to the tissue during processing; or inability to capture a small sample in the plane of section. In most cases, this represents an insignificant number of samples and does not impact the interpretation of the study; however, it may have impact if the missing tissues are from potential target organs or if the number of missing tissues is excessive. When reviewing a study, one should be aware of any missing tissues. If they are of potential impact, it is worth following up with the study pathologist and histology personnel to determine why they were missing and if that data is retrievable. A few laboratories identify the missing tissues in the Methods section, but many do not, so it is important to know where that information can be found. Missing tissue information may be found in summary format in a deviation form in the study file and sometimes in the main study report. If the lab did not generate a missing tissue deviation for the study, the information can also be obtained piecemeal from the Summary and Individual Data Tables that are generated by most, if not all, pathology databases. In the Summary Data Table, the number of tissues (N) examined are listed by group; and in the Individual Data Tables, missing tissues are listed as “not examined” under each animal.

The Pathology Report, Peer Review, and Pathology Working Group

51

5  Results and Discussion The Results and Discussion section presents the test article-related mortality, organ weight changes, macroscopic findings, ­microscopic findings, and the dose levels at which they were present; it is similar to text in the summary but with more detail and discussion around each finding. If there were multiple scheduled necropsies in the study (e.g., interim, terminal, or recovery), the presence or absence of findings at each interval should be indicated and finding resolution or reversibility discussed if appropriate. The text in this section provides the detail needed to understand the findings, to help make the determinations about adversity, and to potentially indicate whether the findings represent direct or secondary effects of the compound. In addition, select findings considered to be incidental may be discussed here to explain why they are not test article-related; these discussions typically only involve incidental findings or procedural-related deaths that are perceived as prominent or likely to generate debate. As a general rule, any discussion about test article-related findings is limited to what is known; speculation about pathogenesis or mechanism should be limited unless there is high confidence in the information. 5.1  Mortality

Mortality is typically discussed first, and, ideally, it is clearly stated in the first sentence if there was test article-related mortality and what dose levels were affected. Following this, all unscheduled deaths in the study should be discussed individually and should be presented in text or table format by animal number, dose level, and the study day the animal died or was euthanized. If it is determined, the cause of moribundity or death is noted for each animal, and it is indicated whether the unscheduled death was incidental or test article-related. The study pathologist will indicate that a particular animal’s cause of moribundity or death was “undetermined” if there are no clinical signs, macroscopic findings, or microscopic findings suggestive of a particular cause of death. If test article-­related macroscopic and microscopic findings in unscheduled deaths are similar to those in animals surviving to the scheduled necropsy, they can all be described together in the subsequent results text and data tables; if not, they should be presented separately in this subsection. In studies with relatively few unscheduled deaths, the information can readily be presented in text format alone; example text follows below in Box 4. Please note that the first sentence is written so that it can be brought up to the summary section. In studies where there are a large number of deaths and/or multiple causes of death, presentation of the data in a text table is often preferable. Similar to the previous text example, the table would be preceded by a short summary statement with an initial sentence or two that can be carried up to the summary section (see Box 5).

52

Ted A. Birkebak and Peter C. Mann

Box 4 Example description of mortality results

Box 5 Example description of a mortality presenting data in tabular form 5.2  Organ Weights

In presenting the organ weight results, all organs with test article-­related findings and the dose levels in which they occurred are ­typically listed in the first sentence or two, which can be brought up to the summary. If there is a recovery period, potential reversibility should be addressed here as well. Following that, specific organ weight changes can be presented in text or table format; text tables are generally easier to digest, particularly if there are multiple dose groups or organs affected or if there are multiple intervals (e.g., interim, terminal, or recovery). If there are multiple affected organs, they should be presented in the same order in which they are presented in the macroscopic and microscopic finding subsections. Regardless of format, the percent increase or decrease from the control values should be presented for both absolute and relative mean values of organs with treatment-related effects. Incidental variances from control values that

The Pathology Report, Peer Review, and Pathology Working Group

53

Box 6 Example discussion on organ weight data

are considered worth attention should be discussed separately and clearly defined as not test article-­related. When discussing incidental variances from control values, some pathologists describe them as “higher or lower” rather than as “increased or decreased” compared to controls, as they feel that the latter terms imply a test article-related effect rather than an incidental change. At the end of the section, there is typically a statement indicating that all other differences were considered to be incidental and within normal expected variation for the particular species used in the study; this is generic text that is also present in the macroscopic and microscopic finding sections and addresses the many changes that were not discussed in detail in the report body. An example using a text table presentation of the results follows in Box 6. 5.3  Organ Weights: Reviewing

There is guidance regarding which organ weights should be collected and which organ weight ratios are most useful in detecting changes (Michael et al. 2007; Haley 2017). However, it is important to recognize that there is no standardized threshold for considering an organ weight change to be biologically or toxicologically significant. In general, many look for a minimal change of 10% from the mean absolute or relative (to body weight [BW], brain weight, or both depending on the organ) control group values. However, it is not uncommon to disregard group differences that are much larger than this in organs that are prone to variation due to differences in maturity (e.g., reproductive organs and thymus), due to propensity for agonal congestion (e.g., spleen in the dog or lungs in general), or due to difficulty in reproducibly trimming the

54

Ted A. Birkebak and Peter C. Mann

tissue at necropsy (e.g., pituitary). Considerations ­that can aid in determining if the organ weight change is test article-related include: the change is consistent within a group and not due to an outlier; similar changes are present in both absolute and relative values; there appears to be a dose response; there is a similar change in both sexes; or there is a potentially correlative test article-related macroscopic or microscopic finding. While a concurrent change in absolute and relative weight values is frequently suggestive of a test article-related change, it is not necessary to see both. Test article-­ related changes in terminal body weight and differences in maturity (particularly with nonrodents) can alter absolute organ weight values, so the relative organ weight can be the more informative parameter when changes in body weight are present. So in the context of test article-related decreased body weight, it is possible to consider an organ weight to be increased based on an increased relative weight value and an unchanged to slightly decreased absolute organ weight value. Importantly, some organ weights are more closely correlated to body weight and others to brain weight, so one should not blindly rely on a single relative weight value. In general, body weight ratios are most valuable with liver and thyroid, and brain weight ratios are most valuable with adrenal, ovary, spleen, and thymus (Bailey et al. 2004). With other organs, there is not a tight correlation to brain or body weight, and the weight ratios are less informative. 5.4  Macroscopic Findings

All organs with test article-related macroscopic findings and the dose levels in which they occurred are typically listed in the first sentence or two, which can then be pasted into the summary section. If there is a recovery period, potential reversibility should be addressed here as well. Following that, specific findings can be described in text or table format; text tables are generally easier to digest if there are multiple findings or multiple affected organs. If there are multiple affected organs, they should be discussed in the same order in which they are presented in the microscopic finding subsection. Example text follows below in Box 7. For test article-related findings, terminology used in this section should be descriptive; interpretative statements should not be

Box 7 Example discussion on macroscopic findings

The Pathology Report, Peer Review, and Pathology Working Group

55

present or should be limited in scope. Interpretive statements are best presented in the discussion of correlating organ weight or microscopic findings. For example, noting the liver as enlarged is a descriptive statement. Noting that liver enlargement is due to hepatocellular hypertrophy would be an example of an interpretive statement. As seen in the following text, this interpretation is more appropriate when the macroscopic change is correlated to organ weight changes, microscopic findings, or both. 5.5  Macroscopic Findings: Reviewing

In general, macroscopic findings are rarely specific and do not typically add information that leads to stand-alone conclusions. They are usually correlative changes that support organ weight (e.g., gross liver enlargement associated with increased liver weights) or microscopic finding data (e.g., red discoloration that correlates with microscopic hemorrhage) and are useful in directing sample collection for microscopic examination (e.g., identification of masses in a carcinogenicity study). Macroscopic findings are nonspecific because there is limited repertoire of changes that can be recognized at the macroscopic level and they are, by convention, descriptive rather than diagnostic. The principal findings are usually changes in size (increased or decreased), a limited palette and distribution of color changes (usually red, dark or pale and diffuse, mottled or focal), changes in texture (firm, gelatinous or soft), or the presence of masses or nodules; it is rare that one or any combination of these changes are exclusively indicative of a specific process. As an example of the nonspecific nature of most macroscopic findings, red focal discoloration or mottling is a common macroscopic finding in the lungs of rats. It can be associated with incidental causes including agonal distress, postmortem congestion, or extravasation of red blood cells associated with euthanasia by CO2 narcosis or with toxicologically relevant hemorrhage, inflammation, or neoplasia; microscopic examination is always required to determine the cause. Finally, it is important to note that many observations noted at necropsy can lack microscopic correlates. Because of this lack of specificity, macroscopic findings that do not have organ weight or microscopic correlates should be viewed critically.

5.6  Microscopic Findings

As in the macroscopic finding subsection, all organs with test article-­related microscopic findings are summarized in the first sentence or two, which can then be pasted into the summary section. If multiple organs are affected, tissues with the most significant findings are mentioned first. Typically for each organ, there will be a short paragraph that begins with the test article-related findings diagnosed, the range of severity present, and the affected dose levels; this initial sentence is routinely brought up to the summary. Following that, a description of the actual changes and the incidences of the findings observed at each severity grade will be described; the description is particularly important if there are unusual findings. If the microscopic findings correlate with organ weight changes or macroscopic findings, this is a good place to

56

Ted A. Birkebak and Peter C. Mann

note the correlation. The text can also include discussion as to whether the test article-related microscopic finding is considered to be adverse, its potential reversibility, and if it may be related to the pharmacologic activity of the test article. In instances where there are multiple findings or affected dose groups, text tables are useful for conveying incidence and severity information; this is particularly true if the finding is an increased incidence or severity of a finding also present in controls, or if the finding is present at multiple intervals (e.g., interim, terminal, or recovery). Following that,

Box 8 Example discussion on microscopic findings

The Pathology Report, Peer Review, and Pathology Working Group

57

findings considered to be secondary to treatment are presented, and then incidental findings that were unusual or require further discussion can be presented. An example utilizing text table presentation is shown below in Box 8. 5.7  Microscopic Findings: Reviewing

The key components of the microscopic findings subsection are the actual findings or diagnoses and the accompanying descriptions. The finding diagnoses should use terminology present in the International Harmonization of Nomenclature and Diagnostic Criteria (INHAND) guides, and each finding should include the same components and have the same basic organization: tissue, process, character, topography or location, distribution, and severity. Using this structure, an infiltrate of mononuclear cells located in the epicardium of the heart would be diagnosed as: heart, infiltrate, mononuclear cell, epicardium, minimal. The INHAND guides address proliferative and nonproliferative findings from every organ system for rats and mice. In practice, much of the diagnostic terminology for rodents is applicable to nonrodents also, but specific guides for nonhuman primate, dog, mini pig, rabbit, and fish are currently in preparation. The guides are available to all at the Society of Toxicologic Pathology website (www.toxpath. org) and provide a wealth of information to both pathologists and non-pathologists. For each organ system, the guides contain preferred terminology for each pathologic process, diagnostic synonyms that may be used, diagnostic criteria, representative images, descriptions, and text and references that are helpful in discussions regarding adversity, reversibility, or pathogenesis. Another good source for rodent information is the Standardized System of Nomenclature and Diagnostic Criteria (SSNDC) Guides which are also available at the Society of Toxicologic Pathology website (www.toxpath.org). Descriptions are an important complement to the INHAND finding diagnoses presented in the results and discussion section. They should add enough detail to allow the reader to recognize the diagnostic criteria that were used to justify the diagnoses presented in the text; a non-pathologist should be able to go to the appropriate INHAND guide and see similarities between the description in the guide and that present in the results section of their report. Because INHAND compliant diagnoses are concise by design, the descriptions often convey additional information that may be used by the study pathologist in determining the potential adversity of the findings.

5.8  Conclusion

A Conclusion section may be present in the phase report; it is not required and is not always necessary. If present, it is usually the same text present in the concluding statement of the Summary. No new information or data should be presented in the conclusion.

58

Ted A. Birkebak and Peter C. Mann

6  Individual and Summary Data Tables The actual pathology data will be presented in tabular form; the formats of these tables vary slightly based on the Laboratory Information Management System (LIMS) in use by the laboratory, but the tables contain the same information regardless of the program. The most common tables are individual animal datasheets/reports, organ weight tables, macroscopic finding tables, and microscopic finding tables. 6.1  Individual Animal Datasheets/ Reports

These reports are organized by animal number, and each contains all the anatomic pathology data collected for every animal on the study. Specifically, the animal identification and fate status, all necropsy findings, organ and terminal weight values, and all microscopic findings are usually present in the report. In some, correlations between macroscopic and microscopic findings are also presented. The upper portion or header of each report typically contains animal identification and fate status information that indicates the dose group, study interim, and study day of death for each animal (i.e., scheduled or unscheduled death). The content and formatting of the report varies by test facility and LIMS, but the body of the report may include terminal body weight values along with absolute, and sometimes, relative organ weight values, and then macroscopic and microscopic findings are presented by organ. The report should indicate tissues that were unremarkable macroscopically and microscopically and, in some programs, tissues that were not processed or examined. There are two key differences between the macroscopic and microscopic findings present in the individual animal reports and other summary tables. First, findings may be truncated to fit in the summary tables, so select modifiers or locators present in the individual animal datasheet/report may not be present in the summary table. Usually, this is not a problem, but the information may be useful on an animal by animal basis or in the generation of summary documents. As an example, the macroscopic finding of “Discolored, red” in an individual animal datasheet/report can be truncated to “Discolored” in the corresponding summary table. If that animal had treatment-related microscopic hemorrhage, it may be useful to know whether that macroscopic discoloration was red and consistent with that hemorrhage, or pale and not likely to be treatment-related. Second, the individual animal datasheet/report will also contain tissue comments that are related to the macroscopic and microscopic findings and are not present elsewhere in the report. These comments can range from indicating whether tissues were not suitable for evaluation to descriptive clarifications of the findings. Figure  1 below is an example of an individual ­animal report. Note the tissue comment following the microscopic finding for femur.

The Pathology Report, Peer Review, and Pathology Working Group

59

Fig. 1 Individual Animal Data Report. Note the tissue comment for the femur indicating a fragment of cartilage was found in the joint. This information would not be available in summary tables. (Table generated by Pristima®, trademark of Xybion Medical Systems)

6.2  Organ Weight Tables

In addition to being present in the individual animal datasheets/ reports, organ weight data is also presented in summary and individual value tables. The summary organ weight tables present mean absolute and relative organ weight values by group and commonly include group mean, standard deviation, number of samples, and statistical significance. The percent change from the mean control value for each organ can be included as well and is ­recommended as it makes data review easier. Most of the pathology database tables are customizable, so if percent change is not

60

Ted A. Birkebak and Peter C. Mann

Fig. 2 Summary of organ weight and organ weight ratios. (Table generated by Pristima®, trademark of Xybion Medical Systems)

present, it should be available upon request. Figure 2 below is an example summary organ weight table for the kidney and liver of males. In this particular example, absolute and relative values for each organ are conveniently on the same page; other databases may report absolute values separately from relative weight values. Individual organ weight tables list the individual absolute and relative organ weight values by group; mean values and standard deviation for each group may also be present. These tables are most useful for identifying outliers or determining if a particular organ weight change correlates to another finding in a specific animal. Figure 3 below is an example of an individual organ weight table. 6.3  Macroscopic Tables

Macroscopic data can be presented by individual animal, with a format similar to that described with the individual animal datasheet/report described above, and in summary incidence tables that present the incidence of a tissue finding within a group. Because individual macroscopic data is already presented in the individual animal datasheet/report, a separate individual macroscopic data table is not always present or needed. The summary macroscopic incidence table is more useful and presents the incidence of macroscopic findings in each tissue by dose group for easy comparison. In a typical table, the header of the table indicates the group and number of animals examined in each group; the body of the table indicates the number of animals per group with no macroscopic findings, the number of each tissue examined per group, the number of each tissue per group that was unremarkable, the number of each tissue per group that was affected or abnormal, and the incidence of each specific finding per tissue. Figure 4, an example table, follows.

The Pathology Report, Peer Review, and Pathology Working Group

61

Fig. 3 Individual organ weight and organ weight ratios. (Table generated by Pristima®, trademark of Xybion Medical Systems)

Fig. 4 Incidence of macroscopic observations. (Table generated by Pristima®, trademark of Xybion Medical Systems) 6.4  Microscopic Tables

Depending on the LIMS and preferred tables used by the lab, microscopic data can be presented in matrix tables that present the microscopic findings by group and individual animal, or by summary tables that present the incidence of a tissue finding within a group, often by severity. A matrix table is not always included in the final report, but they can be generated upon request for review purposes by most software packages. These tables are useful when one has a specific tissue and finding in mind and wants to examine a group for outliers. These tables also indicate which tissues were missing, which tissues had gross findings, and which findings were unilateral or bilateral. An example of a matrix table, Fig. 5, follows.

62

Ted A. Birkebak and Peter C. Mann

Fig. 5 Table of individual microscopic findings. −  =  no finding; +  =  finding present; (= unilateral finding; +G = correlated macroscopic finding; ‘= tissue not examined (Table generated by PathData®, trademark of PDS Life Sciences)

The summary incidence table of microscopic findings has an identical format to the summary incidence of macroscopic findings. Due to space constraints in the tables, diagnoses in these summary tables may be abbreviated and may not contain all the modifiers or locators present in the diagnosis, so it is worth examining the individual animal data when there are findings of concern or interest. The tables can illustrate finding incidence with or without severity grades. An example of a table with severity grades, Fig. 6, follows.

7  Terminology that Is Frequently Confusing to Non-pathologists There are many closely related non-organ-specific diagnostic terms or concepts that have implications that might not always be evident to the non-pathologist. There are a few that frequently lead to questions during the report review process; they include terminology describing inflammatory cell infiltrates and inflammation, decreases in cell numbers or size, and increases in cell numbers or size. An excellent discussion about general terminology and processes common to organs can be found in Mann et al. (2012). A discussion on a few of the more common terms that can cause confusion follows.

The Pathology Report, Peer Review, and Pathology Working Group

63

Fig. 6 Summary of severity for selected microscopic observations. (Table generated by Pristima®, trademark of Xybion Medical Systems) 7.1  Inflammatory Infiltrate Versus Inflammation

The distinction between inflammatory cell infiltration and inflammation is a common source of confusion; they are closely related, both can be incidental or spontaneous findings as well as treatment-­ related findings, and there is considerable variability between pathologists in regard to the criteria they use for distinguishing the two. To the reviewer, the difference can be important as infiltration is generally a non-adverse finding, while inflammation can represent an adverse finding dependent on the severity and location. Infiltration refers simply to the presence of inflammatory cells, or leukocytes, in a tissue without any other findings indicative of an inflammatory process. Inflammation is distinguished from infiltration by the concurrent presence of other evidence of an active inflammatory process or damage, such as fibrin deposition, congestion, hemorrhage, edema, margination or diapedesis of vascular leukocytes, degeneration or necrosis of resident tissue elements, and/or associated fibrosis. While this definition seems straightforward, it can be a contentious issue as some pathologists incorrectly avoid the use of the term “inflammation” in favor of the less ominous sounding “infiltration,” and the distinction between the two entities can get blurred if the infiltration is severe or consists of an inflammatory cell infiltrate in an unusual site. If inflammatory cell infiltration is concurrently diagnosed in a tissue with any of the other findings suggestive of inflammation, the reviewer should feel

64

Ted A. Birkebak and Peter C. Mann

comfortable seeking more information on the underlying process or the diagnostic criteria used in the evaluation. 7.2  Decreased Cellularity/ Hypocellularity Versus Depletion/ Atrophy

There are multiple terms that are commonly used to describe a decrease or loss of cells or a decrease in cell size; they are decreased cellularity, hypocellularity, depletion, and atrophy. The most descriptive and least specific of these are decreased cellularity and hypocellularity. Decreased cellularity or hypocellularity are synonyms, and both mean that there are less cells present than normal; they do not imply a mechanism for the decrease, and both are commonly used to describe a situation where the cells are absent due to lack of development or due to altered trafficking of circulating cells (e.g., lymphoid cells). Depletion and atrophy are sometimes used to describe situations where the cells have been lost to degeneration, to necrosis, or to loss of a progenitor cell; if used in this context, they can represent an adverse effect. A combination diagnosis, such as atrophy/hypocellularity, is sometimes used by the pathologist when the mechanism of the decreased cellularity is unclear. As an example, this combination diagnosis can be used to describe decreased germ cells in seminiferous tubules of the testes when it is not clear whether the finding occurred due to cell loss or lack of development. The diagnosis of atrophy is also commonly used to describe a decrease in cell size, due to a decrease in the production of secretory contents or to a decrease in cytoplasmic constituents. When describing a decrease in cell size or a decrease in secretory material, the finding of atrophy is often considered to be a non-adverse finding, in contrast to when it is used to describe the potentially adverse finding of cell loss. All of these usages of the term atrophy are consistent with INHAND guides, so a good written description of the finding in the microscopic findings section is needed to put them in context.

7.3  Hypertrophy Versus Hyperplasia

Lastly, hypertrophy and hyperplasia are similar terms that are commonly used to describe increases in cell size or number. Hypertrophy refers to an increase in cell size and often represents an adaptation of the cells to a physiologic stimulus, such as enlargement of ­myocytes following exercise (myocyte hypertrophy), or hepatocytes in response to a xenobiotic that causes metabolic enzyme induction (hepatocellular hypertrophy). Hyperplasia describes an increase in the number of cells present, regardless of the size of the cells. Hyperplasia should not be automatically considered to be preneoplastic; it can also be part of a normal reparative response or from a biologically appropriate stimulus, such as lymphoid hyperplasia following exposure to an antigen. The INHAND guides are a good starting point when looking for information on whether a particular hyperplastic finding is suspected to be a preneoplastic lesion.

The Pathology Report, Peer Review, and Pathology Working Group

65

8  Adverse, Adaptive, and Exacerbations Anatomic pathology findings are often key to the determination of a no-observed-adverse-effect level (NOAEL), so there is likely to be discussion within the text as to whether test article-related findings are adverse, adaptations, or exacerbations of spontaneous disease. There should be a clear statement regarding any adverse test article-related effect in the pathology subreport if anatomic pathology data alone is sufficient for that decision. If data from multiple datasets within the main study are needed to make that judgment, the adversity statement is best placed in the main study report. The Society of Toxicologic Pathology recently published guidance with 10 recommendations “designed to produce a more consistent approach for determining and communicating adverse findings in the context of nonclinical toxicity studies for use in risk assessment” (Kerlin et al. 2016). This publication outlines the rationale used regarding the typical determination and reporting of adversity by the study pathologist in the pathology report. The decision as to whether a finding is adverse or non-adverse is often not as straightforward as one would like. There are numerous factors that impact the decision, and the criteria for defining adverse effects are not strictly defined (Keller et  al. 2012; Lewis et al. 2002; Dorato and Engelhardt 2005). Broadly paraphrasing these various definitions, a finding is likely to be considered to be adverse if it has a negative impact on function, structure, lifespan, or the ability of the organism to respond to external stressors. With regard to anatomic pathology data, the types of changes that typically fall into this broad definition are degeneration, necrosis, atrophy, inflammation, or neoplasia. However, there are instances when an intuitively adverse finding of single cell necrosis is not considered to be adverse, or when a typically non-adverse finding, such as pigment deposition, is considered to be adverse. So it is best to remember that decisions concerning adversity can be subjective and open to discussion. Becoming familiar with the considerations being made by the study pathologist during this analysis can facilitate this discussion. They are nicely summarized in the results of the 4th ESTP International Workshop (Palazzi et  al. 2016) and include the inherent adversity of the effect, the finding severity, and the effect constellation, or context of the finding. Some findings are inherently considered to be adverse in the absence of any correlative data; examples would include neuron necrosis, malignant neoplasms, or retinal degeneration. Finding severity can be used as the basis for defining adversity; if this is the case, the grading criteria should be as objective as possible and well documented in the report. The context of the finding, or the effect constellation, is frequently an important factor in the determination of adversity for

66

Ted A. Birkebak and Peter C. Mann

findings that are not considered to be inherently adverse. As an example, simple vacuolation of multiple cell types associated with phospholipidosis is often considered to be a non-adverse finding. In contrast, phospholipidosis-associated vacuolation is considered to be an adverse finding when accompanied by inflammation or other evidence of cell injury. There are several factors that should not be considered as part of the determination of adversity in a specific toxicity study, but they are important in the broader discussion around human hazard assessment. There should be no extrapolation across species when considering potential adversity; the only consideration is whether the finding in the study is adverse to the test species in that particular study. Although important, any discussion about species specificity or the relevance of the finding to human should be separate. An example might be test article-related ulceration in rat forestomach due to prolonged local exposure following oral gavage dosing. A human would be unlikely to be susceptible to this type of injury since it does not have a forestomach that stores ingesta in a similar fashion. Regardless of this species difference and lack of risk to humans, forestomach ulceration would be considered to be adverse in rat and would be important in a discussion about the NOAEL for that study. The clinical relevance of this finding would be an important, separate discussion that is often included in a separate “‘overview document” such as an investigator brochure or investigational new drug application (IND). Further, the mechanism of the effect does not play a role in determination of adversity; it does not matter if the effect is pharmacologic or off-target, or if it represents a direct or indirect effect of the test article. Mechanistic discussions or the determination whether the effect is a direct result or secondary to treatment may be important in determining the relevance of the finding to clinical use, but they are a separate discussion from adversity. Finally, the transience or reversibility of a finding is usually not a consideration in the decision as to adversity; however, these factors are definitely a consideration in ­determining clinical risk and may indicate a lower level of concern when the effects are of short duration or are reversible. Occasionally, findings can be identified as adaptations or exacerbations of spontaneous disease without any further discussion about adversity. Adaptive changes are responses by the test species to survive and function while exposed to the xenobiotic. The most common examples of adaptive changes would include hepatocellular hypertrophy associated with enzyme induction or extramedullary hematopoiesis secondary associated with bone marrow suppression or erythrocyte loss. Adaptive changes can be considered to be adverse, so this distinction should not be made as a substitute for a statement about the potential adversity of the finding. Similarly, a test article-related finding may be described as an exacerbation of a spontaneous finding in that species; chronic

The Pathology Report, Peer Review, and Pathology Working Group

67

progressive nephropathy (CPN) in rodents is a common example of this type of finding. Again, labeling a finding as an exacerbation is not an adequate substitute for discussing adversity in the pathology report; depending on severity, exacerbation of CPN can be an adverse finding in rodent and can be suggestive of an underlying increase in renal tubular damage. With both of these types of findings, the first part of the discussion will be about the adversity of the finding with respect to the test species, and the second part of the discussion can revolve around the mechanism or clinical relevance of the findings.

9  How to Get a Better Pathology Phase Report One can increase the likelihood of obtaining a quality pathology evaluation and report by enlisting the pathologist into the protocol development process, providing ancillary information to the pathologist prior to or during the evaluation, using program-­ specific pathologists, requesting “blinded” reviews only when appropriate, and making use of peer reviews. 9.1  Pathologist Input During Protocol Development

Pathologist input into the protocol during its development is an easy way to increase the quality of the final product. For most routine GLP studies, this equates to having another set of eyes checking the study plan to ensure that recommended tissues are being appropriately collected for organ weight determination and for gross and microscopic examination. For exploratory studies or studies with atypical pathology endpoints, the pathologist can assist with identification of appropriate tissues to sample (such as identification of draining lymph nodes when needed); provide input on special tissue collection, special processing, and/or staining procedures; or recommend appropriate grading scales if a specific endpoint has already been identified.

9.2  Providing Ancillary Information

Having access to additional study-related information, such as clinical pathology data, results of ophthalmic examinations, clinical observation data, information about the expected pharmacology/ mechanism of action, and results of previous studies, can help the pathologist with the evaluation and drafting of a comprehensive pathology report. This information should not influence whether a finding is recognized or noted, but it can help in determining relationship to test article administration and whether it is presented as an adverse effect, a primary or secondary finding, or also help determine if it is an on- or off-target pharmacologic effect. As an example, increased pigment deposition in the spleen is a common microscopic finding that is often clarified by evaluation of hematology data. Evidence of increased red blood cell turnover in the hematology data provides a likely identification of the pigment as

68

Ted A. Birkebak and Peter C. Mann

hemosiderin and a potential mechanism for its deposition. Lastly, providing information from preceding studies is particularly useful to both the study pathologist and project toxicologist. It helps the pathologist more easily recognize findings that may be difficult to appreciate. And more importantly, it markedly increases the likelihood that the same diagnostic terminology will be used to describe identical test article-related findings between studies in a program and makes it easier for the toxicologist to generate clear summary documents. 9.3  Using Program-­ Specific Pathologists

Even with the INHAND guide to standardized nomenclature, it is still possible to get slightly different diagnoses for an identical finding when examined by different pathologists. To a pathologist, these slightly different diagnoses describe the same process, but that may not be obvious to the non-pathologist who reviews the report. The non-pathologist might be tempted to consider the differing terms to represent different findings when generating submission documents or an opinion. One way to greatly reduce diagnostic variation is to use program-specific pathologist(s) to read all the studies for a program, but if this cannot be accommodated, sharing of past pathology reports or using a single peer review pathologist for all studies in a program can help maintain diagnostic consistency. Diagnostic variation is not a new issue and is not an indicator of training or competency; histopathology is simply a highly subjective process. When multiple changes are identified and are part of a single pathogenic process, the preferred practice is to diagnose these changes with a single encompassing diagnostic term and not as multiple individual diagnoses. In actual practice, that single pathogenic process is not always recognized by the study pathologist. Alternatively, and probably more commonly, some pathologists or organizations are more comfortable diagnosing the components of a pathogenic process as individual component fi ­ ndings. This difference of reporting a single term that represents a constellation of findings versus the alternative of splitting an overall diagnosis into the multiple component diagnoses probably represents the biggest source of variation that one will see when reviewing pathology reports. A common example that one might see would be diagnosing inflammation as an entity versus splitting it into multiple diagnoses that might include inflammatory cell infiltration, hemorrhage, edema, degeneration, and/or necrosis. A single encompassing diagnosis more efficiently communicates the overall process and is a more interpretive approach. Diagnosis of individual components of a process is usually a more descriptive and less interpretive approach. Neither approach is always the right one, but it is usually easier to understand and convey findings from multiple studies where encompassing diagnoses have been appropriately made. When diagnosing individual components, it is a virtual guarantee that all the individual components of a pathologic process will not occur

The Pathology Report, Peer Review, and Pathology Working Group

69

together in all animals in a group or between studies, so there is always slight variation in the expression of each component change. So, one might see variability in one component of the change between dose levels or between studies and may falsely interpret that to be an actual test article-related effect. Further, the overall pathogenic process is not always apparent to a non-pathologist reviewer when viewing the individual component diagnoses as opposed to a single encompassing diagnosis. As an example, it is not always clear to a non-pathologist that an inflammatory infiltrate in conjunction with edema, hemorrhage, or tissue damage such as necrosis is indicative of active inflammation. Any time one sees multiple test article-related findings in a tissue, it is appropriate to ask if they represent a single process and if that process needs to be clarified in the diagnostic terminology or in the report narrative. 9.4  Proper Utilization of “Blinded” Reviews

One perceived way to increase the quality of the pathology evaluation is to have the pathologist conduct a blinded evaluation, where the microscopic examination is performed while they are unaware of animal identification, treatment, status, or any ancillary data (such as organ weights, etc.). This approach is often requested in an attempt to decrease the potential for any bias by the pathologist. For routine studies, blinded evaluation is not recommended as it decreases the ability of the pathologist to recognize the range of normal background findings and to potentially miss subtle findings. Blinded evaluations are best reserved for a small subset of studies where the target tissues and findings of interest have already been identified and there is a pre-determined grading scale for those findings. That way, the pathologist is focused solely on the findings of interest and not having to sort out other background findings that might cloud the issue. In addition to potentially having a negative effect on quality, blinded evaluations also are more expensive due to effort spent preparing coded slides, recording and decoding data, and the increased time required to actually evaluate the slides. It often takes two to three times longer to read a blinded study. Those that still remain concerned about the pathologist’s bias should be aware that it is a very common practice for most study pathologists and peer review pathologists to employ informal blinding when trying to determine if there is a subtle treatment effect in a specific target tissue or to determine if there has been significant drift in severity grading. In these instances, the pathologist mixes slides from control and treated animals and then attempts to separate them into piles of normal and abnormal without looking at the animal identification. This common exercise helps ensure that the final recorded diagnoses and severities are based only on the morphologic features on the slide rather than any knowledge of treatment, and it ensures consistency and a high level of confidence in the microscopic data.

70

Ted A. Birkebak and Peter C. Mann

10  Pathology Peer Reviews and Pathology Working Groups2 A pathology peer review is a formalized re-examination of a subset of the tissues, other pathology data (organ weights, macroscopic findings, and often clinical pathology data), and the pathology narrative by a second pathologist. It is formalized in that it is part of the study plan, by inclusion in the protocol or by amendment, and the scope of the review is defined per standard operating procedures and/or the protocol. There are two types of pathology peer review, prospective and retrospective. The distinction between the two is based on when they occur with respect to pathology report finalization. Prospective pathology peer reviews are conducted prior to pathology report finalization, and retrospective pathology peer reviews are conducted after pathology report finalization. A specialized type of the retrospective peer review is a pathology working group (PWG) which involves a review of select data by a group of relevant experts and differs further from a pathology peer review in that there is generation of new data and a stand-alone PWG report. 10.1  Prospective Pathology Peer Review

The goals of a prospective peer review are to increase the quality of the pathology data and narrative by ensuring that all target tissues and effect levels were identified, that optimal terminology was used, that severity scoring for the findings was appropriate and consistent, and that the narrative accurately relates the findings. The rationale for conduct and regulatory requirements, logistics, documentation, and a few general thoughts and recommendations for prospective pathology peer reviews are discussed below. The majority of prospective pathology peer reviews are conducted based on organizational preferences or practices, not regulatory requirements. Prospective pathology peer review is only required by the European Agency for the Evaluation of Medicinal Products (EMEA) for carcinogenicity studies and by the USEPA when a test article is being re-registered (European Medicines Agency Committee for Proprietary Medicinal Products 2002; Environmental Protection Agency 1994). Although most regulatory agencies do not require pathology peer reviews for most studies, there is a broadly held conviction that peer-reviewed data is more credible than non-peer-reviewed data. Aside from an increase in confidence in the data, there are pragmatic benefits to pathology peer review that have led many companies to incorporate routine prospective peer reviews into the workflow for both non-GLP and GLP studies. Peer review by a pathologist familiar with the test 2

 At the time of publication, the USFDA released a proposed draft guidance document that may change some of the regulatory recommendations related to pathology peer review.

The Pathology Report, Peer Review, and Pathology Working Group

71

article ensures consistency of diagnostic terminology between studies within a program; this can decrease time and effort needed by the toxicologist to process the findings and generate summary documents. And importantly, it often makes the findings in these documents easier to understand by reviewers. Routine prospective pathology peer reviews also increase the likelihood that an uncommon or subtle finding or process might be appropriately recognized or that a rare spontaneous or incidental finding will be correctly interpreted. Recognition of findings and pathologic processes largely results from experiential learning, and it is not an uncommon event for either the study or reviewing pathologist to recognize a finding or pattern that might be unfamiliar to the other. This sharing of knowledge and experience benefits both of the pathologists, as well as their respective organizations when it leads to higher-quality pathology evaluations in later studies. For many companies, these benefits make routine peer review of pathology data a cost-effective practice even while not a regulatory requirement. 10.2  Prospective Peer Review: Logistics

In terms of logistics, planning for a prospective peer review consists of identifying the peer review pathologist and incorporating the peer review into the protocol and study timeline. The reviewing pathologist should be appropriately trained and have experience with the type of study being conducted and the test species used; this usually equates to an American College of Veterinary Pathologists (ACVP) board-certified veterinary pathologist who has extensive experience as a study pathologist on GLP toxicity studies. However, the reviewing pathologist can have alternate credentials, particularly if he or she has specific experience or expertise with an organ system or pathologic process that is of interest for a particular study/program. The reviewing pathologist can be an employee of the testing facility, the study sponsor, or a third party. Most studies conducted at CROs are peer reviewed by external pathologists that are employed by or contracted by the study sponsors. In regard to timing, the peer review should take place after the study pathologist has completed review of the study slides and has generated a draft report. For GLP studies, this is done prior to review by the quality assessment (QA) unit of the pathology data. Because this is a sequential process, it adds to the timeline for finalization of the pathology phase report. Depending on the size of the study and the methodology of the review (discussed more below), the peer review will likely add 1–2 weeks to the pathology phase report timeline. There can be significant variation in the design and conduct of pathology peer reviews between laboratories and pathology organizations, and this should be considered when identifying a reviewing pathologist. With all, the basic process is the same and consists of the reviewing pathologist examining select tissues from a subset

72

Ted A. Birkebak and Peter C. Mann

of animals while having knowledge of the original diagnoses, comparing diagnoses to the study pathologist’s diagnoses, resolving any significant differences in opinion that might have an effect on the interpretation of the study, and working with the study pathologist to get those changes incorporated into the pathology dataset. Organizational practices vary in which and how many animals and tissues are reviewed, how the reviewing pathologist’s diagnoses are recorded when different from the study pathologist’s, and how changes to the reviewed pathology data are confirmed. 10.3  Prospective Peer Review: Design and Methodology

The most important variable in the design is what percent of animals and which tissues are examined by the reviewing pathologist. With the exception of the guidance from the EMEA for 2-year carcinogenicity studies, regulatory agencies have not provided guidance on how many animals or which tissues should be reviewed in a peer review. For carcinogenicity studies, the EMEA guidance recommends review of all protocol-listed organs from 10% of animals in all control and treated groups, all potential target tissues from all groups, and 10% of all tumors. Of more relevance is a position paper (Morton et al. 2010), endorsed by multiple toxicologic pathology organizations, that recommends the percentage of animals and tissues to be reviewed for the various types of toxicology studies; these are summarized in Table 1 below. These guidelines should be considered to be minimal requirements; SOPs from many laboratories or sponsors allow for examination of a higher percentage of animals, target tissues, or both. It is important for the peer review pathologist to have some latitude to increase the review as needed. The goal is to examine enough tissues to enable identification of findings, but not so many that the study is essentially being re-examined in entirety. The methodology by which diagnostic differences between the study and reviewing pathologist are recorded also varies between organizations, and it is worth having some familiarity with the two methods. The peer review can be performed “on paper” by making annotations on printouts of the original findings or on a computer, by using data downloaded directly from the pathology database into a specialized program or by conducting the peer review directly in the LIMS. Reviews “on paper” usually require less setup time and can accommodate shorter timelines. Reviews performed using computers require slightly more setup time but more easily generate documentation for an audit trail. For every tissue evaluated as part of the peer review, database documentation can list findings reviewed, document the reviewing pathologist’s opinion, document the consensus opinion, and list the action to be taken with regard to the observations. This level of documentation is possible with a paper-based peer review, but the information is not as easily reviewed and reported. Importantly, the regulatory position

The Pathology Report, Peer Review, and Pathology Working Group

73

Table 1 Guidelines for the number of animals that should be reviewed in various types of toxicity studies Type of study

Complete reviewa Target tissuesb

Neoplasms

Rodent toxicity –subchronic

Control and high dose: all (main and Control: at recovery) pathologist Low and mid-dose groups: at least 50% of discretion animals in affected groups and all at High dose: 30% NOEL in main study and all recovery of animals/sex animals in control, high dose, and all affected dose levels from main study

Not addressed in publication

2-year rodent carcinogenicity

All animals in dose groups with neoplastic Control: at findings and at least 30% animals at pathologist affected dose levels and all animals at discretion NOEL for target organs without High dose: 10% neoplastic findings of animals/sex

All

6-month rodent carcinogenicity

Control: at pathologist discretion High dose: 5 animals/sex

All animals in dose groups with neoplastic findings and at least 30% animals at affected dose levels and all animals at NOEL for target organs without neoplastic findings

All

Nonrodent toxicity Control: at –subchronic pathologist discretion High dose: at least 50% of animals/sex

Not addressed Control and high dose: all (main and in publication recovery) Low and mid-dose groups: all animals in affected groups thru the NOEL in main study and all recovery animals in control, high dose, and all affected dose levels from main study

Review of all protocol-listed tissues Any tissue with evidence of a potentially treatment-related finding

a

b

on what level of peer review documentation is needed varies between regions and has recently been a point of discussion by multiple agencies. If these discussions result in a change in the required level of documentation in peer reviews, this may lead to alterations in the methodologies described above. Post pathology peer review quality control (QC) is an aspect of the peer review that is not often considered by the toxicologist; this is a very important step confirming that all agreed upon changes to the pathology data were actually made. When considering the number of tissues that are dealt with, the potential number of changes, and the ubiquitous tight timelines, omissions and errors when entering consensus changes are possible. When selecting a peer review pathologist, one should expect and confirm that the peer reviewer, their organization, or the CRO will conduct a 100% QC of all peer review-mandated changes to the raw data.

74

Ted A. Birkebak and Peter C. Mann

10.4  Prospective Peer Review: Peer Review Statement/ Memo

Following completion of the actual slide and data review and QC, the deliverable for the peer review is a pathology peer review statement or memo that is generated and finalized after the pathology phase report has been signed and finalized. The reviewing ­pathologist needs to review the finalized pathology phase report before finalizing the peer review statement; the signed peer review statement can then be included in the study report, the study file, or both. The statement is signed by the reviewing pathologist and should include the following: ••

Study identification

••

Peer reviewer name, credentials, and affiliation

••

Identification (group and number) of animals with complete reviews

••

Identification of target tissues examined and animals where they were examined

••

Statement that neoplastic and other proliferative findings were examined

••

A statement indicating that study and reviewing pathologist are in agreement on the findings and interpretations in the study

The peer review organization should generate documentation that there was appropriate quality assurance oversight of the peer review process from plan generation thru signing of the peer review statement; this documentation can be maintained in the study file. As there is no GLP guidance on peer review, one should not expect a compliance statement to be part of the documentation. 10.5  Prospective Peer Review: General Thoughts/ Recommendations

Prospective peer reviews are an underutilized tool, particularly by smaller companies that do not have sizable pathology groups. That is probably because the perceived drawbacks of increased cost and time are much easier to recognize than the perceived benefit. The effects on the timeline can be minimized by planning ahead and integrating the review into the study schedule early. In regard to cost, it is hard to prove a negative and put a dollar value on how much time and effort a peer review can save by avoiding potential issues in the first place. A good pathology peer review not only catches potential diagnostic issues, but it also is an opportunity to catch data entry errors or omissions that could detract from the studies robustness. A common question is whether to have the slides sent to the peer reviewer or to have the reviewing pathologist travel to the test site where the study pathologist is located. The intuitive response is to save travel costs and have the slides shipped to the reviewing pathologist. In many cases, this represents a false economy. Unless there are no diagnostic differences or if the two pathologists have worked well with each other previously, the peer review may take longer than if

The Pathology Report, Peer Review, and Pathology Working Group

75

both pathologists are together at the same site. If there are remotely debatable differences in opinion, there could be multiple rounds of phone calls with images being viewed remotely and slides mailed back and forth before a consensus can be reached. This can add significant time to the process. With both pathologists at the same site, slides with differing diagnoses can be reviewed jointly and any differences in opinion resolved quickly. The costs involved with the shipping and inventory of slides, on both sides of the trip, are frequently underestimated, and, rarely, slides can be lost or damaged during transit. In most cases, the recommendation is to send the reviewing pathologist to the test site for the peer review. 10.6  Retrospective Peer Review

A retrospective peer review occurs after the pathology phase report, and, usually, the main study reports have both been finalized. Typically, these reviews are conducted to address a specific finding, diagnostic criteria, or other issue. They may be the result of a regulatory request or as a prelude to convening a pathology working group. The process for initiating the retrospective review will be similar to that for a prospective review. Unlike a prospective review, there must be an audit trail for all changes made to the data, and an amended pathology phase report and main study report will need to be issued to reflect the changes made in the data and conclusions.

10.7  Pathology Working Group (PWG)

Similar to a retrospective peer review, a PWG consists of a review of tissues and data from one or more finalized study reports to address a specific issue or concern. PWGs differ in that they are conducted by panels of pathologists with expertise relevant to the issue or in the field of toxicologic pathology in general. PWGs are of potential use in addressing diagnostic criteria or the potential clinical impact of neoplastic or non-neoplastic findings; addressing regulatory questions or concerns arising from the data; comparing related but differing findings from multiple studies within a program; or mediation of toxicologically significant, unresolved diagnostic differences between study and peer review pathologists. Because PWG reports are generated by a panel of independent experienced pathologists in an unbiased fashion, they can be highly influential documents with regulatory agencies. Specifics into the conduct and documentation of PWGs are discussed below. A PWG panel is typically composed of an odd number of voting members (often five) and a non-voting chairperson. The chairperson will be responsible for identifying and assembling the members of the panel, selecting the tissues to be examined, leading the slide review and discussion, unblinding the results after voting has concluded, recording findings made by the group, and drafting the PWG report. As such, the chairperson should have expertise in the subject area relating to the issue or question to be addressed, should be familiar with all relevant study data, and should have suf-

76

Ted A. Birkebak and Peter C. Mann

ficient experience in the field of toxicologic pathology to guide the slide review and discussion. The panel of voting members can include the original study pathologist, if a single study is examined or if a single pathologist was responsible for data from multiple studies under review; and it usually includes the original peer review pathologist or a reviewing pathologist that has reviewed relevant tissues from all the studies to be examined. The remaining panel members are selected based on their experience in the field or with the specific topic of interest; they can be medical (MD) or veterinary (DVM) pathologists. The conduct of the PWG consists of multiple rounds of tissue evaluation, voting, and discussion. For each tissue, the voting members come to a consensus diagnosis that is recorded by the chairperson. The panel does not examine all tissues from the subject studies; it only examines and discusses tissues relevant to the question or issue being addressed. Further, all slides are coded by the chairperson or their designate prior to the PWG, so the voting members are unaware of any identifying information in regard to treatment status. Following the final vote, the chairperson may unmask this identifying information to facilitate generation of a concluding opinion; prior to this occurring, the diagnoses are considered to be locked and final. The deliverable for the PWG is a stand-alone report that contains background text concerning the issue and reason for the evaluation, the methodology employed by the PWG, the original study diagnoses, the panel’s consensus diagnoses, and the PWG’s conclusions. The PWG is written primarily by the chairperson, edited by all members of the PWG and signed by all members of the PWG. There will also be accompanying QA documentation of the methods and procedures followed. The time needed to complete a PWG can be highly variable. The majority of this time is consumed in identification of a timeslot where all members of the panel can be available for 2–3 consecutive days; the actual conduct of the PWG takes 1–2 days with a day usually lost to travel. The finalized report is usually available to the sponsor within 1–2 weeks after the PWG is convened. As the chairperson, or their designate, will need access to the slides for coding 1–2 weeks prior to convening the PWG, the sponsor should also plan for the cost and time of shipping materials from archives to the chairperson, or their designate. References evaluations: use, issues, and definition(s). Regul Bailey SA, Zidell RH, Perry RW (2004) Toxicol Pharmacol 42:265–274 Relationships between organ weight and body/ brain weight in the rat: what is the best analyti- Environmental Protection Agency (1994) Pesticide registration (PR) notice94–5: requests for re-­ cal endpoint? Toxicol Pathol 32:448–466 considerations of carcinogenicity peer review Dorato MA, Engelhardt JA (2005) The no-­ decisions based on changes in pathology diagobserved-­ adverse-effect level in drug safety

The Pathology Report, Peer Review, and Pathology Working Group noses. http://www.epa.gov/PR_Notices/ pr94-5.html. Last Accessed 2 Oct 2011 European Medicines Agency Committee for Proprietary Medicinal Products (2002) Note for guidance on carcino- genic potential. http://www. ema.europa.eu/docs/en_GB/document_library/ Scientific_guideline/2009/09/ Haley PJ (2017) The lymphoid system: a review of species differences. Toxicol Pathol 30:111–123 Keller DA, Juberg DR, Catlin N, Farland WH, Hess FG, Wolf DC, Doerrer NG (2012) Identification and characterization of adverse effects in 21st century toxicology. Toxicol Sci 126:291–297 Kerlin R, Bolon B, Burkhardt J, Francke S, Greaves P, Meador V, Popp J (2016) Scientific and Regulatory Policy Committee: recommended (“Best”) practices for determining, communicating, and using adverse effect data from nonclinical studies. Toxicol Pathol 44:147–162 Lewis RW, Billington R, Debryune E, Debryune Gamer A, Lang B, Carpanini F (2002) Recognition of adverse and nonadverse effects in toxicity studies. Toxicol Pathol 30:66–74 Mann PC, Vahle J, Charlotte M, Keenan JF, Baker AE, Bradley DG, Goodman TH, Herbert R, Kaufmann W, Kellner R, Nolte T,

77

SusanneRittinghausen TT (2012) International harmonization of toxicologic pathology nomenclature: an overview and review of basic principles. Toxicol Pathol 40(4):7S–13S Michael B, Yano B, Sellers RS, Perry R, Morton D, Roome N, Johnson JK, Schafer K (2007) Evaluation of organ weights for rodent and non-rodent toxicity studies: a review of regulatory guidelines and a survey of current practices. Toxicol Pathol 35:742–750 Morton D, Sellers RS, Barale-Thomas E, Bolon B, George C, Hardisty JF, Irizarry A, McKay JS, Odin M, Teranishi M (2010) Recommendations for pathology peer review. Toxicol Pathol 38:1118–1127 Palazzi X, Burkhardt J, Caplain H, Dellarco V, Fant P, Foster J, Francke S, Germann P, Groeters S, Harada T, Harleman J, Inui K, Kaufmann W, Lenz B, Nagai H, Pohlmeyer-Esch G, Schulte A, Skydsgaard M, Tomlinson L, Wood CAND, Yoshida M (2016) Characterizing “Adversity” of pathology findings in nonclinical toxicity studies: results from the 4th ESTP international expert workshop. Toxicol Pathol 44:810–824 Vishwanathan CT (2005) FDA perspectives on current issues in GLP.  Presentation at Society for Quality Assurance Regulatory Forum, Baltimore, MD

Chapter 3 Routine and Special Techniques in Toxicologic Pathology Pamela Blackshear, Erica Carroll, Sasmita Mishra, Matthew Renninger, and Arun Tatiparthi Abstract Anatomic and clinical pathology assessments are key components of toxicity studies, often providing the pivotal data to determine whether a test article has the appropriate safety and efficacy profile to become a new drug. Standard anatomic pathology assessments that are part of most toxicity studies consist of evaluation of macroscopic observations, organ weights, and microscopic observations; and standard clinical pathology assessments consist of evaluation of hematology, clinical chemistry, coagulation, and urinalysis data. Routine laboratory techniques for standard anatomic pathology assessments include necropsy, with macroscopic observations, organ weight measurements, and tissue collection, and histology to process tissue samples into hematoxylin and eosin (H&E)-stained slides for microscopic evaluation. These routine techniques are part of most toxicity studies. Several special laboratory techniques may be added to studies for cause, either to better characterize changes first observed in the standard assessment or proactively included in the study design to detect or characterize changes anticipated due to the mechanism of action of the test article or changes observed in previous studies with the test article. Key words Computed tomography (CT), Digital pathology, Electron microscopy, Fixation, Histochemical stains, Histology, Imaging, Immunohistochemistry (IHC), In situ hybridization (ISH), Magnetic resonance imaging (MRI), Morphometry, Necropsy, Organ weights, Positron emission tomography (PET), Single photon emission computed tomography (SPECT), Ultrasound

1  Introduction Anatomic and clinical pathology assessments are key components of toxicity studies, often providing the pivotal data to determine whether a test article has the appropriate safety and efficacy profile to become a new drug. Standard anatomic pathology assessments that are part of most toxicity studies consist of evaluation of macroscopic observations, organ weights, and microscopic observations; and standard clinical pathology assessments consist of evaluation of hematology, clinical chemistry, coagulation, and urinalysis data.

Thomas J. Steinbach et al. (eds.), Toxicologic Pathology for Non-Pathologists, https://doi.org/10.1007/978-1-4939-9777-0_3, © Springer Science+Business Media, LLC, part of Springer Nature 2019

79

80

Pamela Blackshear et al.

Routine laboratory techniques for standard anatomic pathology assessments include necropsy, with macroscopic observations, organ weight measurements, and tissue collection, and histology to process tissue samples into hematoxylin and eosin (H&E)stained slides for microscopic evaluation. These routine techniques are part of most toxicity studies. Several special laboratory techniques may be added to studies for cause, either to better characterize changes first observed in the standard assessment or proactively included in the study design to detect or characterize changes anticipated due to the mechanism of action of the test article or changes observed in previous studies with the test article. Routine and special pathology techniques discussed in this chapter include necropsy, histology, histochemical stains, immunohistochemistry, in situ hybridization, digital pathology and morphometry, electron microscopy, and in vivo imaging.

2  Necropsy Necropsy is defined as a postmortem examination of animals, while the more familiar word “autopsy” is typically reserved for a postmortem examination of humans. Necropsy is performed in toxicology studies to examine organs and tissues through macroscopic observations and organ weights and to collect tissues for microscopic observations and other analyses. As with most laboratory techniques, the success of necropsy is dependent on appropriate preparation and planning. Technical staff performing necropsy should be well-trained and follow standard operating procedures to ensure consistency, so that comparisons made between animals and between treatment groups are valid. Veterinary pathologists do not need to be present at necropsy for standard studies if the necropsy technicians are well-­ trained and experienced, but should be available for consultation if any unusual changes are observed (Nikula 2016). Necropsy technicians often have more experience than veterinary pathologists in identifying macroscopic changes, but veterinary pathologists are better trained to understand the impact of changes and to advise on additional considerations, including special tissue collections when unusual changes are observed. Animals are usually fasted prior to euthanasia to allow for collection of a fasted blood sample for clinical pathology and to reduce variability in organ weights and morphology. Liver and kidney weights change during the day, primarily due to changes in carbohydrate, protein, and water content (Rothacker et al. 1988). Increased glycogen in hepatocytes in unfasted animals can increase liver weights and impact the microscopic evaluation. Fasting animals prior to euthanasia will decrease differences in the measurements

Pathology Techniques

81

that are impacted by recent food consumption. Terminal body weights should be collected for calculations of organ-to-body weight ratios. The sequence of animals for euthanasia and necropsy should rotate through the treatment groups (i.e., necropsy one animal from each group, followed by a second animal from each group, and then a third animal from each group, etc.) or be randomized to minimize the variability that time of day may have on organ weights and morphology. Animals should be euthanized following the American Veterinary Medical Association Guidelines for Euthanasia (AVMA 2013) and standard operating procedures. Animals should be exsanguinated following a standard operating procedure prior to necropsy to reduce variability in organ weights and morphology that can result from variable blood content in tissues (Kanerva et al. 1982). In a study evaluating the effect of exsanguination on organ weights, absolute liver and kidney weights in exsanguinated rats were shown to be 23% and 15% lower, respectively, than those from animals of comparable size that were not exsanguinated (Kanerva et al. 1982). While removal of organs at necropsy does result in blood loss from the organs, this loss is not consistent, and exsanguination is needed to reduce the variability caused by differences in blood content in tissues. Once euthanized, macroscopic evaluation, organ weights, and tissue collection must proceed rapidly to prevent postmortem changes and degradation of tissue samples. For example, postmortem liver vacuoles form in hepatocytes and liver weights increase in a time-dependent manner after death from an influx of plasma into the cytoplasm of hepatocytes (Li et al. 2003). Autolysis is the breakdown of cellular structures from endogenous enzymes and begins immediately after euthanasia. Care should be taken to ensure animals are not euthanized until necropsy technicians are ready for tissue collection. If animals are found dead or need to be euthanized when necropsy cannot occur immediately, the carcass should be refrigerated to slow autolysis and then necropsy started as soon as possible. Standard operating procedures should be followed so that all necropsy technicians are consistent in their approach to prosection, macroscopic evaluation, organ weights, and tissue collection. 2.1  Macroscopic Evaluation

The carcass should be evaluated prior to prosection and any macroscopic observations recorded and correlated to clinical observations if applicable. As the external body, body cavities, and organs are evaluated, macroscopic observations should be made with descriptive terms, including location, color, size, consistency, number, and distribution as applicable. A standard lexicon should be used for macroscopic observations to allow for meaningful tabulation and comparison of observations.

82

Pamela Blackshear et al.

2.2  Organ Weights

Care must be taken to remove organs and trim adipose and connective tissue consistently to allow for valid organ weight comparisons. The Society of Toxicologic Pathology (STP) has made recommendations for organs to weigh in standard toxicology studies (Sellers et al. 2007). Liver, heart, kidney, brain, testes, and adrenal gland weights are recommended for all multi-dose general toxicology studies lasting from 1 week to 1 year. Organ weights are not recommended for carcinogenicity studies. Thyroid gland and pituitary gland weights are recommended for all species except mice, because the collection and weighing process may produce artifacts that complicate the microscopic assessment of these small tissues in mice. Spleen and thymus weights are recommended for rodent studies and may also be weighed in non-rodent studies, though interpretation may be confounded by aging changes. Weighing of reproductive organs is most valuable in sexually mature animals, but weighing of female reproductive organs should be considered case-by-case due to variability from cycling. Additional organs may be weighed to characterize changes anticipated due to the mechanism of action of the test article. While organs are typically weighed fresh, fixation-induced organ weight changes have been shown to be consistent for specific organs in rats, suggesting that fixed organ weights may be a valid alternative to fresh organ weights if needed in special circumstances (Kanerva et al. 1983). Differences in body weight between treatment groups may confound interpretation of differences in organ weights. Organ-­ to-­body weight and organ-to-brain weight ratios are calculated to help normalize organ weight data, but use of these ratios assumes that the weight of an individual organ is proportional to either the body or brain weight of that animal. Based on analysis of control rat data, liver and thyroid gland weights are best compared using organ-to-body weight ratios; adrenal gland and ovary weights are best compared using organ-to-brain weight ratios; and neither ratio works well for brain, heart, kidney, pituitary gland, and testes weight (Bailey et al. 2004). Due to considerable variability in organ weights with age and body weight, concurrent controls are important for comparison of data (Gur and Waner 1993). Organ weights from animals that are terminated early or found dead should be excluded from group means. Organ weights are not recommended when there are less than three animals per group due to the difficulty to interpret through the variability that is normally present.

2.3  Tissue Collection

Tissues are routinely collected in fixative to process to slide for microscopic evaluation. As with other necropsy procedures, standard operating procedures should be followed for consistency of tissue collection, and sections should be taken from the same areas of tissues to minimize variability. The STP has made recommen-

Pathology Techniques

83

dations for tissues to collect for microscopic evaluation based on regulatory guidelines (Bregman et al. 2003). If non-standard tissues are collected due to changes anticipated due to the mechanism of action of the test article, the same tissues should be collected from control animals for comparison. If non-standard tissues are collected due to macroscopic changes observed, the same tissues should be collected from one or more control animals that have not yet been necropsied for comparison. Fixatives are used to preserve the architecture of tissues collected for microscopic evaluation. Immersion fixation, in which small sections of tissues are collected into and allowed to soak in a container of fixative for several hours or days while the fixative penetrates the tissue, is the most common fixation method. Perfusion fixation, in which fixative is instilled through the vascular system under pressure, typically through the heart, can be used for more rapid fixation or fixation of larger tissues. Perfusion fixation is most commonly used for neurotoxicity studies. Common chemical fixatives can be divided into cross-linking or coagulant fixatives, or combinations of the two (Grizzle et al. 2008). These chemical fixatives preserve tissues partly due to antiseptic and disinfectant properties which prevent bacterial growth and tissue degradation but also due to their cross-linking or coagulant properties which arrest postmortem degradation termed autolysis. Autolysis, from Greek words “auto” (self) plus “lysis” (splitting), is also known as self-digestion and occurs from cellular leakage or activation of enzymes which then degrade the cellular contents. Unfixed tissue both autolyzes and is acted upon by putrefactive bacteria from the gut or other tissue epithelial surfaces, which degrade cellular architecture and, if pronounced enough, render microscopic evaluation impossible. Common cross-linking fixatives are aldehydes (e.g., formaldehyde, glutaraldehyde) that react with cellular components to form hydroxymethyl side chains, which are thought to preserve cellular architecture by denaturing macromolecules and making them insoluble and by forming cross-links between peptides (Grizzle et al. 2008). Ten percent neutral buffered formalin (NBF), which contains formaldehyde, is the most common fixative used in toxicologic pathology to preserve tissues for light microscopy and is a cross-linking fixative. Coagulant fixatives preserve cellular architecture by coagulating proteins and making them insoluble. Alcohols are the most common coagulant fixatives and may be used if cross-linking fixatives interfere with the intended purpose of tissue collection, such as some immunohistochemical methods. Some epitopes of interest for immunohistochemistry may be destroyed by either cross-linking or coagulant fixatives, necessitating that the tissues are flash-frozen instead. Cellular detail is poor in frozen tissues compared to tissues embedded in paraffin after

84

Pamela Blackshear et al.

chemical fixation. While 10% NBF is the most common fixative for light microscopy, other fixatives may provide better architecture for specific tissues, e.g., modified Davidson’s fixative for testes (Lanning et al. 2002). Fixatives will be further discussed with specific uses later in this chapter. While collection of tissues for microscopic evaluation is a standard part of toxicology studies, tissues may also be collected for isolation of nucleic acids (DNA or RNA) or protein for analysis. Special considerations for these collections include sanitation of surfaces and instruments between tissues and animals to prevent contamination and to remove RNases that degrade RNA. These tissues may be collected frozen or in special stabilizing solutions (e.g., Allprotect® Tissue Reagent for stabilization of DNA, RNA, and protein, or RNAlater for stabilization of RNA). While tissue samples for DNA, RNA, and protein are most easily collected at necropsy, DNA, RNA, and protein can also be isolated from formalin-­fixed paraffin-embedded tissues for analysis (Scicchitano et al. 2009; Matsuda et al. 2011).

3  Histology Histology is defined as the “branch of anatomy that deals with the minute structure of animal and plant tissues as discernible with the microscope” (Merriam-Webster 2018). In the field of drug development or academia, “histology” often refers to a department that performs laboratory procedures that transform tissues removed from animals into specimens on glass slides for microscopic evaluation by a pathologist. In the pharmaceutical industry, the specific purpose of the evaluation is generally to characterize a novel animal model (e.g., diet-induced obesity in the rat) or the effect of a test article, with special focus on toxicity or adaptive changes in the tissues. For optimal microscopic evaluation of histologic specimens, stained slides must be produced of consistently high quality. This requires a series of laboratory procedures to be performed properly by skilled technical personnel who transform tissue specimens into evaluable, and therefore valuable, microscope slides. Although imprecise, the word “histology” is often used to refer to those collective processes that result in the production of microscope slides and will be used in that sense henceforth. For example, depending on the size of toxicity studies, one or more weeks may be necessary in “histology” before all slides are ready for the pathologist’s evaluation. Figure 1 provides a simple flow chart which generalizes the steps each tissue specimen undergoes from sample collection to readiness for evaluation with a microscope.

Pathology Techniques

85

Fig. 1 Histology processes from animal to glass slide. Tissue specimens taken at necropsy undergo a series of precise processes to render them evaluable under a microscope. At necropsy tissues are immersed in fixative (most often formalin) for a specified time. Fixed tissues are trimmed to fit into plastic cassettes for processing (usually automated), embedding in paraffin blocks, microtomy, and staining. High production facilities may have a final quality control step in which slides are reviewed individually for adequate quality. Examples of automated equipment (Leica® 2255 microtome and Leica® TS5025 with CV5030) are shown 3.1  Fixation

Routine histology is performed using tissues immersion-fixed in 10% neutral buffered formalin (NBF). Formalin is a 37–40% solution of formaldehyde, a toxic gas, in water and methyl alcohol; and 10% NBF contains approximately 3.7–4.0% formaldehyde in phosphate-­buffered saline. Most tissues intended for histopathologic (i.e., microscopic) evaluation, regardless of the organ of origin (e.g., heart, haired skin, liver, kidney), should measure no more than 0.5 cm in one dimension before being placed in fixative. Certain tissues (e.g., eyes and rodent brain) are left intact when placed in fixative, because cutting these tissues prior to fixation would introduce excessive artifact. Formalin penetration proceeds at approximately 0.5 mm per hour; therefore, tissues need to be “fixed” for 12–24 hours in a volume ratio of at least 10 volumes of fixative per volume of tissue for adequate preservation (King et al. 1989). Tissues greater than 0.5 cm in thickness (e.g., brain) require longer

86

Pamela Blackshear et al.

periods (48–72 hours) for adequate fixation. Increased temperature, up to 60C, speeds the fixation reaction (likely by increasing the movement of molecules, but it is not used in many labs). Other fixatives, such as modified Davidson’s fixative or Bouin’s solution, often used for male reproductive tissues or eyes, respectively, have different optimal fixation times. 3.2  Trimming

When a tissue arrives in histology, it is “accessioned” (i.e., its arrival is recorded), and upon completion of fixation, it is trimmed. To “trim” or “trim in” a tissue means to cut away excess tissue with a scalpel or sharp razor blade to produce a standardized size, shape, and orientation of specimen. Tissues are typically trimmed to 3–4 mm thickness and placed into small, porous, plastic cassettes for tissue processing. In toxicity studies, tissues for microscopic evaluation are generally predesignated in the study protocol. Histology technicians follow study protocols and standard operating procedures for guidance, for example, whether right, left, or both of paired organs (such as kidneys or eyes) are required, or whether longitudinal or transverse sections should be made. The study protocol specifies which animals and tissues are to be processed, embedded, microtomed, stained, and delivered for microscopic evaluation. Attention to the study protocol by histology personnel is critical because it sometimes dictates that only specific dosage groups be processed first (e.g., controls and high-dose groups or only terminal sacrifice animals), with instructions to process remaining tissues only when instructed by amendment after tissues affected by the test article (known as “targets”) are identified by the pathologist. Other protocols dictate processing and evaluation of tissues identified as targets without an amendment. Histology laboratories typically have a standard trim plan that details the required shape of each routine tissue and the orientation of each tissue when embedded in the paraffin block. Most cassettes will contain more than one tissue or sections of tissue, but not generally more than four. Standard trim plans are followed meticulously by skilled technicians, which is important because trimming each tissue type must be done as uniformly as possible to facilitate comparison of tissues between animals and between treatment groups. Trim plans can be altered to ensure a change observed at necropsy will be represented on the slide, or to help answer specific scientific questions. For example, a protocol for testing the safety of a new drug using beagle dogs that may affect the heart may specify that three sections of the heart be processed (right atrium/ ventricle with right atrioventricular valve, left atrium/ventricle with atrioventricular valve, and interventricular septum with aorta and valve). Cassettes are carefully labeled with, at the minimum, study identification information (generally a number), animal

Pathology Techniques

87

identification number, and gender, with other data depending on the facility and use of the tissues, such as group identification or generation. Formalin is a carcinogen; therefore technicians trimming tissues wear personal protective equipment (PPE), consisting of a laboratory coat, nitryl or latex gloves, closed-toe shoes, and safety glasses, and generally work in a well-lit fume hood or biosafety cabinet containing a sharps container for safe disposal of used razor and scalpel blades. Histology technicians who spend hours a day trimming may wear dosimeters to measure their exposure to formalin. Tissues that have been exposed to infectious agents (pathogens) may require different or longer fixation times, different and additional (secondary) transport containers, and additional PPE. Tissues exposed to infectious agents (bacteria, viruses, fungi) known or suspected to cause disease in humans are designated as requiring protections afforded at biosafety levels of containment 2 through 4. Histology work performed in biosafety containment is beyond the scope of this chapter, but a primary source of information is available in the Biosafety in Microbiological and Biomedical Laboratories, fifth edition, issued by the Centers for Disease Control and Prevention (Chosewood and Wilson 2009). 3.3  Processing

Once tissues have been trimmed and oriented in labeled cassettes, the cassettes are loaded into a tissue processor. Processing consists of a series of timed dehydration steps followed by a “clearing” step and ending with infusion with melted paraffin. Dehydration is necessary because formalin will not form a homogeneous mixture (i.e., it is not “miscible”) with paraffin. Tissues begin in 10% NBF and proceed, for example, to be immersed in 70% ethanol, followed by 80% ethanol, 95% ethanol, a second 95% ethanol, 100% ethanol, and a second and third step in 100% ethanol. Ethanol is usually the dehydrant, but others have been used (e.g., methanol, propanols, acetone) (Llewellyn 2009). Steps are generally from 30 to 60 minutes in duration, are automated, and are optimized for each species (e.g., rodent soft tissues, or large animal bone) into a standardized computer program and validated prior to processing study samples. Once validated, the program is routinely used for that species. Each processor can run programs for several species or tissue types and for immediate or delayed processing. Overnight processing is routine. After the tissue is sequentially dehydrated, it is “cleared” with xylene, which is miscible with paraffin. Tissues are infiltrated and embedded in paraffin because paraffin has approximately the same density as tissue, which facilitates “sectioning” (technical term preferred to “slicing”) the tissue into 4–6-micron-thick sections on a microtome for mounting on glass slides.

88

Pamela Blackshear et al.

3.4  Embedding

Upon completion of tissue processing, the cassettes contain paraffin-­ impregnated tissues. To enable microtomy, the tissues must then be embedded into a block of paraffin that can be shaved, not unlike meat-slicing in a delicatessen. At embedding stations, cassettes rest on a warmed platform. The technician fills a metal mold (about the size of a cassette) with hot liquid paraffin and moves the tissues from the plastic cassette into the mold, being careful to maintain the orientation prescribed in the trim guide. Once the tissues are in the metal paraffin-filled mold, the labeled cassette from which they came is placed on top of the mold, which adheres to it when chilled, and maintains the identity of the tissue and paraffin block. The mold (with its attached cassette) is then placed on a chilled flat surface to solidify. As in all histology procedures, consistency and attention to detail are essential.

3.5  Microtomy

Once cooled, the paraffin-embedded tissue block can be removed from the metal mold and taken to the microtomy station. There, the microtome has a “chuck” which attaches to the cassette and holds the block-embedded tissues securely. The microtomist turns the handle of the microtome, which runs the blade across the face of the block multiple times to trim away the paraffin-only block until each of the tissue sections is exposed. This is called “facing” the block. Some labs will then place the paraffin blocks on a block of ice or other chilled surfaces to further chill the paraffin, which facilitates smooth cutting by the room-temperature microtome blade and minimizes artifacts. Histology artifacts are easy to inadvertently produce, are well published (McInnes 2012), and can interfere with microscopic evaluation, so measures to minimize their production are important to success of a study. The chilled, faced block is returned to the chuck and a ribbon containing a few tissue sections is produced and gently placed on the surface of a temperature-controlled water bath (generally 40–50 degrees centigrade). The microtomist picks up a single section of tissue by dipping an already labeled glass slide into the water and lifting it to pick up the section, ensuring no bubbles or wrinkles, wipes away water without touching the section, and places the slide vertically for drying. Some tissues are so small that they are difficult to detect in an unstained section. In those cases, the microtomist quickly verifies with a microscope that the tissues are, indeed, in the section on the slide.

3.6  H&E Staining

Routine evaluation of tissues under a microscope is done on tissues stained with hematoxylin and eosin (H&E). Hematoxylin, actually a combination of hematoxylin and a linking (“mordant”) substance such as aluminum cation, is therefore positively charged and acts as a basic dye. It binds with basophilic cell components such as nucleic acids (DNA and RNA) in a cell nucleus and stains them blue (Parry 2015). Eosin is anionic and negatively charged. It therefore binds to positively charged cell components (which are

Pathology Techniques

89

acid-loving and therefore eosinophilic), such as amino groups in proteins, which are usually in the cytoplasm or extracellular. Virtually all proteins stain pinkish red, including collagen in connective tissue, scar tissue, the cornea of the eye, as well as muscle fibers and keratin in skin. Staining is typically automated. Racks containing tens of slides are manually placed into a stainer and the program is started. The staining steps vary but are similar per laboratory. Generally, a robotic arm carries a rack into a chamber containing a heating element (an “oven”) and then from bin to bin in a series of timed steps. After staining, the rack of slides is transferred (manually or automatically) to a cover-slipper. Cover slips are required to view slides clearly through a light microscope, especially at 40× objective. As with a tissue processor, the staining programs are optimized for types of tissue (soft, bone, larger animal) and validated so results are consistent between batches and of optimal quality. Dried, stained slides are ready for evaluation under a light microscope. 3.7  Anatomic Pathologist Evaluation

Reporting is discussed elsewhere in this book, but it is appropriate to mention here that the preceding histology processes result in slides that provide much of the information considered by the pathologist for the pathology report, certainly one of the most important aspects in preclinical toxicity testing. The pathologist correlates histopathologic findings with relevant macroscopic observations, organ weights, clinical observations, and, often, clinical pathology (hematology, serum chemistry, coagulation, and urinalysis data). Therefore, slides of tissues that are of consistent fixation, thickness, and staining intensity and primarily free of artifacts (e.g., “Venetian blind” effect due to jerky microtome blade movement, or vacuoles in cerebellar white matter due to excessive processing time in ethanol) facilitate the pathologist’s ability to discern microscopic changes associated with the test article.

4  Histochemical Stains Histochemical stains dye tissue and cell components based on their chemical properties. Hematoxylin and eosin (H&E) is the most common histochemical stain used in toxicologic pathology, but hundreds of other histochemical stains exist and can be used to further characterize changes observed in H&E-stained tissues. A few common histochemical stains are listed below as examples: Fluoro-Jade: Degenerating neurons fluoresce when evaluated with a fluorescent light source. This stain may be helpful in evaluation of test articles suspected to cause neuronal injury (Schmued and Hopkins 2000). Red blood cells also fluoresce under a fluorescent light source. Perfusion of the brain for optimal fixation also

90

Pamela Blackshear et al.

removes most of the red blood cells, providing a better specimen for this type of evaluation. Masson’s trichrome: Connective tissue stains blue; cytoplasm stains red; and nuclei stain brown or black. This stain is often used to evaluate areas of fibrosis. Oil Red O: Neutral triglycerides stain orange. This stain can be used to help determine if clear vacuoles observed in H&E-stained tissues are neutral lipid. Because lipid will be removed in p ­ rocessing, Oil Red O staining needs to be conducted on frozen sections instead of sections from paraffin blocks. Periodic acid-Schiff (PAS): Carbohydrates stain magenta. This stain can be used to help determine if clear vacuoles observed in H&E slides are glycogen.

5  Immunohistochemistry (IHC) While histochemical stains dye tissues based on their chemical properties, immunohistochemical labels localize specific antigens within cells and tissues through the use of antibodies tagged with enzymes that precipitate a chromogen that is visible by light microscopy or tagged with a fluorescent label. IHC is an important auxiliary method used in routine diagnostic work, in research, and for the identification of protein target or biomarker in drug discovery and development. While most IHC target antigens are proteins, other antigenic biological substrates (e.g., carbohydrates, lipids, or nucleic acids) can also be recognized by antibodies. There are several practical applications of IHC in toxicologic pathology, including tissue cross-reactivity studies for therapeutic monoclonal antibodies to determine off-target tissue binding, characterization of cells or cell subtypes that are not distinguishable by H&E staining (e.g., pancreatic islet cells, tumor cells, or lymphocyte subtypes), or identification of immune complex depositions. 5.1  Considerations for IHC

There are many critical steps in performing IHC (O’Hurley et al. 2014). These include preparation of tissue sections (proper handling of the specimen, appropriate fixation, and paraffin block or frozen preparation), IHC procedures (antigen retrieval, selection and preparation of antibody and reagents for staining, and counterstaining), protocol development, and the desired data (e.g., semiquantitative or quantitative) from the IHC slides. It is essential that the specimen preparation, histologic processing, and IHC techniques are rigorously defined and followed for consistency to ensure accuracy and reproducibility of the IHC results. Minimizing variables in a study is critical in being able to achieve reproducibility in IHC. The advent of automated IHC machines has improved the reliability and reproducibility of IHC in the clinical and preclinical settings.

Pathology Techniques

91

5.2  Fixation

The fixative used should be compatible with the specific IHC procedures by identifying if the antibody is suitable for paraffin or frozen samples or both. Fixation for 24–48 hours at room temperature with tissue-to-fixative ratio of 1:1 to 1:20 is recommended based on the type of the tissues (Engel and Moore 2011); however, tissue-to-fixative ratio of 1:10 is adequate for most of the tissues. Tissues for possible future IHC can be transferred into ethanol from formalin if they cannot be blocked within 24–48 hours to prevent excessive cross-linking fixation. Common fixative types include 10% neutral buffered formalin, Bouin’s, Davidson’s, or stored frozen. Over- or under-fixation can lead to decreased availability of antigen/proteins of interest. Samples for frozen tissues should be collected in optimal cutting temperature (OCT) medium. Some antibodies for specific antigens are known to only work in frozen sections due to the retention of confirmation of epitopes. If an antibody works in both paraffin and frozen samples, paraffin is preferred due to better tissue morphology. Fixation with acetone or NBF is also required in frozen sections in certain situations such as evaluating new antibodies or detection of soluble antigens.

5.3  Sectioning and Storage

Paraffin blocks can be stored at room temperature for a longer period of time than frozen blocks. Frozen blocks on the other hand need to be stored at −20 °C or −80 °C. The recommended thickness of the tissue section for IHC is mostly 4 μm, and storing tissue sections at room temperature for more than a week can result in loss of certain antigens. Paraffin coating of sections can improve the storage condition of slides by preventing oxidation to retain epitopes in tissues (Economou et al. 2014). Storage of slides at 4 °C also helps prevent degradation of antigen. Frozen sections are recommended to store at −80 °C.

5.4  IHC Methods

Several factors should be considered before performing the assay (Kim et al. 2016) such as the target of interest, availability of primary antibodies, type of sample processing, antigen retrieval (AR) for fixed tissues, blocking nonspecific staining, optimal antibody concentration, detection methods, protocol development, and antibody interpretation. The goal of the IHC staining procedure is to have specific staining with primary antibody that recognizes target protein, while the isotype control matching the class and type of the primary antibody but lacking specificity to the target shows minimal or no staining, making it useful as a negative control. This requires optimization of methods of the laboratory performing the staining for both commercially available antibodies and proprietary therapeutic antibodies. Because antibody and antigen interaction cannot be seen by the human eye, chromogenic or fluorescence detection method must be used for visualizing the reaction. Chromogenic detection

92

Pamela Blackshear et al.

uses colors developed by reaction between enzymes and substrate, whereas fluorescence detection method uses light emission by fluorophores. A fluorescence detection system needs a specialized fluorescent microscope to evaluate the stain, and it needs separate methods to accomplish the goal. The chromogenic reaction can be achieved by direct method where primary antibody is tagged with enzymes or indirect method using secondary antibodies tagged with enzymes. Secondary antibodies raised against the immunoglobulin of the species from which the primary antibody is derived are applied after the primary antibody binding has occurred. Both direct and indirect methods can be used to detect bound antibodies. Biotin-streptavidin was a commonly used method for visualizing bound antibodies. In recent years, polymer-based detection methods are more popular with higher sensitivity. The use of more than one chromogen system (with sequential staining by primary antibodies) allows for the staining of one tissue section with more than one antibody. 5.5  IHC Protocol Development

Tissue controls are selected by choosing tissue/structures which are known to be positive for the protein target of interest and tissues known to be negative for target of interest. For frozen sections, the post-fixation methods (acetone or formalin) may have an impact on antibody or soluble antigens. For paraffin, the antigen retrieval methods (heat-induced versus proteolytic-induced epitope retrieval) need to be considered. For primary antibody dilution, a 3–4 series dilution must occur (1 dilution below, 1 dilution within, and 1 dilution above the recommended dilution range provided by the antibody vendor in the data sheet). When using secondary antibodies, one must ensure they are tagged with enzymes that react with the selected substrate to produce the desired color. This color can be quantitated using a microscope or software-­ associated digitized images. The last step is to counterstain the tissue to have contrast between positive stain and background. Automated IHC systems are recommended if a large number of samples are to be tested. The following discusses the steps involved in protocol development.

5.6  Antigen (or Epitope) Retrieval (AR)

Formaldehyde-based fixation causes cross-linking of amino groups on adjacent molecules resulting in masking of epitopes within tissues. Antigen retrieval is necessary to unmask or make the epitopes available for binding the antibody (Shi et al. 2007). Determination of the most appropriate AR technique is a critical step and choices include heat and various enzymes. Heat-induced epitope retrieval (HIER) is the most widely used method for antigen retrieval and is most often performed using a microwave oven or pressure cooker in varying conditions including pH 6–10. Generally, using a pressure cooker or microwave oven, the temperature is set at 120 °C at full pressure or 750–800 W, respectively, and typically for 10 min-

Pathology Techniques

93

utes. Alternatively, in enzymatic retrieval method, tissue sections are incubated in either trypsin or proteinase for 10–20 minutes at 37 °C. In general, AR is not necessary for frozen sections, unless they have been post-fixed with acetone or NBF. 5.7  Protein Blocking

Protein blocking is required to reduce unwanted background staining due to nonspecific binding of Fc portion of primary or secondary antibodies to the tissues (Vogt Jr. et al. 1987). An ideal agent for the protein blocking is 5–10% normal serum from the same species as species of origin of the secondary antibody. Other agents include 0.1–0.5% bovine serum albumin, gelatin, or nonfat dry milk. Recently commercial mixes of synthetic peptides are being widely used. Incubation time for the blocking step can vary from 30 minutes at room temperature to overnight at 4 °C.

5.8  Endogenous Enzyme Blocking

When using peroxidase detection systems, diluted 3% hydrogen peroxide is widely used for blocking endogenous peroxidase activity in tissues (Radulescu and Boenisch 2007). Tissues with high blood content or with intense granulocytic inflammatory infiltrate, bone marrow, and lymphoid tissue have high endogenous peroxidase activity which requires strong suppression. Tissues rich in endogenous alkaline phosphatase such as kidney, intestine, osteoblasts, lymphoid tissue, and placenta are blocked in frozen tissue with 10 mM levamisole (Garba and Marie 1986). Endogenous biotin which is more prevalent in liver and kidney compared with other tissues can be blocked by incubating the tissue section in avidin and biotin solution beforehand (avidin solution for 15 minutes followed by biotin solution for 15 minutes at room temperature).

5.9  Antibody Selection and Validation

Antibodies used in IHC are either polyclonal or monoclonal. Polyclonal antibodies contain multiple clones of antibodies that are produced in experimental animals following repeated stimulation of antigen to target various epitopes and therefore have higher levels of binding to a single antigen. Due to their recognition of multiple epitopes, polyclonal antibodies can help amplify the signals of proteins with low expression and are highly sensitive. Relative to polyclonal antibodies, monoclonal antibodies are highly specific and react to a single epitope in an antigen, which greatly reduces crossreactivity issues. The use of monoclonal antibodies permits better reproducibility than the use of polyclonal antibodies. Traditionally monoclonal antibodies are produced in mouse or rat; however, recently rabbit monoclonal antibodies are becoming more popular. Interpretation of IHC stain patterns in control tissues should be done carefully considering appropriate location, intensity, and signal/noise ratio. Selection of an appropriate primary antibody should consider specificity of antibody, suitability of the antibody for paraffin or frozen samples, ability of the antibody to react with the species

94

Pamela Blackshear et al.

being tested, origin of the antibody from a species other than the one being tested, cellular localization of target, product citations/ references, availability of positive/negative control tissues, and antibody supplier database (O’Hurley et al. 2014). If commercially available antibody is not available, customized antibody production may be needed to perform IHC. Antibodies produced for clinical IHC diagnostic are labeled as in vitro diagnostics (IVD); however, IHC antibodies used for preclinical laboratory animal tissue are classified as research use only (RUO). The production of RUO antibodies is not regulated; therefore, it is essential that RUO antibodies are optimized prior to use in IHC studies. These steps include an antibody dilution series to identify the optimal concentration for antigen-antibody interaction that produces the least background staining in positive control tissues. Ideally each newly manufactured lot of primary antibody should be optimized for variability in inter-lot antibody performance. 5.10  Detection System

Detection is the process of detecting proteins after binding of antibody to antigen (Pekmezci et al. 2012). Commonly used detection systems include labeled primary or secondary antibody with enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (AP), labeled biotin-streptavidin methods, and polymer-­ based detection systems. In comparison to other IHC methods, a polymer-based amplification method typically has at least 50-fold greater sensitivity (Fig. 2). Biotin-binding conjugates are normally limited to carrying 1–3 HRP molecules per protein in order to maintain enzymatic activity. Given the small size of the HRP molecule (44 kDa), the sensitivity of detection system is increased by using polymeric HRP (poly-HRP) conjugated to secondary antibodies to detect proteins present in very low amounts (picogram quantities) (Fig. 3). The outcome of an IHC depends on the selection of optimal methods for signal amplification for the target molecule. An AP-based detection system is preferred for tissues rich in endogenous peroxidase, such as bone marrow or lymphoid tissue. Likewise, a peroxidase based-detection system may be used for tissues with endogenous alkaline phosphatase but AP is easily destroyed by high-temperature antigen retrieval. A biotin-free synthetic polymer system is recommended for tissues with high endogenous biotin such as liver and kidney. A choice of several chromogens is available to achieve the best color based on tissue type and/or counterstain. In general, diaminobenzidine (brown) or 3-amino-­ 9-ethyl carbazole (red) is routinely used for peroxidase, and 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/tetranitroblue tetrazolium (TNBT) (purple/black) is used for alkaline phosphatase. Recent advancement in multiplex IHC allows simultaneous detection of multiple antigens in a tissue independently with adequate signal amplification. Monoclonal antibodies are validated to enable detection of more than one antigen in one section of tissue.

Pathology Techniques DAB + H2O2

a

DAB + H2O2

b

Brown Color

95

Brown Color

Enzyme

Enzyme Primary Antibody

Secondary Antibody Antigen

Antigen

Indirect Staining

Direct Staining

c

DAB + H2O2

d

Brown Color

DAB + H2O2

Brown Color Amplification

StreptavidinEnzyme Complex Dextran backboneEnzyme (Polymer)

Biotinylated Secondary Antibody Primary Antibody

Primary Antibody

Secondary Antibody Antigen

Antigen Biotin-Streptavidin Staining

Polymer Staining

Illustration of Types of IHC Staining Fig. 2 (a) Antigen binds to primary antibody conjugated with an enzyme. (b) Antigen binds to primary antibody followed by binding of secondary antibody conjugated with an enzyme. (c) Antigen binds to primary antibody followed by binding of biotinylated secondary antibody and streptavidin-enzyme complex. (d) Antigen binds to primary antibody followed by binding of secondary antibody and polymer conjugated with several enzymes to amplify the reaction. Last step in all four methods is to add substrate diaminobenzidine (DAB) with H2O2 to produce color reaction

The benefits of multiplexing include efficient use of small and rare tissue samples, co-expression and spatial organization of many targets without disrupting the tissue integrity, and quick turnaround time. 5.11  Counterstaining

Counterstaining provides contrast to the chromogens used as the principal stain, making the stained structure easily visible using the microscope. Hematoxylin is the most commonly used counterstain in IHC. Various other counterstains are used in multiplex IHC where multiple antibodies are used to identify multiple targets (Stefanovic et al. 2013). Counterstain should be selected carefully, taking into consideration the chromogens used for detection and how well they blend in or contrast with the chosen counterstain. The ideal combination has sufficient contrast. Other commonly

96

Pamela Blackshear et al.

Fig. 3 Histiocytes in the red pulp of a mouse spleen with brown cytoplasmic labeling for F4/80. Tissue was fixed in 10% NBF for 48 hours, enzymatic antigen retrieval was performed, and the tissue was incubated with a rat monoclonal anti F4/80 antibody and DAB chromogen

used counterstains are methylene blue, methylene green, or toluidine blue. 5.12  Quantitative Data from IHC Slides

IHC provides highly specific qualitative and quantitative information in relation to the expression of particular molecules within a tissue (Taylor and Levenson 2006). If the IHC data is to be accurately interpreted in the context of the study design, a pathologist must also evaluate routine (H&E-stained) sections—the purpose of this is to define the pathologic process(es) present and then qualitatively/semi-quantitatively evaluate the IHC slides with a light microscope to define the staining as “on-target” or “off-­ target.” This approach recognizes clear differences between experimental and control groups; however, more subtle differences often require quantitative comparison of the protein expression using image analysis. This can be achieved by whole-slide scanning of IHC-stained slides to capture digital images. The area of interest in each slide to be analyzed is outlined and the total staining in that area is determined by vendor-provided software. For multiplex IHC, multicolor stained tissues are analyzed. Due to a massive amount of information in a single multiplex IHC slide, a spectral image analysis method is used to interpret the data, in which each chromogen can be isolated and quantitated separately. The study pathologist, using his or her broad training and expertise, integrates the quantitative IHC data of the target molecules with the morphologic findings obtained from H&E-stained tissues and other study data. Inclusion of quantitative IHC data can allow the study pathologist to provide a more comprehensive and sensitive assessment of chemical, pharmaceutical, biopharmaceutical, or medical device-related changes of interest, which can be a critical component of the overall safety assessment of some studies.

Pathology Techniques

5.13  Commonly Used IHC Antibodies

97

B220 (B cell), Bcl2 (anti-apoptotic protein), cytokeratin AE1/ AE3 (epithelial cell), CD3 (all T cells), CD4 (T cell), CD8 (T cell), CD31 and von Willebrand factor (vasculature), CD34 (hematopoietic stem cells), CD68 and F4/80 (macrophage), chromogranin A (neuroendocrine cell), cleaved caspase 3 (apoptosis), collagen I–IV (connective tissue), Iba-1 (microglia), IgG and IgM, insulin and glucagon (pancreatic cells), Ki67 (mitotic cell), nestin (neural stem cell), and vimentin (mesenchymal cell).

6  In Situ Hybridization (ISH) ISH is a technique that allows for the localization and identification of messenger RNA (mRNA) in fixed tissues. In situ is Latin for “on site or in position.” Hybridization is the process of chemical bonding. As a molecular technique, DNA or RNA hybridization is the process of joining complementary strands of nucleic acids (deoxyribonucleic acids, DNA, or ribonucleic acids, RNA) in order to provide supplemental information concerning gene targets in support of proof-of-concept and/or safety and efficacy studies. ISH uses nucleic acid probes that are complementary to the nucleic acid sequence of interest. Nucleic acid probes are designed similarly to polymerase chain reaction (PCR) primers except only the anti-sense strand is used. Nucleic acid probes are short segments of single-stranded complementary DNA (cDNA), oligonucleotides, or RNA, which are complementary to a messenger RNA (mRNA) of interest in the tissue. RNA probes are very sensitive and are considered the best nucleic acid choice for probes, given the abundance of RNA compared to its homologous DNA (Pringle 1995). RNA targets also reveal more information about cellular activity for a specific gene. However, single-stranded RNA is very susceptible to degradation by RNase which is ubiquitous and requires everything (glassware, plastics, solutions, etc.) used in the ISH process to be “RNAse-free.” Oligonucleotides and cDNA probes are less sensitive and thus do not require RNase-free conditions. Protocols for the in situ hybridization procedure can be divided into four main categories: pretreatment, pre-hybridization, hybridization, and post-hybridization procedures. However, the specifics and intricacies of each category are dependent on probe type and detection method. Since RNA probes are the most sensitive nucleic acid probes and non-radioactive (non-isotopic) procedures are more commonly used, the following will concentrate on protocol specifics related to non-isotopic RNA in situ hybridization. Manual procedures take 2–3 days to perform. However, with automated equipment, commercial reagent kits, and probes, the procedure can be performed in 10–12 hours. Tissue collection, probe preparation, and reagent and labware preparation are key components that contribute to the successful outcome of ISH.

98

Pamela Blackshear et al.

6.1  Tissue Collection and Controls

Tissue collection requirements for ISH are similar to IHC (maximum 24–48 hours of fixation) with the added requirement, for working with RNA probes, that all specimen-handling procedures be performed with gloved hands, and that labware be sterile and/ or treated with commercially available products to inactivate RNases. Tissues can be frozen or immersion-fixed in 4% paraformaldehyde or 10% formalin and embedded in paraffin. For best results with frozen sections, tissues should be placed in optimal cutting temperature compound (OCT) and precooled isopentane. Frozen sections require a post-cryotomy fixation step with 4% paraformaldehyde at 4 °C. Formalin-fixed, paraffin-embedded (FFPE) sections require removal of paraffin (deparaffinization) by immersing the tissue-mounted slides in xylene followed by decreasing concentrations of ethyl alcohol to rehydrate the tissue sections and post-fixation with paraformaldehyde. Positive and negative control tissues and probes should be included with each protocol to confirm that hybridization occurred, assess probe specificity for the target of interest, and distinguish nonspecific background effects. It is recommended to have a positive control probe that recognizes a highly expressed gene target such as a housekeeping gene (e.g., cyclophilin A, GAPDH, or 18S RNA). Figure 4 is a positive control tissue that contains the target of interest. Figure 5 is a negative control probe that targets a sequence not present in the tissue/cells of interest (e.g., bacterial gene).

6.2  Pretreatments

One or more pretreatments with proteolytic enzymes, acid, salt washes, and acetylation are required to increase accessibility of the target in the tissue sections, prevent nonspecific binding, and block nonspecific background signals. Controlled digestion of tissue ­sections is performed with proteolytic enzymes such as proteinase K, pronase, or pepsin. Concentration of proteolytic enzymes and

Fig. 4 In situ hybridization using ACD™ cyclophilin A probe and RNAscope ®2.5 LS assay kit on formalin-fixed paraffin-embedded rabbit ovary section. Cyclophilin A mRNA expression is present in the granulosa and theca cells of the ovarian follicle and is demonstrated by brown granules

Pathology Techniques

99

Fig. 5 In situ hybridization using ACD™ DAPB probe and RNAscope ®2.5 LS assay kit on formalin-fixed paraffin-embedded rabbit ovary section. DAPB is used as a negative control probe as it recognizes bacterial RNA

duration of digestion are dependent on tissue type and fixation method (Pringle et al. 1990). Proteinase K is the most commonly used proteolytic enzyme for ISH due to its ability to digest nucleases. Mild acid treatment with 0.1 M HCL and salt washes help to prevent nonspecific binding of the probe (Cox et al. 1984). Acetylation with acetic anhydride helps to reduce binding of probe to charged molecules in the tissue section, thereby reducing nonspecific background signal (Hayashi et al. 1978). 6.3  Pre-­ hybridization, Hybridization, and Post-hybridization

Tissue sections are acclimated to hybridization conditions by incubating in a pre-hybridization solution. Pre-hybridization solutions contain components (e.g., formamide, double-stranded salmon sperm DNA, dextran) that maximize thermal stability and specificity of RNA hybrids. Thermal stability of RNA hybrids is characterized by the mean thermal denaturation temperature (Tm) which is based on sodium chloride concentration, percentage of guanine and cytosine residues in the target sequence, and percentage of formamide and is used to determine optimal hybridization temperature (Pringle 1995). The most common hybridization temperature range is between 55 and 62 °C. Prehybridization is followed by hybridization with the addition of the probe to the pre-hybridization solution. Salt washes after the hybridization step help to reduce nonspecific binding and to remove mismatched hybrids.

6.4  Detection

Multiple options for detection systems are available and depend on the type of signal-generating molecules (haptens) linked to the probe (O’Leary et al. 1995). Probes can be linked with radioactive or non-radioactive signal-generating molecules to create a signal that can be detected visually in the tissue sections. Although radioactive probes are most sensitive, non-radioactive probes are more commonly used due to safety, monitoring, and disposal concerns

100

Pamela Blackshear et al.

associated with the use of radioactive substances (Mullink et al. 1995). Non-radioactive molecules (haptens) linked to probes include biotin, digoxigenin (DIG), and tyramide. Digoxigenin is a plant-based molecule associated with low background. All of these molecules can be amplified to help boost signal detection. Radioactive probes are visualized using liquid emulsion and photography or autoradiography film. Non-radioactive probes use colorimetric or fluorescence methods that require the addition of an anti-hapten monoclonal or polyclonal antibody (e.g., anti-­ digoxigenin, anti-biotin, or anti-fluorescein antibody) and a secondary antibody conjugated to an enzyme or fluorochrome (e.g., biotinylated secondary antibody against the primary antibody) conjugated with peroxidase or alkaline phosphatase similar to methods used in immunohistochemical staining in single or multistep process depending on level of sensitivity desired. For alkaline phosphatase-linked antibodies, an enzymatic color precipitation reaction is performed, such as 5-bromo-4-chloro-3-indolyl phosphate (an enzyme substrate) combined with nitro blue tetrazolium, a purple-black chromogen (BCIP/NBT). Slides are finally counterstained with a stain complementary to the chromogen that allows for maximum visual contrast (e.g., hematoxylin). 6.5  Application

In situ hybridization can establish the site, temporal distribution, or morphological distribution of RNA expression in tissues and provide information concerning adaptive responses as compared to IHC which gives information concerning newly synthesized or stored proteins (Pringle 1995). ISH can provide information under circumstances when the gene product is not detectable or available such as when antibodies are not available or if the protein product is mutated, rapidly degraded, or rapidly released from the cell. RNA ISH and IHC can be combined to study the transcriptional versus translational regulation of gene expression. When combining non-radioactive RNA ISH and IHC, ISH is performed first, followed by IHC in order to preserve RNA targets. Commercial ISH and IHC reagents, probes, and automation make these applications more practical and cost-effective when higher throughput volume is required for addressing specific questions regarding a gene target and its product. ISH can be used in a variety of research areas such as cancer, neuroscience, inflammation, and gene therapy and used to identify biomarkers or targets within molecular signaling pathways such as Jak-Stat and Wnt signaling pathways. An ISH probe can be made from any gene target but is most frequently used in situations where an antibody for a particular target is unavailable or not working.

Pathology Techniques

101

7  Digital Pathology and Morphometry Whole slide scanners that use a camera to scan a glass slide and create a high-resolution digital image of the whole slide are commonly used in pathology. Software enables a user to then view this digital image on a monitor in a similar manner as an individual would evaluate a glass slide, moving from area to area and increasing or decreasing the magnification of their view as desired. While most scanners create an image with one focal plane, specialized scanners create a stack of images at multiple focal planes that allow an individual to “focus up and down” on the digital image. Whole slide images have most commonly been used to share and discuss lesions across multiple locations without shipping glass slides, but image management systems have become more sophisticated, making primary evaluation or peer review of entire toxicology studies feasible and possibly more efficient than evaluating glass slides. Image management systems can allow for rapid retrieval and comparison of digital slides between specific animals or treatment groups and even “blinding” of treatment groups without the inefficiency of pulling and sorting glass slides from slide boxes. Primary evaluation or peer review using digital images is not currently accepted as GLP-compliant by regulatory agencies, but peer review using digital images is becoming more common. Challenges to GLP compliance include the lack of guidelines on practical conditions, such as level of scanning magnification, acceptable focus, use of multiple stacks, etc. (Gauthier et al. 2019). Digital images also allow for morphometry, or quantitative measurements of features on the slides, such as counts of specific cell types (e.g., numbers of specific types of inflammatory cells) and measurement of areas of interest (e.g., size of pancreatic islets, length of wounds, or thickness of granulation tissue). These measurements are often made on slides that have been labeled with immunohistochemistry or in situ hybridization. Specific algorithms have been developed to automate detection and quantification of specific changes (e.g., bile duct hyperplasia, renal tubular dilation, or foci of necrosis). Multiple efforts are ongoing to attempt to develop software that can analyze all tissues from a toxicology study and identify which slides are normal so the pathologist can focus evaluation on slides that are “not normal.” While this would greatly speed the microscopic evaluation of tissues, this software will likely require many years of testing before gaining regulatory acceptance. Images that are used for data generation (to make a diagnosis or for morphometry) are raw data that must be authenticated and archived (Tuomari et al. 2007). Images that are illustrative and not used for data generation are not raw data and do not have to be archived (Tuomari et al. 2007).

102

Pamela Blackshear et al.

8  Electron Microscopy The resolution possible with light microscopy limits useful magnification to approximately 1500×. The range of magnification with light microscopy makes it ideal to evaluate larger areas of organs and tissues as well as the morphology of individual cells and some subcellular structures. Electron microscopy has the ability for much greater resolution and useful magnification of over 100,000×, which allows for evaluation of subcellular detail including individual organelles, but does not allow an efficient survey of large areas of organs and tissues. Due to this, electron microscopy is best used to further characterize changes first observed by light microscopy. For example, if vacuolation is observed in hepatocytes with evaluation of H&E-stained slides, electron microscopy could be used to determine whether those vacuoles are due to neutral lipid accumulation, phospholipidosis, or distension of an organelle with accumulation of material or organelle injury. Electron microscopy can be used to evaluate subcellular changes to help determine the mechanism of injury observed with light microscopy. Two main techniques in electron microscopy are scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM allows for three-dimensional evaluation of surfaces and is rarely used in toxicologic pathology for drug development. TEM is more similar to light microscopy, in that it is evaluation of a very thin section through the tissue providing a two-dimensional view. Sections for TEM are generally less than 100 nm thick. The best-quality images for TEM are obtained if samples are collected for the specific purpose of electron microscopy. Ultrastructural changes occur within just a few minutes after euthanasia, so samples for electron microscopy should be prioritized for collection before samples for light microscopy. Glutaraldehyde is one example of a fixative suitable for electron microscopy. Glutaraldehyde provides more extensive cross-linking than formalin and therefore better preservation of architecture for evaluation of ultrastructure with electron microscopy (Grizzle et al. 2008). Glutaraldehyde is slower to penetrate than formaldehyde, so tissues fixed in glutaraldehyde must be less than 0.5 mm thick (Grizzle et al. 2008) instead of less than 5 mm thick for samples collected in formalin for light microscopy. While the best preservation of subcellular architecture is obtained in samples collected specifically and appropriately for electron microscopy, samples that were collected in formalin can be processed for electron microscopy. While formalin-fixed tissues will have more artifactual changes than ­tissues immediately collected in a more appropriate fixative for electron microscopy, sufficient morphology is typically preserved to further characterize changes observed with light microscopy.

Pathology Techniques

103

9  In Vivo Imaging Imaging in the biomedical field has made great progress with its in vivo and ex vivo applications in pharmacological and toxicological studies. Anatomical, molecular, and functional imaging can provide valuable information to help evaluate the efficacy, safety, and toxicity of drugs in development. Both in vivo and ex vivo imaging provide great tools to noninvasively assess the morphology of tissues in preclinical animal models. Imaging offers high-­ resolution and virtual 3D slicing of the scanned region of interest which can be an added advantage to pathologists. In vivo imaging methods present the advantage of longitudinal follow-up of the same animal for the duration of the study which provides baseline and control data. This helps in utilizing fewer animals for the study and eliminates staggered study designs. Animal enrollment can be evidence-based on the imaging data for randomization and/or therapeutic intervention. Computed tomography (CT), magnetic resonance imaging (MRI), ultrasound (US), and positron emission tomography (PET)/single photon emission computed tomography (SPECT) are widely used imaging modalities in the clinic and considered translational biomarkers in preclinical drug discovery. Toxicological pathologists can use imaging (ex vivo or in vivo) along with conventional histopathology to assess disease progression and toxicological effects. With the use of appropriate analysis software, the 2D or 3D images can be quantitatively assessed for changes in function or morphometry of the tissues. 9.1  Computed Tomography (CT)

CT imaging is an x-ray based medical imaging modality that is widely used in both clinical and preclinical studies. An image is created when x-rays that pass through the scan subject are then detected by a charge-coupled camera. X-rays passing through the biological tissue are partially absorbed and this is called “attenuation” which determines the density of the imaged tissue. In CT imaging, the x-ray source and detector are placed on a gantry that rotates around the scan subject to collect 2D projection images from different angles. These 2D projection images are post-­ processed by a computer to reconstruct the 3D information of the scan subject. Both in vivo and ex vivo CT imaging are rapid, high-resolution 3D imaging techniques that help to visualize and characterize pathophysiological changes in a tissue. Different tissues have different degrees of x-ray absorption based on internal composition. For example, bone has more attenuation than soft tissue and air, which makes CT imaging of bone a valuable anatomical reference tool in musculoskeletal research. While primarily used for anatomical evaluations, CT imaging can be used to evaluate blood flow and

104

Pamela Blackshear et al.

perfusion through organs and tumors with the use of iodine- or barium-based contrast agents that enhance the imaging signal. Dynamic contrast-enhanced CT (DCE-CT) is used in both preclinical and clinical studies to measure vascular changes in the brain, liver, kidney, lung, and tumors. In vivo longitudinal imaging helps to identify the onset of the disease along with noninvasively monitoring the pathophysiology associated with the disease. For example, an orthotopic lung cancer model was created in athymic nude mice by injecting the H441 cells (human pulmonary adenocarcinoma) into the left lung lobe of the athymic mice. Twenty-one days post-inoculation, the animals were randomized into three groups (vehicle, Gemzar® 75 mg/kg, Gemzar® 150 mg/mg) based on the tumor burden measured by in vivo CT imaging. Starting from day 21 the animals were treated intraperitoneally with 0.2 mL of Gemzar® (75 and 150 mg/kg) or vehicle (saline) once every 3 days, with a total of four treatments. Longitudinal in vivo CT imaging was performed weekly to monitor the disease progression and tumor growth from day 21 to day 49. Animals treated with Gemzar® had regression of tumor, compared to vehicle-treated animals, as shown in Fig. 6, which was validated with histopathologic evaluation. High-resolution ex vivo CT imaging, also called as micro-CT, is used in preclinical studies and can produce an image at as high as 1- to 2-μm resolution. One application of micro-CT is high-­ resolution bone imaging that can be used as a 3D morphometric tool by pathologists to complement traditional 2D histomorphometry. Recently micro-CT imaging has also been used in developmental and reproductive toxicology studies for fetal skeletal assessments as an alternative to traditional alizarin-red staining methods (Solomon et al. 2018; Wise et al. 2013). Figure 7 is a comparison of rat fetus scanned at 35-um pixel size followed by staining with alizarin-red. Skeletal features that are visible from stained fetuses can be visualized and assessed using microCT. Additionally micro-CT images can be used for measuring ­ bone lengths and bone mineral density useful for toxicological studies. The primary advantages of CT imaging are speed, high resolution, and good signal-to-noise ratio. The major disadvantage is the effects of prolonged radiation on the tissue or organs; therefore care needs to be taken in designing the in vivo animal studies. 9.2  Magnetic Resonance Imaging (MRI)

MRI is another widely used imaging modality known for its high soft-tissue contrast. MRI generates a static magnetic field around the scan subject and uses the body’s inherent magnetic moment of atomic nuclei. The most abundant atomic nuclei in the body are hydrogen protons, and hence protons of water and fat are observed using proton imaging. MR imaging can be used for other atomic

Pathology Techniques

105

Fig. 6 Tumors are easily visible in the left lung shown with arrows. (a) Note the ability of CT imaging to evaluate similar levels of the lung in all mice simultaneously. (b) Corresponding histologic sections demonstrating typical tumor pattern. (c) Longitudinal change in tumor volume between vehicle and Gemzar treated groups as measured with CT imaging

106

Pamela Blackshear et al.

Fig. 7 Alizarin-red staining (left) compared to the 35-μm micro-CT 3D rendering (right)

nuclei like helium-3 (3He), carbon-13 (13C), fluorine-19 (19F), sodium-23(23Na), phosphorus-31(31P), and xenon-129(129Xe). Gradient coils are used to create a gradient field for spatial encoding of the MR signal in X, Y, and Z directions. A transmit coil is used to excite atomic nuclei from a static state by applying radio frequencies called excitation pulses or RF pulses. When excited atomic nuclei return to a static state, called relaxation, they emit a resonant frequency that is read by the receive coil, called the MR signal. This MR signal is processed to identify the spatial orientation of the atomic nuclei, which form an image. In preclinical studies, animal MRI has been used for both in vivo and ex vivo imaging to measure drug-induced pathophysiological changes in organs like the brain, liver, kidney, and cardiovascular system due to its high soft-tissue contrast and resolution. Dynamic contrast-enhanced MRI (DCE-MRI) is used to improve the contrast between normal and abnormal tissues. Gadolinium-, manganese-, or iron oxide-based contrast agents are widely used, and when these agents are given intravenously, blood flow, vascular permeability, and vascular density can be measured in tissues like tumors or liver parenchyma. Magnetic resonance histology (MRH) has been developed as an ex vivo imaging technique to nondestructively visualize and quantify fixed tissue specimens at high resolutions which can help identify appropriate regions to be sampled for further standard histopathology (Johnson et al. 2002; Ramot et al. 2017) evaluation. In Alzheimer’s disease, brain atrophy is one of the pathophysiological conditions indicating neuronal loss and behavioral impairment. In a study of rTg4510 mouse Tauopathy model (transgenic mouse rTg4510 overexpresses the Tau gene), in vivo MRI imaging

Pathology Techniques

107

Fig. 8 T1 weighted in vivo MRI brain images of rTg4510 transgenic mouse at 3 and 8 months of age (a and b, respectively). Decrease in hippocampal and cerebral cortex volumes and increase in ventricular volume can be observed in the 8-month-old mouse brain image

was implemented to measure volumes of multiple brain regions like the cortex, hippocampus, lateral ventricles, and cerebellum to assess the onset of the disease with age. Figure 8 shows an in vivo MRI brain image of rTg4510 transgenic mouse showing decrease in hippocampal and cerebral cortical volume and increase in ventricular volume from 3 to 8 months indicating brain atrophy with aging. Unlike CT imaging, MRI does not require ionizing radiation and has high soft-tissue contrast making it valuable in assessing both anatomical and functional changes in soft tissues. However, the high initial capital cost of the MRI equipment prohibits access to many researchers. MRI also takes long scan times reducing the scanning throughput and adding stress to the animals due to extended anesthesia. 9.3  Ultrasound (US)

Ultrasound imaging uses high-frequency sound waves to generate signals for creating an image. The sound waves are generated by a probe, called a transducer, by converting the electrical signals to sound. When the probe is placed against a body, the sound wave travels within the body and gets reflected back as an echo by the internal body structures. The reflected echo is read by the probe. The sound waves are sent in pulses at defined time intervals which help to identify the reflected echo’s strength and time of travel for virtually “constructing” the internal body structure. The reflected sound waves are converted to electrical signals and processed by a computer to generate an image. High-frequency sound waves provide high-resolution images, but the depth of penetration decreases with increase in frequency. Ultrasound is used for both anatomical and functional imaging in preclinical research. Different modes of ultrasound imaging are used for different applications like B-mode for anatomical imaging,

108

Pamela Blackshear et al.

M-mode for functional imaging, Doppler-mode for blood perfusion imaging, and others. Since fluids like blood transmit rather than reflect sound waves, gaseous microbubbles are used as a contrast agent to increase the echo signal. Ultrasound imaging, which has high temporal resolution and capability of capturing real-time images, is a valuable imaging modality to assess function of heart by measuring parameters like left ventricular chamber volume, left ventricular wall thickness, and ejection fractions. Applications of ultrasound imaging in preclinical research include cardiovascular, cancer, musculoskeletal, and developmental toxicology studies (Foster et al. 2011; Graham et al. 2005; Li et al. 2008). A preclinical rat model of isoproterenol-induced cardiac hypertrophy is shown in Fig. 9, in which ultrasound was used. The B-mode imaging was used to visualize the myocardium and the M-mode for cardiac function. Isoproterenol induced infarct-like lesions in rat myocardium and rapidly impaired the left ventricular function. Ultrasound imaging is gaining acceptance in both clinical and preclinical research due to lack of ionizing radiation, real-time imaging, noninvasiveness, and high spatial and temporal sensitivity. Ultrasound devices are relatively inexpensive compared to other imaging modalities and hence are widely available. Some disadvantages of ultrasound imaging are its low signal-to-noise ratio and depth of penetration, which is inversely proportional to resolution.

Fig. 9 Isoproterenol induces cardiac hypertrophy. B-mode and M-mode imaging show the changes in LV wall thickness due to isoproterenol compared to vehicle treatment up to 15 days post-dosing

Pathology Techniques

9.4  Positron Emission Tomography (PET)/Single Photon Emission Computed Tomography (SPECT)

109

PET and SPECT are considered molecular imaging modalities; both use radionuclides as a source for imaging. In PET imaging a short-lived radiolabeled tracer is given intravenously to the test subject and is then traced with a PET camera. The radionuclide is produced at a cyclotron followed by chemical synthesis to produce the radiopharmaceutical or radiolabeled tracer. The radionuclide decays by emitting positrons which collide with electrons naturally found in the body (annihilation). Each collision generates a pair of gamma photons (electron and positron) that have energies of 511 keV and 180° apart which are then picked up by a pair of scintillator sensors of the PET camera. A PET system has a stationary gantry that is loaded with multiple PET cameras that detect the positron annihilation events from the injected radiolabeled tracer. The captured annihilation events are then processed by a computer to generate a 3D image. PET imaging is mostly used to observe changes in metabolism associated with drug or disease states. Special tracers are used to assess sites of metastases, receptor occupancy, or drug metabolism. The most commonly used tracer is fluorodeoxyglucose (18F-FDG), which is used for tissue metabolism studies and is now the standard radiotracer used for PET neuroimaging and cancer patient management in clinics (Kelloff et al. 2005). SPECT imaging uses radionuclides that decay with emission of single gamma rays and typically have longer half-lives than PET radionuclides. A gamma-emitting radionuclide is given intravenously to the test subject, and as the radionuclide decays, it emits gamma rays that are detected by a gamma camera (typically scintillation camera). A SPECT system consists of one or two gamma cameras mounted in a rotating gantry, similar to a CT scanner, and acquires multiple 2D images from different angles which are processed to obtain the 3D distribution of the radionuclide. The 2D images represent emission events from the radionuclide decay indicating the amount of blood flow within the imaged region. Typical preclinical SPECT radionuclides include technetium-99 m (99mTc), iodine-123 (123I), and indium-111 (111In) used for imaging cancer xenografts in mice and rat hearts following ischemiareperfusion and for imaging dopamine transporters in rat brain (Scherfler et al. 2002). PET and SPECT are combined with CT to enable correlation of anatomical and functional information for better localization of the decay events. PET/CT is more widely used in clinic but the short half-lives of the radionuclide require close proximity access to a cyclotron. Since the half-lives of radionuclides used in SPECT/ CT are longer, they can be obtained from central radiopharmacies instead of a cyclotron. SPECT/CT also has the capability of imaging multiple tracers labeled with different radionuclides, thereby allowing the simultaneous study of multiple molecular or cellular events.

110

Pamela Blackshear et al.

The advantage of PET and SPECT is that they detect biochemical changes that typically precede anatomical changes (Maronpot et al. 2017); however, access to radionuclides and radiochemistry labs is needed, which may limit their use. Another limitation is exposure to radiation from the radionuclides in both PET and SPECT.

10  Conclusion Anatomic and clinical pathology assessments often provide the key data needed to decide whether a test article should be developed into a new drug. Standard assessments (evaluation of organ weights, macroscopic and microscopic observations, and hematology, clinical chemistry, coagulation, and urinalysis data) often provide the only pathology information needed to achieve study objectives and decide the fate of test articles. Many special laboratory techniques are available that can further characterize changes when needed, but are impractical to use routinely because they require focus on a single tissue or lesion and/or have resource needs that would be prohibitive to use except when warranted. Veterinary pathologists and pathology laboratory technicians are valuable resources to help determine when and how to apply specific pathology techniques. References AVMA (2013) AVMA guidelines for the euthanasia of animals: 2013 Edition Bailey SA, Zidell RH, Perry RW (2004) Relationships between organ weight and body/brain weight in the rat: what is the best analytical endpoint. Toxicol Pathol 32(4):448–466. https://doi.org/ 10.1080/01926230490465874 Bregman CL, Adler RR, Morton DG, Regan KS, Yano BL (2003) Recommended tissue list for histopathologic examination in repeat-dose toxicity and carcinogenicity studies: a proposal of the Society of Toxicologic Pathology (STP). Toxicol Pathol 31(2):252–253. https://doi. org/10.1080/01926230390183751 Chosewood LC, Wilson DE (eds) (2009) Biosafety in microbiological and biomedical laboratories, 5th edn. Centers for Disease Control. https:// www.cdc.gov/labs/pdf/CDC-BiosafetyMicrob iologicalBiomedicalLaboratories-2009-P.PDF. Accessed 20 Dec 2018 Cox KH, DeLeon DV, Angerer LM, Angerer RC (1984) Detection of mrnas in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev Biol 101(2):485–502

Economou M, Schoni L, Hammer C, Galvan JA, Mueller DE, Zlobec I (2014) Proper paraffin slide storage is crucial for translational research projects involving immunohistochemistry stains. Clin Transl Med 3(1):4. https://doi. org/10.1186/2001-1326-3-4 Engel KB, Moore HM (2011) Effects of preanalytical variables on the detection of proteins by immunohistochemistry in formalin-fixed, paraffin-embedded tissue. Arch Pathol Lab Med 135(5):537–543. https://doi. org/10.1043/2010-0702-RAIR.1 Foster FS, Hossack J, Adamson SL (2011) Micro-­ ultrasound for preclinical imaging. Interface Focus 1:576–601 Garba MT, Marie PJ (1986) Alkaline phosphatase inhibition by levamisole prevents 1,25-dihydroxyvitamin D3-stimulated bone mineralization in the mouse. Calcif Tissue Int 38(5):296–302 Gauthier BE, Gervais F, Hamm G, O’Shea D, Piton A, Schumacher VL (2019) Toxicologic pathology forum: opinion on integrating innovative digital pathology tools in the regulatory

Pathology Techniques framework. Toxicol Pathol 47:436. https:// doi.org/10.1177/0192623319827485 Graham KC, Wirtzfeld LA, MacKenzie LT, Postenka CO, Groom AC, MacDonald IC, Chambers AF (2005) Three-dimensional high-­frequency ultrasound imaging for longitudinal evaluation of liver metastases in preclinical models. Cancer Res 65(12):5231–5237. https://doi. org/10.1158/0008-5472.CAN-05-0440 Grizzle WE, Fredenburgh JL, Myers RB (2008) Fixation of tissues. In: Bancroft JD, Gamble M (eds) Theory and practice of histological techniques, 6th edn. Elsevier, Philadelphia Gur E, Waner T (1993) The variability of organ weight background data in rats. Lab Anim 27(1):65–72. https://doi. org/10.1258/002367793781082368 Pringle JH (1995) Non-isotopic detection of RNA in situ. IRL Press at Oxford University Press, Oxford; New York Hayashi S, Gillam IC, Delaney AD, Tener GM (1978) Acetylation of chromosome squashes of Drosophila melanogaster decreases the background in autoradiographs from hybridization with [125I]-labeled RNA. J Histochem Cytochem 26(8):677–679. https://doi. org/10.1177/26.8.99471 Johnson GA, Cofer GP, Fubara B, Gewalt SL, Hedlund LW, Maronpot RR (2002) Magnetic resonance histology for morphologic phenotyping. J Magn Reson Imaging 16(4):423–429. https://doi.org/10.1002/jmri.10175 Kanerva RL, Alden CL, Wyder WE (1982) The effect of uniform exsanguinatin on absolute and relative organ weights, and organ weight variation. Toxicol Pathol 10(1):43–44 Kanerva RL, Lefever FR, Alden CL (1983) Comparison of fresh and fixed organ weights of rats. Toxicol Pathol 11(2):129–131 Kelloff GJ, Hoffman JM, Johnson B, Scher HI, Siegel BA, Cheng EY, Sullivan DC (2005) Progress and promise of FDG-PET imaging for cancer patient management and oncologic drug development. Clin Cancer Res 11(8):2785– 2808. https://doi.org/10.1158/1078-0432. CCR-04-2626 Kim SW, Roh J, Park CS (2016) Immunohistochemistry for pathologists: protocols, pitfalls, and tips. J Pathol Transl Med 50(6):411–418. https://doi.org/10.4132/ jptm.2016.08.08 King JM, Dodd DC, Newson ME, Roth L (1989) The necropsy book. Arnold Printing Corporation, New York Lanning LL, Creasy DM, Chapin RE, Mann PC, Barlow NJ, Regan KS, Goodman DG (2002) Recommended approaches for the evaluation of testicular and epididymal toxicity. Toxicol Path

111

30(4):507–520. https://doi. org/10.1080/01926230290105695 Li X, Elwell MR, Ryan AM, Ochoa R (2003) Morphogenesis of postmortem hepaocyte vacuolation and liver weight increases in Sprague-­ Dawley rats. Toxicol Pathol 31(6):682–688 Li Y, Garson CD, Xu Y, French BA, Hossack JA (2008) High frequency ultrasound imaging detects cardiac dyssynchrony in noninfarcted regions of the murine left ventricle late after reperfused myocardial infarction. Ultrasound Med Biol 34(7):1063–1075. https://doi. org/10.1016/j.ultrasmedbio.2007.12.009 Llewellyn B (2009) Dehydrants. In: Stains file –the internet resource for histotechnologists. http:// stainsfile.info/StainsFile/prepare/process/ dehydrants.htm. Accessed 19 Dec 2018 Maronpot RR, Nyska A, Troth SP, Gabrielson K, Sysa-Shah P, Kalchenko V, Ramot Y (2017) Regulatory forum opinion piece∗: imaging applications in toxicologic pathology-­ recommendations for use in regulated nonclinical toxicity studies. Toxicol Pathol 45(4):444–471. https://doi. org/10.1177/0192623317710014 Matsuda Y, Fujii T, Suzuki T, Yamahatsu K, Kawahara K, Teduka K, Kawamoto Y, Yamamoto T, Ishiwata T, Naito Z (2011) Comparison of fixation methods for preservation of morphology, RNAs, and proteins from paraffin-­ embedded human cancer cell-implanted mouse models. J Histochem Cytochem 59(1):68–75. https://doi.org/10.1369/jhc.2010.957217 McInnes E (2012) Artifact in histopathology. In: McInnes E (ed) Background lesions in laboratory animals- a color atlas. Saunders Elsevier, Edinburgh Merriam-Webster Dictionary (2018). https:// www.merriam-webster.com/dictionary/histology. Accessed 20 Dec 2018 Mullink H, Vos W, Jiwa NM, Horstman A, Rieger E, Meijer CJLM (1995) Combination of non-­ radioactive in situ hybridization and immunochemistry. IRL Press at Oxford University Press, Oxford; New York Nikula KJ (2016) Regulatory forum opinion piece: an experienced pathologist need not always be present at necropsy for small molecule or biotherapeutic safety studies. Toxicol Pathol 44(1):12–13. https://doi. org/10.1177/0192623315617034 O’Hurley G, Sjostedt E, Rahman A, Li B, Kampf C, Ponten F, Lindskog C (2014) Garbage in, garbage out: a critical evaluation of strategies used for validation of immunohistochemical biomarkers. Mol Oncol 8(4):783–798. https:// doi.org/10.1016/j.molonc.2014.03.008 O’Leary JJ, Browne G, Bashir MS, Landers RJ, Crowley M, Healy I, Lewis FA, Doyle CT (1995)

112

Pamela Blackshear et al.

Non-isotopic detection of DNA in tissues. IRL Press at Oxford University Press, Oxford; New York Parry N (2015) A beginner’s guide to haematoxylin and eosin staining. In: https://bitesizebio. com/13400/a-beginners-guide-to-haematoxylin-and-eosin-staining/. Accessed 20 Dec 2018 Pekmezci M, Szpaderska A, Osipo C, Ersahin C (2012) The effect of cold ischemia time and/or formalin fixation on estrogen receptor, progesterone receptor, and human epidermal growth factor receptor-2 results in breast carcinoma. Pathol Res Int 2012:947041. https://doi. org/10.1155/2012/947041 Pringle JH, Ruprai AK, Primrose L, Keyte J, Potter L, Close P, Lauder I (1990) In situ hybridization of immunoglobulin light chain mRNA in paraffin sections using biotinylated or hapten-­ labelled oligonucleotide probes. J Pathol 162(3):197–207. https://doi.org/10.1002/ path.1711620305 Radulescu RT, Boenisch T (2007) Blocking endogenous peroxidases: a cautionary note for immunohistochemistry. J Cell Mol Med 11(6):1419. https://doi.org/10.1111/j.1582-4934. 2007.00185.x Ramot Y, Schiffenbauer YS, Maronpot R, Nyska A (2017) Compact magnetic resonance imaging systems-novel cost-effective tools for preclinical drug safety and efficacy evaluation. Toxicol Sci 157(1):3–7. https://doi.org/10.1093/toxsci/ kfx024 Rothacker DL, Kanerva RL, Wyder WE, Alden CL, Maurer JK (1988) Effects of variation of necropsy time and fasting on liver weights and liver components in rats. Toxicol Pathol 16(1):22– 26. https://doi. org/10.1177/019262338801600103 Scherfler C, Donnemiller E, Schocke M, Dierkes K, Decristoforo C, Oberladstatter M, Wenning G (2002) Evaluation of striatal dopamine transporter function in rats by in vivo beta-[123I] CIT pinhole SPECT. NeuroImage 17(1):128–141 Schmued LC, Hopkins KJ (2000) Fluoro-Jade: novel fluorochromes for detecting toxicant-­ induced neuronal degeneration. Toxicol Pathol 28(1):91–99 Scicchitano MS, Dalmas DA, Boyce RW, Thomas HC, Frazier KS (2009) Protein extraction of formalin-fixed, paraffin-embedded tissue enables robust proteomic profiles by mass spectrometry.

J Histochem Cytochem 57(9):849–860. https://doi.org/10.1369/jhc.2009.953497 Sellers RS, Morton D, Michael B, Roome N, Johnson JK, Yano BL, Perry R, Schafer K (2007) Society of toxicologic pathology position paper: organ weight recommendations for toxicology studies. Toxicol Pathol 35(5):751– 755. https://doi. org/10.1080/01926230701595300 Shi SR, Liu C, Taylor CR (2007) Standardization of immunohistochemistry for formalin-fixed, paraffin-embedded tissue sections based on the antigen-retrieval technique: from experiments to hypothesis. J Histochem Cytochem 55(2):105–109. https://doi.org/10.1369/ jhc.6P7080.2006 Solomon HM, Murzyn S, Rendemonti J, Chapman S, Skedzielewski T, Jucker BM et al (2018) The use of micro-CT imaging to examine and illustrate fetal skeletal abnormalities in Dutch Belted rabbits and to prove concordance with Alizarin Red stained skeletal examination. Birth Defects Res 110(3):276–298. https://doi. org/10.1002/bdr2.1168 Stefanovic D, Stefanovic M, Nikin Z (2013) Romanowsky-Giemsa as a counterstain for immunohistochemistry: optimizing a traditional reagent. Biotech Histochem 88(6):329–335. https://doi. org/10.3109/10520295.2013.785595 Taylor CR, Levenson RM (2006) Quantification of immunohistochemistry--issues concerning methods, utility and semiquantitative assessment II. Histopathology 49(4):411–424. https://doi.org/10.1111/j.1365-2559. 2006.02513.x Tuomari DL, Kemp RK, Sellers R, Yarrington JT, Geoly FJ, Fouillet XLM, Dybdal N, Perry R (2007) Society of toxicologic pathology position paper on pathology image data: compliance with 21 CFR parts 58 and 11. Toxicol Pathol 35(3):450–455. https://doi. org/10.1080/01926230701284509 Vogt RF Jr, Phillips DL, Henderson LO, Whitfield W, Spierto FW (1987) Quantitative differences among various proteins as blocking agents for ELISA microtiter plates. J Immunol Methods 101(1):43–50 Wise LD, Winkelmann CT, Dogdas B, Bagchi A (2013) Micro-computed tomography imaging and analysis in developmental biology and toxicology. Birth Defects Res C Embryo Today 99(2):71– 82. https://doi.org/10.1002/bdrc.21033

Chapter 4 Pathology of the Liver and Gallbladder Robert R. Maronpot and David E. Malarkey Abstract The liver is a major metabolic organ, and the first site of contact of xenobiotics follows oral ingestion or administration. The primary functional unit of the liver is the hepatic lobule. Gradients for metabolizing enzyme and oxygen tension in the hepatic lobule determine toxification and detoxification of ingested xenobiotics with immediately toxic agents causing cell damage in the periphery of the hepatic lobule, while enzymatic generation of toxic metabolites typically affect centrilobular hepatocytes. A wide spectrum of spontaneous degenerative changes occurs in the liver, some changes are age-, species-, and strain-­ dependent, and the job of the pathologist is to assess the significance of a potential treatment-induced liver changes against the background changes in a particular study. Because the liver has a high regenerative capacity following insult, proliferative changes, including benign and malignant neoplasms, are common, especially in the rodent liver. These proliferative changes also occur with an age-, species-, and strain-­ related background, and the pathologist must weight any proliferative response against this background incidence. Unique to the liver is a class of proliferative lesions designated as foci of cellular alteration which are localized presumptively preneoplastic responses. Several cell types in addition to hepatocytes are present in the liver, and each needs to be addressed by the pathologist in safety assessment of liver changes. Key to understanding the judgment involved in documenting and categorizing the broad spectrum of liver lesions is the pathology narrative where criteria for assessing hepatopathology is spelled out by the pathologist. Key words Hepatic lobule, Cell death, Apoptosis, Fatty change, Degeneration, Hypertrophy, Hyperplasia, Foci of cellular alteration, Hepatocellular adenoma, Hepatocellular carcinoma

1  Introduction The purpose of this chapter is to present and discuss some practical considerations in understanding and interpreting common hepatic responses in rat and mouse toxicity and safety assessment studies as diagnosed and described by the study pathologist. We have deliberately avoided elaborating on morphological features and subtle diagnostic criteria as these are well documented in manuscripts and other textbooks. The liver is a multifunctional and biochemically diverse organ capable of rapid responses to insult and stimuli to maintain optimal function. Because of its high metabolic activity and exposure to Thomas J. Steinbach et al. (eds.), Toxicologic Pathology for Non-Pathologists, https://doi.org/10.1007/978-1-4939-9777-0_4, © Springer Science+Business Media, LLC, part of Springer Nature 2019

113

114

Robert R. Maronpot and David E. Malarkey

exogenous xenobiotics via the portal blood supply, the liver is a frequent site of toxicity. As the first major organ to be exposed to ingested material, the liver provides protection to other organ systems by “first pass” removal of potentially toxic materials but may also be at increased risk for hepatic injury from xenobiotics and/or their metabolites.

2  Structure and Function The liver is a major body organ located in the cranial aspect of the abdomen and represents 1–4% of adult body weight in most species. It is comprised of individual lobes in and contains a gallbladder in all common laboratory species except rats. There is a dual blood supply to the liver with approximately 75% of blood coming from the gastrointestinal tract via the portal vein and 25% from the hepatic artery. The functional microscopic units of the liver are multiple hepatic lobules where the dual blood supplies get mixed and enter at the portal area of each lobule. While a three-­ dimensional structure of the liver has been described (Teutsch et al. 1999), most pathologists typically describe their microscopic findings based on a two-dimensional description of the hepatic lobule. The hepatic lobule unit can be viewed from different perspectives. The classic hepatic lobule considered in two dimensions as viewed in typical histological sections is used by most pathologists to localize lesions. This classic lobule is a polygonal structure with peripherally located portal triads and a central hepatic vein (Fig. 1). The portal triad consists of a portal vein, a hepatic artery, and a bile duct. Extraneous small bile ducts, hepatic arteries, and lymphatic venules also comprise the portal tracts. Mixed portal vein and hepatic arterial blood flows from the portal region to the sinusoids and then the central vein and hepatic vein and ultimately enters the posterior vena cava moving on to the heart with ultimate systemic distribution throughout the body. An alternative depiction of the microscopic functional unit of the liver is the hepatic acinus (Fig.  1), where blood flow is defined as flowing from the portal triads at each pole of the unit to the central vein. This unit is based upon the gradients of metabolic functions and oxygen tension. A mixture of oxygenated and nutrient-rich blood enters zone 1 which includes the periportal hepatocytes, zone 2, the middle zone of hepatocytes, and zone 3, which includes those cells closest to the central vein and possessing the lowest oxygen tension. A third type of view of the hepatic unit is the portal lobule, which is based upon bile flow (Fig.  1). Bile flow is opposite the flow of blood in the acinus and classic lobule. These alternative depictions are useful in understanding the dynamics of hepatic physiology and responses.

Pathology of the Liver and Gallbladder

115

Fig. 1 Idealized drawing of liver lobules identifying the structure of functionally different hepatic lobules. The classic lobule is defined as peripherally located portal triads each consisting of hepatic artery, portal vein, and bile duct and a central hepatic vein. The hepatic acinus (blue shading) is a functional depiction based on blood flow from the portal areas to the central vein. This hepatic acinus may also be depicted based on oxygen tension gradients (multicolored shading) with the highest oxygen gradient in the portal zone (zone 1). The portal lobule (pink shaded triangle) is based on bile flow from the central vein to the portal bile duct

3  Structure and Cellular Components of the Hepatic Lobule Hepatocytes, the primary cellular component of the liver, occupy positions throughout each hepatic lobule. They are arranged in linear plates extending from the portal triad to the central vein and are separated by vascular sinusoids. There are functional gradients (Fig.  2) in hepatocyte enzymatic content depending upon their lobular position and with respect to oxygenation and blood supply (Teutsch et  al. 1999). In general, centrilobular hepatocytes are slightly larger than portal hepatocytes, due to their abundant intracellular endoplasmic reticulum, and have a high proportion of drug metabolizing enzymes. Functional gradients also apply to various non-parenchymal cell types in the liver. Other cells of the liver include biliary cells that form bile ductules and a spectrum of sinusoidal-lining cells including endothelial cells, Kupffer cells, and stellate cells. The sinusoidal endothelial

116

Robert R. Maronpot and David E. Malarkey

Fig. 2 Depiction of an hepatic lobule showing gradient zones of enzymatic content with the highest level of several endogenous and inducible drug metabolizing enzymes in zone 6. Oxygen tension is greatest in zone 1 with a diminishing gradient moving toward the central vein (CV). (Figure adapted from Teutsch et al. 1999)

Fig. 3 Diagrammatic representation of the cellular and structural components of an endothelial lined fenestrated hepatic sinusoid showing positions of hepatocytes and other cellular components of the liver

cells are fenestrated and can use pinocytosis to aid in the transfer of cell-free components from blood to the perisinusoidal Space of Disse bringing molecules in direct contact with the hepatocytes (Fig.  3). Kupffer cells are from the mononuclear phagocytic cell system, are situated on the sinusoidal endothelial lining, function as macrophages which engulf and digest cellular debris and foreign substances, and are the source of cytokines during hepatic injury.

Pathology of the Liver and Gallbladder

117

Pit cells, a type of natural killer large granular lymphocyte, as well as dendritic cells, that are regulators of liver immunity, line the perisinusoidal space and are among the first lines of defense. A small population of stellate (Ito) cells located along the sinusoidal surface stores lipids and vitamin A and, if activated, initiates the production of collagen seen in hepatic fibrosis. The liver is the major filter of blood from the gastrointestinal tract prior to its systemic circulation and functions to metabolize and detoxify chemicals that are then secreted into the bile. Endogenous and inducible metabolizing and detoxifying enzymes are concentrated in a gradient within each hepatic lobule (Fig. 2). The liver also carries out metabolism of fats, proteins, and carbohydrates; stores glycogen, minerals, and vitamins; and synthesizes plasma proteins and clotting factors. A variety of hepatic lesions identified by the pathologist during microscopic examination, including necrosis, inflammation, cholestasis, steatosis, vascular changes, and neoplasia, can cause perturbations in any one or more of the diverse functions of the liver.

4  How Pathologists Diagnose and Document Liver Lesions and the Importance of the Pathology Narrative In most toxicity and safety assessment studies, the pathology findings are tabulated as individual diagnoses with specific modifiers by treatment groups. Studies differ in what strains and species are used, in the range of doses and duration of exposures, and with differing study objectives. Thus, there may be significant differences among studies in how the study pathologist diagnoses, grades, documents, and interprets tissue changes. Consistent with a best practice approach to conducting a pathology evaluation, a pathology narrative provides the basis for the judgments made by the study pathologist for a given study and is an effective means of communicating the interpretative aspects of a given pathology evaluation (Morton et  al. 2006). Descriptions in the pathology narrative can convey to the reader the degree to which a spontaneous or treatment-related lesion conforms to classical published morphological features and how severity grading was applied for well-defined as well as for subtle lesions. Pathologists may differ in the threshold they use for detecting and recording lesions during the evaluation of a safety assessment study. There are age-related common background spontaneous lesions seen in control animals of all types of studies. Some pathologists, therefore, establish a threshold for the occurrence of such lesions and document the presence of commonly seen lesions only when they exceed the background level in incidence and/or s­ everity, or if the lesions occur at an earlier age than is typically seen. What constitutes an acceptable background level of a common lesion is

118

Robert R. Maronpot and David E. Malarkey

based to some degree on historical control data and the age of the animals but mostly depends on the experience and judgment of the study pathologist. To help ensure consistency within and across studies, laboratories with multiple pathologists may establish a policy or common practice for defining spontaneous age-associated background lesions. Since background lesions may be exacerbated by treatment, understanding the diagnostic threshold used by the study pathologist is important and ideally should be defined in the narrative portion of the pathology report for certain lesions that are considered treatment-related. With respect to the documenting lesions in the liver, pathologists may be “lumpers or splitters” in how they categorize lesions. In addition to indicating the severity of a liver lesion, the pathologist will typically identify the anatomical or lobular distribution of a given lesion and also indicate if the lesion is acute, subacute, or chronic. Thus, what is basically the same type of lesion may have several different modifiers. In the tabulation of a given lesion with multiple different modifiers, there is an opportunity to combine what are basically similar lesions for assessing a potential treatment effect. Subcategorizing a lesion excessively with too many modifiers along with different severity grades may mask what is a true effect. For example, in a given dose group, one diagnosis may be “liver, inflammation, acute, neutrophilic, moderate,” while another diagnosis is “liver, inflammation, subacute, mixed inflammatory cell, minimal.” Since these subcategories probably actually reflect inter-animal variability of the same process, it would be reasonable to combine the two categories for purposes of assessing a significant treatment-related response. In this instance, a combined diagnosis of “liver, inflammation” with a description of the spectrum of features associated with that diagnosis could be presented in the pathology narrative. In addition to considerations regarding combining and dividing lesions based on documenting subtle variations in lesion characteristics, it is important to maintain consistency in categorizing lesion variations and lesion severity in a chronic study to determine if lesions progress in extent or severity or if there is evidence of lesion recovery following cessation of treatment. Evidence for the progression or regression of treatment-­ related lesions can be explained in the pathology narrative.

5  Discussion of Hepatic Responses in Rats and Mice Detailed morphological descriptions of rat and mouse hepatic responses with associated photographic examples are documented in many publications (Thoolen et  al. 2010; Harada et  al. 1989, 1999; Maronpot et  al. 1987; Eustis et  al. 1990; NNLA 2019; Bannasch and Zerban 1990; Cattley et al. 2013; Deschl et al. 2001; Foster 2018; Frith et al. 1994; Greaves 2012; Haschek et al. 2010)

Pathology of the Liver and Gallbladder

119

as well as in other earlier publications. The following discussion of commonly occurring rat and mouse liver responses will focus on how the study pathologist documents and interprets tissue changes rather than detailed morphologic features and diagnostic criteria.

6  Liver Enlargement (Hepatomegaly and Hepatocyte Hypertrophy) Non-neoplastic liver enlargement can be a relatively common occurrence in rodent toxicity studies, and liver weight is a sensitive metric to identify a treatment-related liver response, whether that response is due to overt toxicity or to microsomal enzyme induction. The pathologist may look at toxicokinetics or pharmacokinetics (TK or PK) and/or absorption, distribution, metabolism, and excretion (ADME) data to provide an interpretative perspective of a particular liver response. Liver weights are typically expressed as absolute weights and as weight expressed relative to body weight. The latter is usually more useful, particularly if there are systemic effects that affect body weight. Relative liver weight increases of 15–20% may not present with easily recognized morphological changes during histopathological evaluation but may still be a significant response to treatment. Relative liver weight increases of greater than 20% usually have an altered morphological feature such as hepatocellular hypertrophy or hepatic inflammatory cellular infiltrates to define the likely cause of the increased weight. However, depending upon the type of microscopic change, for example, marked centrilobular hypertrophy, even very low levels of relative liver weight increases may be supported by microscopic effects. Generally, liver weight increases at 90 days along with concurrent hepatocellular hypertrophy, and degenerative changes such as cytoplasmic vacuolization and/or hepatocyte necrosis are multiple contributing factors in the risk of development of hepatocellular tumors in the rat and mouse. Liver weight increases serve as a sensitive quantitative response to liver toxicity or hepatic enzymatic induction and represent an important component in the assessment of liver responses to xenobiotic exposure (Hall et al. 2012; Allen et al. 2004; Maronpot et al. 2010).

7  Clinical Chemistry Just as liver weight measurements should be considered in evaluating effects on the liver, several routine serum measurements used in conjunction with microscopic examination of stained liver sections can provide an indication of the degree of liver toxicity (Wiedmeyer 2018). Alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) have their highest tissue levels in the liver, and elevated serum levels indicate hepatocellular leakage or injury. Aspartate

120

Robert R. Maronpot and David E. Malarkey

aminotransferase (AST) and alkaline phosphatase (ALP) occur in several tissues and are thus less specific for the liver, but serum increases in these analytes in conjunction with elevated serum ALT and/or SDH help to confirm liver damage. Additional clinical chemistry analytes that may be useful in assessing liver integrity and function include serum bilirubin, total bile acid, glutamate dehydrogenase (GDH), and gamma-glutamyl transferase (GGT). During the course of conducting toxicity and safety assessment studies in rodents, routine clinical chemistry analyses are carried out. Generally, serum enzymes levels greater than twofold above normal will have a correlated morphological microscopic change provided the liver histology sample is taken close to the time serum was collected for analysis. Sometimes twofold elevations of a liver-­ associated analyte show statistical significance that suggests liver toxicity, even if this degree of elevation falls within laboratory historical control ranges. In these instances, the study pathologist is charged with determining if there is a morphological change that correlates with the statistically flagged analyte. In the absence of identifying a morphological change that could explain the statistically flagged value, some other explanation may be needed to account for the clinical chemistry finding such as diet, serum hemolysis, or normal variability.

8  Frequently Occurring Non-neoplastic Liver Lesions in Rodent Studies 8.1  Hepatodiaphragmatic Nodule

This is a rounded, herniated nodular protrusion of normal hepatic parenchyma that was occupying a defect in the diaphragm. It is considered a developmental anomaly occasionally seen in rats, especially F344 rats, and may be seen in fetuses and should not be considered a treatment-related lesion (Eustis et al. 1990).

8.2  Cytoplasmic Alterations

Cytoplasmic alterations that occur in the rodent liver include glycogen accumulation or depletion, fatty change, phospholipidosis, and hydropic and cystic degeneration. While the nonspecific diagnostic term of “cytoplasmic vacuolization” may be used for hepatocyte glycogen accumulation, fatty change, and phospholipidosis, definitive proof for a specific diagnosis requires special staining (Thoolen et al. 2010). However, an experienced pathologist can recognize morphologic features consistent with fatty change or glycogen accumulation on a hematoxylin and eosin (H&E)-stained section of the liver. Hydropic and cystic degeneration are generally diagnosed based on H&E-staining.

8.2.1  Glycogen Accumulation and Depletion

Abnormal glycogen retention or depletion is unusual but suggests a functional failure to normally metabolize glycogen and should be documented when present. Glycogen accumulation is characterized by clear or rarefied cytoplasm surrounding a centrally located

Pathology of the Liver and Gallbladder

121

nucleus and can be demonstrated by special staining (periodic acid Schiff (PAS) with and without diastase). Most pathologists expect some degree of cytoplasmic vacuolation in hepatocytes consistent with glycogen and do not diagnose it unless there is an apparent difference among treatment groups. Being nocturnal feeders, glycogen is stored in rodent hepatocytes and is gradually mobilized as a source of energy throughout the day (Greaves 2012; Thoolen et al. 2010; Eustis et al. 1990; Harada et al. 1999; Malarkey et al. 2005). Thus, glycogen accumulation in hepatocytes is more readily apparent in non-fasted animals and may be seen to some degree in animals that are fasted overnight. Animals with decreased food intake, such as moribund animals or those with restricted diet, are prone to have depleted their glycogen stores. The dynamics of glycogen accumulation and its subsequent mobilization (depletion) are important conditions to be aware of, and a reason for randomizing the sacrifice timing of treated and control animals in a toxicity study (Malarkey et al. 2005). 8.2.2  Fatty Change

Lipidosis and steatosis are synonyms for fatty change which reflect perturbation of lipid metabolism and can be a treatment-related change (Thoolen et al. 2010; Cattley and Popp 2002; Evans and Lake 1998; Greaves 2012; Haschek et al. 2010). Macrovesicular and microvesicular fatty change are two forms of fatty change that are diagnosed separately but can occur together. Both represent abnormal storage of fat within hepatocytes, both are potentially reversible, and both may occur along with the presence of cytoplasmic glycogen and hepatocellular lesions such as necrosis. When accompanied by significant inflammatory foci, it is referred to as steatohepatitis. Macrovesicular fatty change is an imbalance between uptake of blood lipids and hepatocyte secretion of lipoproteins, while microvesicular fatty change is usually considered a more serious hepatic dysfunction and may reflect a mitochondrial perturbation. An experienced pathologist can recognize characteristic morphological features in hematoxylin and eosin-stained sections to provisionally diagnose macrovesicular and microvesicular fatty change, but confirmation with an oil-red-O or Sudan black stain is needed for definitive diagnosis. Since fatty change is potentially reversible, consistency in diagnosis and severity grading is important in studies that include a reversibility cohort.

8.2.3  Phospholipidosis

Phospholipidosis is a drug-induced storage disease involving the lysosomes. Phospholipidosis is a response seen following treatment with cationic amphophilic compounds and represents a lipid disorder where xenobiotic and phospholipid complexes are deposited in hepatocyte lysosomes. It can resemble microvesicular fatty change in an H&E-stained section. Definitive diagnosis of phospholipidosis requires either electron microscopy (for the presence of lamellated and crystalloid inclusions) or immunohistochemical

122

Robert R. Maronpot and David E. Malarkey

staining for lysosomal-associated membrane protein (LAMP) and acidophilin (Chatman et al. 2009; Halliwell 1997). 8.2.4  Hydropic Degeneration

Sometimes referred to as cloudy swelling, hydropic degeneration refers to a cytoplasmic swelling attributed to perturbation of cell membrane integrity (Thoolen et al. 2010). It is typically associated with exposure to toxic xenobiotics, may have a centrilobular or periportal localization, and is often a precursor to cell death.

8.2.5  Cystic Degeneration

A traditional synonym for this change is spongiosis hepatis. It is usually localized and somewhat focal and is formed by cystic enlargement of perisinusoidal stellate (Ito) cells. This change is more common in rats than in mice and is more often seen in older animals (Eustis et  al. 1990; Harada et  al. 1999; Thoolen et  al. 2010). Although this is a spontaneous age-associated change, there is some evidence for xenobiotic induction of cystic degeneration, and, thus, it is recommended to document this change whenever present.

8.3  Amyloidosis

Extracellular deposits of amyloid, a form of misfolded insoluble protein, is rare in rats but seen in some strains of mice as a spontaneous aging lesion (Harada et al. 1999) and may be influenced by husbandry factors (Lipman et  al. 1993). It is usually a systemic disease manifested in several organs. In the liver, it appears in perisinusoidal and periportal locations as well as in blood vessels. It can be identified microscopically with Congo red staining and polarizing birefringence. The occurrence of amyloid is either primary or secondary to chronic illness, and it may be exacerbated by treatment or stress. It is usually diagnosed as a systemic disease rather than specifically in the liver.

8.4  Mineralization

Hepatic mineralization is rare and may reflect a dietary-calcium disturbance (NNLA 2019). Dystrophic mineralization often accompanies necrosis, inflammation, or neoplasia. It can be identified with Alizarin Red or von Kossa special stains for calcium. When secondary to a pronounced necrotic or inflammatory lesion, it may not be separately diagnosed but could be mentioned in the pathology narrative.

8.5  Pigment

Hepatic pigment includes lipofuscin, iron, porphyrin, bile, or possibly an unusual xenobiotic metabolite (Thoolen et  al. 2010). It typically reflects an ongoing process. Lipofuscin is commonly referred to as “wear and tear” pigment, is age-associated, can occur as a background change, and may be exacerbated by treatment. Iron pigment is normally very low to undetectable in the adult liver. When present it is most often related to hemosiderin deposition from the breakdown of erythrocytes, and thus a treatment-­

Pathology of the Liver and Gallbladder

123

related cause should be considered. Since definitive diagnosis of a specific pigment requires special staining, a generic diagnosis of pigment and an indication of its location and severity are typically sufficient in initial histopathological evaluation. Should there be evidence of a dose-related or treatment-related response, definitive special staining is warranted. 8.6  Hepatocellular Hypertrophy

As a distinct form of cytoplasmic change, hepatocellular hypertrophy is a common adaptive metabolic response following administration of some xenobiotics. It is identified by hepatocytes that are larger than normal and have a finely granular, pale staining cytoplasm. There is usually a lobular localization with centrilobular hepatocellular hypertrophy most commonly seen. Since there is often a dose-related response, severity grading based on the degree of hepatocyte enlargement and lobular extent of the hypertrophy is usually helpful. Hepatocellular hypertrophy is typically reflected by increased relative liver weight. Common etiologic agents include enzyme inducers (e.g., phenobarbitone), peroxisome proliferators (e.g., fibrates), and AhR receptor agonists (e.g., dioxins). While this change is reversible following adaptation to a higher level of hepatic homeostasis or following cessation of exposure to the causative agent, severe and prolonged hypertrophy can result in occlusion of sinusoidal space, oxygen deprivation, and hepatocyte death with the latter most evident around central veins. Since hepatocellular hypertrophy is initially an adaptive physiological response, it is usually not associated with any elevation of circulating hepatic enzymes reflective of liver damage. However, when severe and prolonged and associated with centrilobular cell death, some elevation of serum ALT and AST can be present, and there is increased risk of development of hepatocellular tumor development in mouse (Allen et al. 2004; Hall et al. 2012).

8.7  Increased Mitosis

Mitogenesis: Increase in the presence of hepatocyte mitotic figures may be documented in safety assessment studies and, when present above a very low threshold, is usually given a severity grade and indication of its lobular localization. There are different situations where increased mitoses will be present in the liver, and it is important to try to determine the underlying cause. Some xenobiotics are inherently mitogenic, and their administration results in increased numbers of mitotic figures in routinely stained liver sections. Upon withdrawal of treatment with a mitogenic agent, there is typically an increase in hepatocellular apoptosis as the liver cellular mass adjusts back to a normal physiological number of hepatocytes. An increase in mitotic figures occurs as a recovery response after prior loss of hepatocytes from necrosis. Hence, it is important to determine if the response is secondary to earlier necrosis. During

124

Robert R. Maronpot and David E. Malarkey

pregnancy there is an increase in hepatocellular mitosis presumably to provide maternal energy support during gestation. 8.8  Cell Death

There has been considerable dialogue in recent years centered around recommended nomenclature for cell death (Elmore et al. 2016; Elmore 2007). Depending upon the lexicon used by pathologists and organizations, there are preferences for use of “single cell necrosis” by some and “apoptosis” by others for the same change seen in H&E-stained sections. Special caspase stains are used to more definitively identify the programmed cell death pathway referred to as apoptosis. In general, the term necrosis is appropriate when small to large groups of contiguous dead cells often accompanied by secondary inflammation are present.

8.8.1  Apoptosis

A diagnosis of hepatocyte apoptosis implies there is an active cell death of individual hepatocytes in the absence of accompanying inflammation. The morphologic features in H&E-stained sections are often sufficiently characteristic to make the diagnosis of apoptosis without the need for special stains. The diagnosis typically has some modifiers indicating location within hepatic lobules and severity of the response. A very low level of hepatocyte apoptosis may occur spontaneously, and some pathologists do not diagnose apoptosis unless it exceeds a threshold. If believed to be an important factor in a study, the pathology narrative can be used to define the threshold for diagnosis and to describe the location and extent of the observed apoptosis.

8.8.2  Necrosis

Documentation of necrosis in safety assessment studies generally includes important modifiers that localize the cell death and provide an indication of its extent and severity (Cattley et al. 2013; Thoolen et al. 2010). Necrosis can be focal, multifocal, random, or generalized. When generalized, there may be a zonal distribution (i.e., centrilobular, midzonal, or periportal) suggesting a mechanistic toxicological injury (see below). Differential distribution among hepatic lobes reflecting the lobar pattern of portal blood flow from the gastrointestinal tract may be discerned. In typical studies, at least two and sometimes three different liver lobes are sampled for histopathology, and an occasional single focus of microscopic hepatocellular necrosis may occur in one or two of the sampled lobes as a background lesion. Usually, these are very small focal necrotic lesions, may be found in controls as well as in treated animals, are documented by the study pathologist with indication of a minimal severity, have a random lobular localization, and may be mentioned in the pathology narrative as background changes. It is recommended to document focal necrosis even if considered a background lesion since there may be an exacerbation associated with treatment. Many pathologists using a diagnosis of focal necrosis then use a

Pathology of the Liver and Gallbladder

125

severity score to reflect multifocality. As would be expected, larger areas of necrosis are more serious indication of hepatotoxicity, and their lobular location may reflect their pathogenesis. The most common location of hepatic necrosis in safety assessment studies is centrilobular and can often be attributed to toxicity from a metabolite of the test agent generated by the high centrilobular content of endogenous or inducible metabolizing enzymes (Fig.  4). A good example of this is acetaminophen toxicosis. Alternatively, centrilobular necrosis can also be a response to tissue anoxia since centrilobular hepatocytes reside in a low oxygen gradient area. Periportal necrosis (Fig. 5) typically reflects more direct damage from hepatic toxins carried there in the portal blood. Midzonal necrosis (Fig.  6) is not commonly seen but may be a response to specific toxins. Regardless of its lobular localization, necrosis may be accompanied by congestion, hemorrhage, inflammation, and biliary stasis and can extend between lobules as a bridging necrosis. Since necrosis may reflect a dose-related response, severity grading is important.

Fig. 4 Centrilobular necrosis. Blue granular areas surrounding the central vein represent necrotic liver parenchyma. This is a common site of necrosis because of a low gradient of oxygen (relative anoxia) and the presence of CYP metabolizing enzymes in this area of the lobule. In severe cases, the areas of necrosis may extend into the mid-lobular areas and also may form a bridging necrosis by extending to centrilobular areas of adjacent lobules

126

Robert R. Maronpot and David E. Malarkey

Fig. 5 Periportal necrosis. Blue granular areas representing necrosis surround the portal triads and sometimes may extend between adjacent portal areas to form a bridging necrosis. This pattern of necrosis may be seen with exposure to highly reactive agents such as acrolein and elemental phosphorus

8.9  Inflammation

Hepatic inflammation is generally documented as a focal, multifocal, or generalized infiltration of specific inflammatory cells and categorized under a diagnosis of “infiltration, neutrophilic” or “infiltration, mixed leukocyte” rather than being diagnosed as “hepatitis”, which is a nonspecific term for inflammation of the liver. Small foci of mononuclear cell infiltrates, often without any obvious associated cell death, are common background lesions in rodent livers, and most pathologists only document these focal mononuclear cell infiltrates when they occur above a specific threshold. Since their occurrence and frequency can be exacerbated by treatment, it may be necessary to adjust the threshold for diagnosis appropriately to better identify any potential treatment-­ related effect. Inflammatory cell infiltration: The specific cell type of infiltrating inflammatory cell is usually defined during histopathology evaluation (Cattley et  al. 2013; Thoolen et al. 2010; NNLA 2019). Neutrophilic infiltrates indicate an acute response to liver injury and necrosis, but occasional lymphocytes and monocytes may also be present in what is a primary neutrophilic infiltrate. Mononuclear infiltrates are typically

Pathology of the Liver and Gallbladder

127

Fig. 6 Midlobular necrosis. Blue granular areas representing necrosis and localized mid-way between portal areas and central veins are uncommon but have been seen after administration of ferrous sulfate and may be associated with exposure to some phytotoxins

associated with a low level of obvious cell death and usually represent a more chronic response. A mixed neutrophilic and mononuclear response with relatively equal components of each cell type would be expected in a chronic, active inflammatory process. Since the dose of a given hepatotoxic agent and the timing after initial insult might influence the morphological features of the inflammatory infiltrate, consideration should also be given to combining related inflammatory responses in assessing dose-related responses and determining no adverse effect levels. Hepatic fibrosis can occur as a response to prolonged repeated hepatotoxicity. 8.10  Vascular Changes

Hemorrhage and congestion can accompany hepatic necrosis and inflammatory responses and is often present as a relatively minor secondary response. In those situations, hemorrhage and congestion may not be separately diagnosed but rather described in the pathology narrative. Angiectasis: Angiectasis is a cystic or cavernous localized widening of hepatic sinuses that is occasionally observed in chronic studies (Thoolen et al. 2010). Because it is often macroscopically visible on natural and cut surfaces of the liver, the technician may diagnose it as a

128

Robert R. Maronpot and David E. Malarkey

gross lesion at the time of necropsy or even tissue trimming. It may be present secondary to hemodynamic changes in blood flow associated with hepatic neoplasia. It has been induced by treatment. 8.11  Hyperplasias

While any one of the different cell types that reside in the liver may undergo a diffuse or localized increase in number, the most frequent type of hyperplasia involves hepatocytes. Bile duct epithelium and oval cell hyperplasias are most commonly diagnosed in chronic studies, occur spontaneously, and may be exacerbated by treatment. Rarely there can be Kupffer cell hyperplasia in rats and mice and stellate (Ito) cell hyperplasia in mice.

8.11.1  Hepatocellular Hyperplasia

There is a wide spectrum of morphological features of hepatocellular hyperplasia (Thoolen et al. 2010). Most hyperplasias are focal or multifocal rather than diffuse. Because of their focality and uncommon occurrence, they are sometimes misdiagnosed as foci of cellular alteration. Foci of cellular alteration are classified based on their phenotype in H&E-stained sections (see below) and are much more common than hyperplasias. In contrast to foci of cellular alteration, more extensive regions of hepatocellular hyperplasia have been reported as regenerative and non-regenerative hyperplasia. Non-regenerative hyperplastic responses span multiple hepatic lobules and are seen primarily in chronic studies. Non-­ regenerative hyperplastic nodules are rare, while a diagnosis of regenerative hepatocellular hyperplasia is associated with previous or concurrent hepatocellular damage.

8.11.2  Foci of Cellular Alteration

Hepatic foci of cellular alteration are typically classified based on the cytoplasmic tinctorial H&E features (Harada et al. 1989; Eustis et al. 1990; Thoolen et al. 2010). They occur as an age-associated and strain-related change with some foci of cellular alteration occurring spontaneously, while other phenotypes are more likely a response to treatment (Tables 1 and 2). Commonly occurring spontaneous foci of cellular alteration can be exacerbated by treatment and thus appear earlier in treated animals during the course of a study (Table 3). Foci may be induced but are a common background change, especially in rats. They are rarely seen in any rat strains in short-­ term (≤90  day) toxicity studies, but in Fischer rats, there is an almost 100% incidence in both males and females by 2 years of age. In contrast, 28% and 38% incidences in male and female Sprague Dawley rats, respectively, have been documented at 24–26 months of age (Newsholme and Fish 1994). Basophilic foci are most common in F344 and female Sprague Dawley rats. The importance of foci of cellular alteration relates to their presumptive preneoplastic potential based on their early appearance following treatment with known rodent liver carcinogens, their

Pathology of the Liver and Gallbladder

129

Table 1 Foci of cellular alteration in rats Rarely seen in control animals at 90 days of age Occurs in 75–100% of animals by 2 years of age  Numbers per liver ~ 500/cm3  Males – Basophilic and clear cell phenotypes most common  Females – Basophilic phenotype most common Incidence varies by sex and strain – F344 > SD rats

Table 2 Foci of cellular alteration in mice Rarely seen in control animals at 90 days of age Occurs in 15–30% of animals by 2 years of age  Numbers per liver ~100’s  Eosinophilic and clear cell phenotypes most common Incidence varies by sex and strain  More common in males

appearance before development of hepatic adenomas and ­carcinomas (Fig. 7), and the morphological continuum between large foci of cellular alteration and hepatocellular neoplasia. The most common, spontaneously occurring foci of cellular alteration (in decreasing order) are basophilic foci, eosinophilic foci, clear cell foci, and mixed cell foci. The occurrence is species and strain dependent in rats and mice and rarely recognized in humans (Thoolen et al. 2010) (Table 4). They are reported to be in about 30% of control mice with the eosinophilic and clear cell phenotypes most common. They increase as the animals age and can be more numerous and appear earlier following exposure to known hepatocarcinogenic agents. Because foci of cellular alteration are a common early response to known rodent carcinogens and because of their morphological similarity to hepatocellular adenomas, they are presumptively preneoplastic. Consequently, in routine safety assessment studies, they are always diagnosed and may be given a severity grade based on size and number of foci on routine H&E sections. Additionally, stereological methods may be employed to assess for preneoplastic growth. They may be present in liver that also contain hepatocellular adenomas and carcinomas. In some short-term rodent models, quantitative stereology has been used to evaluate immunohistochemically stained foci of cellular alteration as an experimental means to identify potential hepatocarcinogens (Tsuda et al. 2003) (Fig. 7).

130

Robert R. Maronpot and David E. Malarkey

Table 3 Predisposing factors influencing the incidence of liver tumors in various speciesa Mouse

Rat

Dog

Man

Infectious hepatitis

+





+

 Hepatitis viruses







+++

  Helicobacter hepaticus

+







Cirrhosis

+

++

+++

++++

 Alcohol







+

 Toxins

+

+





 Immune-mediated







+

Hepatocarcinogens

+++

+++

?

++++

 Aflatoxin

+

+

?

+

b

The “+” indicates that the factor has been shown to play a role in liver cancer development with “+” through “++++” indicating the frequency and/or magnitude this factor plays in hepatocarcinogenesis in various species. “−” indicates not a known or major factor b Aflatoxin is the only unequivocal human hepatocarcinogen, while there are dozens of known rodent hepatocarcinogens a

Fig. 7 This figure depicts the proposed process of rodent hepatocarcinogenesis whereby early, initiated hepatocytes may be detected using immunohistochemistry for glutathione-S-transferase-pi (GST-pi), an immunohistochemical (IHC) marker of pre-neoplastic and neoplastic hepatocytes. GST-pi expression can be demonstrated in individual cells, foci of altered hepatocytes (FAH), and adenomas, which can progress and become hepatocellular carcinomas (HCCs). The occurrence of progress is also evidenced by the development of adenomas arising from FAH and HCCs arising from adenomas. Also note that there are hundreds to thousands of FAH in adult rodents, conveying that the vast majority of FAH do not progress

131

Pathology of the Liver and Gallbladder

Table 4 Comparative incidences of hepatocytic proliferative lesionsa Mouse

Rat

Dog

Man

Hyperplasia

+

+

+++

+

Preneoplastic lesions

+++

++++



+

 Foci of altered hepatocytes

++

++





 Large/small cell lesionsa







+

Hepatocellular adenoma

++++

+

+

+

Hepatocellular carcinoma

++++

++

+

+

Hepatoblastomab

+++





+

This table is an overview of the frequency and the comparative occurrences of various hepatocytic proliferative lesions in various species. The “−” (not or rarely encountered) and “+” through “++++” indicating the frequency and/or magnitude of hepatocarcinogenesis in each species b Hepatoblastoma differs in man and mouse, where in man it is a disease of childhood and in mouse it is a late-onset neoplasm of adults a

8.11.3  Bile Duct Hyperplasia

Bile duct hyperplasia is an age-associated change commonly seen in chronic rat and mouse studies (Thoolen et  al. 2010), but it can also be observed in other common laboratory species. Some pathologists only record bile duct hyperplasia when it exceeds their threshold for diagnosis. When present it may be associated with a minimal peribiliary mononuclear cell infiltrate that may also be below a threshold for diagnosis for some pathologists. As for any relatively common background lesion, any threshold for diagnosis can be described in the pathology narrative. When bile duct hyperplasia is diagnosed, it may be given a severity grade since it has some morphological features suggestive of progression to cholangiofibrosis and cholangiocellular neoplasia.

8.11.4  Oval Cell Hyperplasia

Hyperplastic oval cells appear to arise from terminal ductal cells of bile ducts and, thus, are related to biliary hyperplasia. Oval cell hyperplasia is relatively rare, is seen following severe hepatotoxicity, and is also seen as an initial and high incidence response to some known hepatocarcinogens (Deschl et  al. 2001; Foster 2018). Even when part of a constellation of other hepatic changes, it is recommended to be diagnosed and given a severity grade.

8.12  Other Non-­ neoplastic Changes

Several other non-neoplastic hepatic changes are occasionally seen and are described in multiple publications and texts (Greaves 2012; Cattley et al. 2013; Foster 2018; Frith et al. 1994; Harada et al.

132

Robert R. Maronpot and David E. Malarkey

1999; Haschek et al. 2010). These are not covered here but include nuclear and cytoplasmic inclusions, intracellular and extracellular crystals, karyocytomegaly, chronic passive congestion, thrombosis, infarction, endothelial cell hypertrophy, endothelial cell hyperplasia, and biliary cysts.

9  Frequently Occurring Neoplastic Liver Lesions in Rodent Studies Morphological features of rat and mouse liver tumors are well documented in the literature (Harada et al. 1989, 1999; Eustis et  al. 1990; Thoolen et  al. 2010; Maronpot et  al. 1989). Assessment of liver tumor responses in chronic rodent studies utilizes incidence, latency or time to appearance, presence of multiple tumors in a given liver, and concurrent and historical control data in determining the strength of evidence in categorizing the test agent’s carcinogenicity. Hepatocellular tumor responses are the most common tumor target sites in long-term rat and mouse studies with important strain and species differences in background incidences of adenomas and carcinomas. Rats are generally more resistant to spontaneous hepatocellular tumor development than mice, whereas up to 49–90% of some strains of mice are susceptible. There is some experimental evidence indicating regression of hepatocellular neoplasms in rodents that are chemical dependent (Malarkey et al. 1995). A statistically significant increased incidence and/or earlier than normal appearance of adenomas and carcinomas is regarded as a positive response in a 2-year chronic study. In addition to hepatocellular neoplasms, biliary cells may give rise to cholangial tumors. Mixed hepatocellular-cholangial tumors also occur in rodents, and benign and malignant endothelial cell tumors can originate in the liver. 9.1  Hepatocellular Adenoma

Diagnostic features of hepatocellular adenomas are well documented in the literature (Harada et al. 1989, 1999; Eustis et al. 1990; Thoolen et al. 2010; Maronpot et al. 1989). The older terminology of “neoplastic nodule” has essentially been replaced by hepatocellular adenoma. Since hepatocellular adenomas have a well-established background incidence, especially in some strains of rats and mice, assessment of a treatment-driven response will be based on factors including reduced latency, increased incidence, and increased multiplicity. Since the diagnostic borderline between very large foci of cellular alteration and hepatic nodular hyperplasia and hepatocellular adenomas is often not distinct, it is helpful to evaluate any hepatocellular adenoma response in light of the severity grade of foci of cellular alteration based on the incidence and multiplicity of these

Pathology of the Liver and Gallbladder

133

putative preneoplastic lesions. Tinctorial and morphological features of hepatocellular adenomas can be described in the pathology narrative as these features vary depending upon the test agent. 9.2  Hepatocellular Carcinoma

There are a few clear examples that show the development of hepatocellular carcinoma arising within hepatocellular adenomas. This finding supports the belief that there is a morphological continuum between adenomas and carcinomas. Indeed, for a given neoplasm, arriving at a confident diagnosis of carcinoma may be challenging, especially if based on a relatively small area in what is otherwise a large adenoma. Furthermore, in studies using known hepatocarcinogens, some individual animals have both adenomas and carcinomas in their livers, supporting progression of neoplastic growth but not necessarily excluding the possibility of independently arising carcinomas. Molecular biological evidence also supports independently arising liver tumors (Hoenerhoff et al. 2009, 2011). Consequently, many pathologists recommend combining the incidence and multiplicity of adenomas and carcinomas for assessing a carcinogenic response. When dealing with a situation where there are multiple adenomas or carcinomas or both adenomas and carcinomas present in the same liver, it is recommended that information be captured and discussed in the pathology narrative. Hepatocellular carcinomas may present in several different morphological types including solid, glandular, trabecular (the most common type), and anaplastic, with frequent pulmonary metastases (25–50%). Different morphological types of carcinomas are generally combined when tabulating tumor frequency with descriptions of their morphological features provided in the pathology narrative.

9.3  Hepatoblastoma

Hepatoblastomas are poorly differentiated hepatic neoplasm seen primarily in male mice and consisting of primitive-appearing basophilic cells that often arise within or adjacent to hepatocellular adenomas. Hepatoblastomas are typically not combined with either adenomas or carcinomas in carcinogenicity studies. A statement of how diagnoses are recorded when a hepatoblastomas arise within hepatocellular adenomas can be addressed in the pathology narrative. There has been molecular evidence that hepatoblastoma have distinctive cancer gene mutations not present in the associated hepatocellular adenoma (Kim et al. 2005). As is the case with hepatocellular adenomas and carcinomas, hepatoblastomas can occur spontaneously in untreated male and female mice but have not been reported in rat (Turusov et al. 1973). Metastasis in males may be as high as 36% (Turusov et al. 2002).

134

Robert R. Maronpot and David E. Malarkey

9.4  Cholangioma and Cholangiocarcinoma

Cholangiomas and cholangiocarcinomas are rare ( mouse alveolus). The alveolar parenchyma comprises 90% of the total volume of the mammalian lung. The most prominent structures in the lung parenchyma are the alveolar ducts and alveoli. Alveolar ducts arise from the most distal (terminal) bronchiolar airways, i.e., respiratory or non-respiratory bronchioles, depending on the species. The walls of alveolar ducts are composed of a linearized arrangement of alveoli. The proximal alveolar duct branches into secondary alveolar ducts, each of which ends in a blind outpouching composed of two or more small clusters of alveoli called alveolar sacs. The alveoli arise from the respiratory bronchioles and the alveolar ducts. The alveolar wall consists of three components: epithelial

Pathology of the Respiratory System

321

Fig. 4 Illustration of normal alveolar parenchyma (gas-exchange region of the lung)

cells (lining the alveolar air space), endothelial cells (lining the capillaries), and interstitial connective tissue (lying between epithelial and endothelial cells). The major alveolar cell types are the epithelial type I and type II cells, the pulmonary endothelial cells, interstitial cells, and alveolar macrophages (Fig. 4). Type I epithelial cells constitute approximately 10% of all cells found in the alveolar region, and type II epithelial cells constitute about 15%. Tight junctions are present between these epithelial cells that lie on a continuous basement membrane, as do endothelial cells. In many places, the basement membranes of both epithelial and endothelial cells are fused, forming an extremely thin air–blood barrier. In other areas, the cells are separated by interstitial tissue that consists of scant amounts of connective and elastic connective tissue, resident fibroblasts, and myofibroblasts and lesser numbers of interstitial macrophages and other mononuclear cells. Alveolar type I epithelial cells are flat, highly differentiated cells that do not divide; they cover approximately 90–95% of the alveolar

322

Jack R. Harkema and James G. Wagner

surface. Since these cells have a large surface area, they are highly susceptible to injury. The main function of the type I epithelial cell is maintenance of a barrier to prevent leakage of fluid and proteins across the alveolar wall into the air spaces, while allowing gases to cross the air–blood barrier freely. Alveolar type II epithelial cells are cuboidal in shape, located in corners or niches between capillaries, and contain lamellar bodies in which surfactants are stored. The functions of type II cells include the synthesis, storage, and secretion of pulmonary surface-­active material (surfactants); the reepithelialization of the alveolar wall after lung injury; and transepithelial solute transport to limit the volume of and perhaps regulate the composition of alveolar fluid. Capillaries, lined by endothelial cells (30–40% of all lung cells), are of the closed type without openings or fenestrations. Intercellular junctions between endothelial cells, however, are less tight than the epithelial junctions. Therefore, unlike other tissues, the major permeability barrier preventing leakage of vascular fluids into the alveolar air space is the alveolar epithelium rather than the capillary endothelium. Fibroblasts and myofibroblasts in the pulmonary interstitial tissue play a major role in disease processes that result in fibrosis. Resident alveolar macrophages are the major phagocytizing cells in the distal lung. They principally reside in the alveolar airspace (alveolar macrophages) (Fig. 4) or interstitial tissue (interstitial macrophages). In some animals, including humans (but not rodents or dogs), small numbers of macrophages have also been identified in the capillary bed (intravascular macrophages). Alveolar macrophages serve as the major cellular defense mechanism in the deep lung by phagocytizing and removing inhaled materials including fine inert dust particles and microbial agents. Alveolar macrophages also remove cellular debris resulting from cell death and/or inflammation and excess surfactant fluids lining the alveoli. Macrophages may leave the air surface by way of the conducting airways (mucociliary escalator), interstitium, or lymphatic vessels. These important alveolar cells are also extremely important in orchestrating anti-inflammatory (M2 or alternative phenotype) or inflammatory (M1 or classical phenotype) responses to inhaled agents by secreting various chemical mediators (e.g., resolvins). Blood reaches the lungs through two separate systems, the pulmonary and bronchial. The pulmonary system differs from other organs in that it is derived from the low-pressure pulmonary arteries, supplying high volumes of poorly oxygenated blood from the right heart for gas exchange by the pulmonary capillaries in the distal portion of the respiratory bronchioles, alveolar ducts, and alveoli. The bronchial system is a high-pressure arterial system derived from the aorta and carries oxygenated blood to meet the metabolic needs of the larger airways, visceral pleura, and large pulmonary vessels.

Pathology of the Respiratory System

323

Lymphatics are confined to the extra-alveolar interstitium, i.e., peribronchial, interlobular, and pleural interstitial tissue. Lymph flows centripetally through a subpleural network that is joined by perivascular and peribronchial lymphatics at the hilus. Afferent lymphatic vessels from the lungs drain into the lymph nodes, and then into the thoracic, right, and left lymphatic ducts, and the bloodstream. The sympathetic and parasympathetic divisions of the autonomic nervous system provide motor (efferent) innervation to the lungs, including bronchial smooth muscle, blood vessels, submucosal glands, and lymphatics. Sensory (afferent) innervation is maintained by way of several types of chemo- and mechanoreceptors that respond to inhaled irritants and other stresses.

3  Toxicologic Pathology of the Respiratory System The focus of the remainder of this chapter will be on the pathological basis of respiratory toxicology. We will emphasize how chemical-induced pathology of the respiratory system is identified, analyzed, described, and interpreted by pathologists in the context of toxicology studies whether they are designed to determine compound safety or mechanisms of toxicity. We will first review some general cellular and biochemical mechanisms of respiratory toxicity that are important to understand site-specific pathology in the respiratory system. The reader is referred to other publications for more detailed descriptions of the pathology of the respiratory system caused by toxicant exposure (Harkema et al. 2006, 2013; Renne et al. 2009; Wallig et al. 2017). 3.1  General Cellular and Biochemical Mechanisms of Respiratory Toxicity

Toxic damage to the respiratory system is a complicated series of interdependent cellular and biochemical events. Targeted toxicant-­ induced respiratory tissue injury may be initiated by mechanisms such as direct cell membrane injury (e.g., oxidative damage to epithelial cell constituents mediated through the formation of reactive oxygen species) or indirect toxicity mediated through metabolism of the xenobiotic agent and the formation of a reactive intermediate(s) (toxic metabolite) that can damage intracellular targets (e.g., endoplasmic reticulum, mitochondria). Pathological responses of airway and alveolar cells to toxicant exposures are highly dependent on the physicochemical properties of the chemical compound and its exposure regimen (route, duration, deposition, and clearance). Toxicant-induced cell injury is often further amplified through secondary events such as cellular inflammation (e.g., influx of leukocytes into sites of damaged tissue), activation of cellular mediators (e.g., cytokines, chemokines), release of lysosomal enzymes, and initiation of complement cascades. For example, toxicant-­

324

Jack R. Harkema and James G. Wagner

induced activation of alveolar macrophages can result in the production of reactive oxygen species, which contributes to more tissue oxidant levels and less antioxidants, resulting in oxidative stress which creates further cytotoxicity and secondary inflammation. Inflammatory, or M1, macrophages which are activated by interferon-γ and other chemical mediators (e.g., TNFα) are important orchestrators of acute, type 1 inflammation. In contrast, alternatively activated macrophages (M2 cells), which are activated by cytokines such as IL-4 and IL-13, have an anti-inflammatory role and are important in type 2 immune responses and tissue repair processes. M2 alveolar macrophages may, however, participate in the pathogenesis of chronic respiratory diseases, including chronic obstructive pulmonary disease (COPD) and asthma. 3.1.1  Direct Toxicity

Many agents produce respiratory injury by a direct interaction of the chemical compound with an airway or alveolar cell target (direct toxicity). Examples of direct acting toxicants are oxidant and irritating gases such as oxygen, ozone, chlorine, phosgene, ammonia, and hydrochloric acid. These gases, as well as acidic or alkaline irritants, may produce cytolytic changes directly at their site of primary interaction, such as the epithelial cell’s plasma cell membrane. This could result in lipid peroxidation of the outer cell membrane leading to cellular degeneration and eventual cell death. Injury mediated by oxygen reactive species is an important mechanism in respiratory toxicology caused by ozone (O3), the commonly encountered air pollutant in photochemical smog. This gaseous compound is so highly reactive that it likely produces oxygen free radicals, aldehydes, and hydroxyperoxides through ozonolysis of substances in the surface fluids lining airways and alveoli. Reactive oxygen species form free radical reactions. It is these reaction products (e.g., aldehydes) that are thought to cause the direct damage to proteins, lipids, and oligonucleotides of lung cells, like epithelial cells lining the luminal surface of the airways. Common consequences of exposure to irritating toxicants that directly damage the airway epithelium are (1) loss of surface cilia or the entire ciliated epithelial cell (resulting in loss of mucociliary function), (2) followed by acute inflammation (sterile inflammation because no infectious agent is involved), and (3) subsequent adaptive epithelial responses such as a reparative increase in the number or size of epithelial cells (hyperplasia and hypertrophy, respectively) or a change in the normal epithelial phenotype (metaplasia) resulting in either mucous cells or squamous cells that are not normally part of the epithelial lining layer (mucous or squamous metaplasia) (Fig. 5). Inhaled direct toxicants that reach the alveolar region at high concentrations may induce acute or delayed leakage of vascular fluids into the alveolar air spaces due to injury to both capillary endothelial cells and alveolar epithelial cells (Fig.  6). Following inhalation of phosgene, hydrochloric acid,

Pathology of the Respiratory System

325

Fig. 5 Common adaptive changes to airway lining cells (surface epithelium) after repeated toxicant exposures

ammonia, or smoke, a latent period of several hours may occur prior to the appearance of lung damage. Life-threatening loss of gas exchange due to massive pulmonary edema may then develop, even in the absence of further exposure to the offending agent (see detailed description of pulmonary edema formation later in this chapter; Fig. 7). 3.1.2  Indirect Toxicity (Metabolic Activation)

Another type of chemical-induced respiratory injury is mediated through the metabolism of air- or blood-borne xenobiotics. Three different mechanisms may be involved in this indirect toxicity. In the first scenario, the parent compound itself reaches the respiratory tract, either through the blood following systemic administration or as an inhalant. The compound then undergoes metabolic activation to the proximate toxin. Interaction with the target is often manifested as covalent binding of the reactive metabolite to cell macromolecules. Activation by microsomal mixed-function oxidases, especially the cytochrome P450 enzymes, is a key element in the process; on the other hand, protective systems such as intracellular levels of GSH and enzymes involved in maintaining reducing equivalents within the cell are crucial components in protection. Cell types that contain high concentrations of cytochrome P450s are particularly important in the detoxification of xenobiotics; however, these cells

326

Jack R. Harkema and James G. Wagner

Fig. 6 Illustration of alveolar injury after acute toxicant exposure (alveolitis)

are vulnerable to injury caused by the reactive metabolites they form. Examples of chemicals that cause respiratory damage following in situ metabolic activation include phenacetin, ­bromobenzene, 4-ipomeanol, butylated hydroxytoluene, CCl4, 3-methylindole, acetaminophen, and 3-methylfuran. In mice, both 3MI and 4-ipomeanol cause club cell necrosis; 3MI also causes necrosis of olfactory epithelium. Both the club cells and sustentacular cells in the olfactory epithelium contain large amounts of smooth endoplasmic reticulum that contain xenobiotic metabolizing enzymes. The endogenous generation of toxic metabolites can injure or kill these metabolizing epithelial cells. The second mechanism of indirect toxicity involves uptake of a systemically administered foreign compound by the liver, or any other organ, where the agent is metabolized to a highly reactive and toxic metabolite. This metabolite may cause liver injury but also may escape into the circulation via the hepatic veins and

Pathology of the Respiratory System

327

Fig. 7 Cell and tissue responses in pulmonary edema

inferior vena cava. The best studied examples of chemicals causing this form of lung damage are pyrrolizidine alkaloids. The pyrrolizidine alkaloid monocrotaline (MCT) is found in the plant Crotalaria spectabilis. Human toxicity has occurred from consuming contaminated grain or herbal teas made from such plants, whereas animal toxicity can occur following grazing on such plants. At high doses, MCT rapidly causes severe liver injury and death. At lower doses, it produces mild liver injury and delayed pulmonary injury characterized by pulmonary hypertension. MCT is bioactivated in the liver to pyrrolic metabolites by the cytochrome P450 enzyme system. The “putative” reactive metabolite dehydromonocrotaline (MCTP) is toxic to both the liver and lung. The third mechanism of indirect toxicity involves what has been called “futile redox cycling.” Paraquat, a herbicide, is selectively taken up by alveolar epithelial cells via an energy-dependent transport system. It is not metabolized but undergoes cyclic oxidation and reduction with concomitant production of reactive oxygen species such as superoxide anion, hydrogen peroxide, and hydroxyl free radicals. Direct evidence for the formation of lipid peroxides in paraquat toxicity remains more elusive. This is partly due to the pulmonary antioxidant defense mechanisms, including vitamins C and E, which make it difficult to obtain reliable measurements of peroxidative processes. A second event in paraquat toxicity is excessive oxidation, glutathione oxidation, and eventual depletion of cellular reducing equivalents, particularly of NADPH. The

328

Jack R. Harkema and James G. Wagner

extent to which this mechanism contributes to the development of toxic lung damage is unknown. Acute pulmonary edema may occur within a few hours of ingestion of paraquat, and pulmonary fibrosis and death typically occurs 1–3 weeks after ingestion of lethal amounts of paraquat. 3.1.3  Immune-Mediated Toxicity

Both physical and immunological mechanisms are important in pulmonary defense against chemical agents. However, immune responses in the upper conducting airways or lung may also result in adverse effects as the result of a hypersensitivity reaction or immunosuppression. Hypersensitivity diseases are the most common types of immune-mediated respiratory diseases caused by inhaled agents. There are four types of hypersensitivity reaction – type I (anaphylactic), type II (cytotoxic), and type III (Arthus type) are antibody-mediated reactions, whereas type IV (delayed hypersensitivity) is a cell-mediated reaction. The most common types of hypersensitivity reaction documented in the respiratory tract are types I and III. Exposure to pulmonary sensitizers, either foreign proteins or simple chemicals that act as haptens, at sufficiently high concentrations, induces the formation of specific antibodies. Type I hypersensitivity is primarily manifested as rhinitis (inflammation of the nasal mucosa) or asthma (inflammation and constriction of the pulmonary conducting airways; Fig. 8). Initial exposure to an allergen or sensitizer induces production of IgE antibodies, which bind to mast cells and ­basophils. Subsequent exposure to the allergen cross links the cell-­ bound IgE and triggers mast cell degranulation with release of

Fig. 8 Key pathologic features of hyperreactive small airways (bronchioles) in asthma

Pathology of the Respiratory System

329

vasoactive amines and other mediators. Platelet activation factor causes platelet aggregation and release of histamine, heparin, and vasoactive amines, thus further amplifying the response. Eosinophils and neutrophils, attracted by eosinophil chemotactic factor of anaphylaxis and neutrophil chemotactic factors, release hydrolytic enzymes that cause tissue necrosis. Chemicals that produce a type I reaction include toluene diisocyanates, trimellitic anhydride, and platinum salts. However, the type I reactions due to small chemical molecules may be mediated by CD8+ T cells, IL-5 secretion, and eosinophils through an IgE-independent mechanism. Numerous pharmaceutical agents have been implicated in type I reactions, but β-lactam antibiotics and sulfa-containing drugs are the most common causes of drug-induced type I reactions. Penicillin is thought to cause approximately 75% of fatal anaphylactic reactions in the United States. Type III hypersensitivity is manifested as hypersensitivity pneumonitis, also called “extrinsic allergic alveolitis,” and results from deposition of antigen–antibody complexes plus complement in the lungs, which in turn causes inflammation. Chemicals that produce type III hypersensitivity include trimellitic anhydride and mercury. Organic dusts containing spores of thermophilic bacteria, true fungi, or animal proteins are the most common cause of hypersensitivity pneumonitis in people. Examples such as farmer’s lung, humidifier lung, mushroom picker’s lung, and pigeon breeder’s lung are named for the settings in which the antigens are encountered. The initial alveolitis may progress to chronic fibrotic lung disease with granulomas. Type IV hypersensitivity is likely involved, along with the type III response, in the formation of granulomas. Type IV hypersensitivity occurs when sensitized T lymphocytes induce a cell-mediated response after a latent period. An example of a primary type IV response is the granulomatous reaction induced by beryllium in dogs and people. In rats, however, the granulomas induced by beryllium are considered foreign-body type granulomas, and beryllium-specific T cells are not found. Immunosuppression due to inhaled air pollutants has been documented in humans through epidemiological studies and in experimental animals, primarily through bacterial infectivity models, for agents that include oxidant gases such as ozone, nitrogen dioxide, and sulfur dioxide, tobacco smoke, benzene, toluene, and metals such as arsenic, cadmium, nickel, zinc, and lead. Effects on the physical and innate (nonspecific) lung defense mechanisms frequently cannot be separated in these studies, but various studies have demonstrated adverse effects on the mucociliary apparatus, pulmonary surfactant, and macrophage function. As an underlying potential mechanism for immunosuppression, decreased macrophage function, including phagocytosis and antigen presentation, has been the most commonly studied and documented suppressive effect on the immune system of experimental animals exposed to

330

Jack R. Harkema and James G. Wagner

oxidant gases, aerosols, and particulate air pollutants. Effects on the adaptive immune system have been documented less frequently, but immunosuppression in experimental animals exposed to dioxin has resulted in decreased cell-mediated immune responses and depressed antibody production in response to T-dependent antigens. 3.1.4  Respiratory Toxicity of Inhaled Particles

Pulmonary toxicity and responses to inhaled particles are influenced by the inhaled dose of the particulate material, the size and surface area of the particles, chemical composition of the particles, and the dynamics of their deposition, retention, and clearance in the lung. For inhaled drugs, the delivered particle form, pharmacologic mechanisms may modulate the expression of airway or alveolar toxicity. Responses to inhaled particles may include adaptive physiological responses or inflammatory and immune-­mediated responses, cell injury, and repair. For particulate chemicals, metabolism may also affect toxicity. Highly soluble particles leave the lung rapidly, and the dose is delivered in a pattern that is like that of inhaled gases. In contrast, poorly soluble particles persist in the lung after cessation of exposure, thus delivering a protracted dose to the lung. Historically, crystalline silica (quartz) and coal dust have been the most important inhaled, nonfibrous particles causing occupational lung disease (see the section below on pulmonary fibrosis). After a single inhalation exposure to particles, the amount of particulate material in the lung decreases with time due to ­dissolution and/or mechanical clearance. Dissolution is a major route of clearance of soluble particles. Phagocytosis by alveolar macrophages and clearance via the mucociliary escalator or lymphatics are major defense mechanisms for removal of poorly soluble fine or coarse particles from the lung. However, these mechanisms of airway or alveolar defense may be compromised by highly toxic particles. Phagocytized silica particles, for example, are toxic to alveolar macrophages that result in continuous cell injury and cell death as new monocytes/macrophages are recruited to the lung. As part of this cytotoxic response, macrophages release cytokines that stimulate fibroblasts to replicate and synthesize collagen. In addition, silica also causes activation and release of mediators by viable macrophages, including interleukin-1, tumor necrosis factor (TNF), fibronectin, lipid mediators, oxygen-derived free radicals, and fibrogenic cytokines. The macrophage cytotoxicity and ensuing inflammatory and fibrotic responses retard particle clearance can result in pulmonary fibrosis (scarring of the lung) and the formation of large silicotic fibrous nodules that can compromise normal gas exchange. Depending on the physicochemical properties of the particle, concentration, duration of exposure, and species exposed, nonneoplastic parenchymal responses to particles may include infiltrates of alveolar macrophages, alveolar epithelial hyperplasia, inflammation,

Pathology of the Respiratory System

331

bronchiolization, proteinosis, and fibrosis. After cessation of exposure, lesions may regress, persist, or progress. Figure 9 provides a diagrammatic representation of the acute and chronic pulmonary pathology of rats that received short- and long-term inhalation exposures to high concentrations of inert fine particles, like carbon black or diesel engine exhaust particles. Numerous studies in rats using inert, insoluble, fine particles, particularly titanium dioxide and carbon black, have led to a consensus that, for these nonfibrous particles, the quantity of material in the lung is the key determinant of the lung pathology. Furthermore, evaluation of data across multiple studies indicates that a lung burden of approximately 0.1 mg/g lung is required for findings to be detectable in histologic sections by light microscopy. When that burden is achieved in rats, increased numbers of alveolar macrophages are observed. Chronic and reversibility studies have shown that with exposures resulting in lung burdens of these relatively “inert” particles below approximately 1 mg/g lung, the adaptive increase in alveolar macrophages does not progress to more complicated lesions and is reversible. When lung burdens

Fig. 9 Cell and tissue responses in the lungs of rats after subacute and chronic inhalation exposures to high airborne concentrations of inert, carbonaceous fine particles (e.g., carbon black, diesel engine exhaust particles)

332

Jack R. Harkema and James G. Wagner

exceed approximately 1 mg/g lung, inflammation, epithelial hyperplasia, and fibrosis are observed, and these findings may persist or progress after cessation of exposure. Very large lung burdens, approximately 50–100 mg/g lung, result in “lung overload” in which macrophage-mediated clearance is reduced and chronic lung injury (e.g., fibrosis) progresses after cessation of exposure. There are numerous non-genotoxic, poorly soluble particles, such as titanium dioxide, talc, and carbon black that induce lesions in laboratory rats that are exposed under conditions resulting in overload of macrophage-mediated clearance. Chronic inhalation of these poorly soluble particles by rats can result in pulmonary inflammation, fibrosis, alveolar epithelial hyperplasia, bronchiolization, squamous metaplasia, and squamous cysts. Neoplastic lesions that occur late in life (usually between 24 and 32 months of age) include squamous epitheliomas, bronchiolar–alveolar adenomas, squamous cell carcinomas, and bronchiolar–alveolar adenocarcinomas. In contrast to rats, mice and hamsters develop less severe lesions and do not develop lung tumors even with particle lung burdens that are like that in the rat. These findings have raised questions concerning the appropriate use of data from rats, exposed under conditions resulting in clearance overload, for hazard identification in humans. The preceding discussion of particle toxicity applies to particles greater than 100 nm in size. Particles in the nanoscale size (less than 100 nm in at least one dimension) are referred to as nanomaterials if they are engineered, man-made materials, and as ultrafine particles if they are air pollutants. Most ultrafine particles are produced by incomplete fuel combustion in engines and industrial furnaces; however, natural sources include volcanic activity and sand storms. Concern for the potential toxicity of nanoparticles initially came from epidemiological data showing a relationship between exposure to ultrafine particulate air pollution and increased cardiovascular and respiratory morbidity and mortality in sensitive populations. Subsequent studies in animals using air pollution condensates, as well as a number of in vivo and in vitro studies using manufactured nanoparticles, have helped describe potential mechanisms of nanoparticle-related toxicity (Hubbs et al. 2011, 2013). Inhalation is the major route of entry into the body for nanoparticles. Nanoparticles have a high surface area per unit mass and thus have a large catalytic surface for formation of free radicals that drive oxidative stress, which is especially important for nanoparticles with bound transition metals and metal-based nanoparticles (such as silver and cadmium nanoparticles) which can be highly toxic. Additionally, the large surface per unit mass of nanoparticles may lead to adsorption of organic toxicants in air pollution and increase their interaction with cells. Soluble metals and aromatic hydrocarbons on the surface of nanoparticles may interact with lung lining fluid and undergo cyclic redox reactions

Pathology of the Respiratory System

333

that produce reactive oxygen species. Inhaled nanoparticles may agglomerate in the lung lining fluid or become coated by opsonins. Agglomerated or opsonized nanoparticles are phagocytosed by alveolar macrophages and may induce oxidative stress and inflammatory processes similar to those induced by other respirable particles. Nanoparticles that are not phagocytosed by alveolar macrophages efficiently enter lung epithelial cells and fibroblasts in the interstitium of the alveolar septa or may cross the alveolar–capillary barrier to enter the circulation, whereby they are distributed to extrapulmonary organs. Nanoparticles deposited on the olfactory mucosa may translocate to the brain through or around the axons of olfactory sensory cells (olfactory nerves) to the olfactory bulb. When nanoparticles gain entrance to cells, they may induce oxidative stress and cause protein, DNA, and membrane injury, as well as activate inflammatory and growth factor cascades and the oxidant-induced transcription factor nuclear factor-κB, and these processes potentially lead to inflammation and fibrosis. Inflammation and oxidative stress in the lung indirectly promote atherothrombosis and atherosclerosis through adverse effects on the endothelium, cardiac blood flow, platelet activation, and coagulation. 3.1.5  Toxicity and Responses to Inhaled Fibers

Fibers are a special type of particle defined as having a length-to-­ diameter ratio greater than 3 to 1. Fibers of toxicological concern are asbestos (chrysotile, crocidolite, anthophyllite, amosite, actinolite, and tremolite), synthetic vitreous fibers (SVF; glasses and ceramics in fibrous form), and fibers at the nanoscale (e.g., nanotubes). For asbestos- and SVF-related respiratory toxicity, there are four interrelated key factors – dose, particle dimensions, surface properties, and bio-persistence (length of time the fiber can persist in the tissue or organ). These largely determine the toxicity of the inhaled fiber. Iron ions on amphibole types of asbestos react with lung lining fluid and generate active oxygen species that induce toxicity and oxidant stress that may lead to DNA damage. There is also a direct relationship between bio-persistence, which is determined by fiber length and chemical composition, and toxicity. Longer, thinner fibers are more toxic because they are not cleared by alveolar macrophages and persist in the lung. Fibers longer than approximately 15 μm are particularly toxic and bioactive. They are not completely engulfed by macrophages, leading to release of lysosomal contents, cytotoxicity, oxidant stress, and stimulation of inflammatory and growth factor pathways. Inhaled asbestos can cause asbestosis, which is a fatal interstitial fibrosis, lung cancer, and pleural disease consisting of pleural fibrosis, plaques, and mesothelioma. Fibers must be thinner than 0.5 μm to translocate to the pleural surface. Inhaled asbestos and erionite, which is a non-asbestos, long fiber, are the only known causes of mesothelioma in humans.

334

Jack R. Harkema and James G. Wagner

Nanofibers are a diverse group of materials. Their potential for toxicity will likely be determined by dimensions, surface properties, bioreactivity, and bio-persistence. Carbon nanotubes are among the most commonly manufactured nanomaterials. Inhaled carbon nanotubes have been shown to penetrate the alveolar macrophages, the alveolar wall, and the pleura in mouse studies. They induce an inflammatory response, oxidant stress, granulomatous pneumonia, and interstitial fibrosis. In vitro experiments have shown that carbon nanotubes can induce mitotic disruption. The toxicology of nanomaterials is a relatively new field, and further studies are needed to develop screening strategies to evaluate potential hazards and set safe exposure limits. 3.2  Site-Specific Toxicity of the Respiratory System 3.2.1  Toxicant-Induced Pathology of the Nasopharyngeal and Laryngeal Airways

The distribution of damage in the nasal airways is particularly dependent on regional deposition of inhaled chemicals in the upper respiratory tract and on tissue or cell susceptibility to chemical agents (Fig. 2). Regional deposition of inhaled chemicals is dependent on airflow and, in the case of particles, on particle characteristics such as size (diameter) and aerodynamic shape. Inhaled coarse particles (2.5–10 μm in diameter) are predominately deposited in the nasal airways, while the smaller-diameter fine particles (0.1–2.5 μm) are deposited predominantly in the alveolar parenchyma. Ultrafine particles (nanoscale particles 6 months

4–14 weeks

Picut et al. 2017a, 2015b; Barrow 2007 a Age ranges are for general purposes only b Ages for male rat—Female rats have peripubertal stage 33–37 days and early adult stage 38–46 days

Rabbit

5–12 weeks

786

Catherine A. Picut and Amera K. Remick

Table 2 Morphologic maturationa of selected tissues in Sprague Dawley rat Birth Pancreas, endocrine 1 week Bone marrow (cellularity) Parathyroid glands Skeletal muscle Thymus 2 weeks Adrenal gland Cornea Liver Lymph node, mesenteric Spinal cord Sternum 3 weeks Femur Kidney Lung Skin Pancreas, exocrine Pituitary gland Retina Thyroid gland 4 weeks Bronchus-associated lymphoid tissue Lymph node, axillary Lymph node, mandibular Prostate Stomach/intestine Vagina 5 weeks Brain Epididymis Gut-associated lymphoid tissue Mammary gland (sex dimorphism) Nasal-associated lymphoid tissue Ovary 6 weeks Spleen Testis When tissues have subanatomic features and architecture similar to adult a

Pathology of Juvenile Animals

787

full adult size (e.g., bones reach full length; epididymis tubules are fully expanded with sperm). Physiologic maturation is also not a simple concept, because it could refer to either the time of maturation of one metabolic enzyme system (e.g., CYP enzymes in the kidney), or all physiologic functions (e.g., maximum glomerular filtration rate, maximum concentrating ability, etc. in the kidney). The physiological maturation in the liver and kidney are of particular importance since these organs often have direct consequences on bioavailability, clearance, and biotransformation of drugs, and can therefore complicate the toxicity profile in other organ systems. Even in organs that are not traditionally considered metabolic organs, like the mammary gland, the underdeveloped tissue may affect metabolic and clearance mechanisms that affect toxicity of chemicals (see DMBA metabolism in XVIII.  Integumentary System: Mammary Gland). The timing of “maturation” has been reported in agency guidelines for some human organ systems that undergo significant postnatal development and include: •









Kidneys and gastrointestinal system, where functional maturation is first reached at approximately 1  year of age (Radde 1985; Walthall et al. 2005) Lungs, where most alveolar maturation occurs in the first 2 years of life (Burri 1997) Immune system, where adult levels of IgG and IgA antibody responses are not achieved until 5–12 years of age (Miyawaki et al. 1981) Reproductive system and the brain, where maturation is not completed until adolescence (Zoetis 2003; Rice and Barone 2000) Skeletal system, where maturation continues well into adulthood for 25–40 years (Zoetis 2003)

Based on the fact that maturation is a dynamic process involving morphologic and physiologic changes, the reader should expect to see wide variation in the laboratory animal and human literature as to when “maturation” of organ systems occur.

6  Liver The liver is poorly developed at birth. At birth, the hepatic cords are loosely arranged and are at least three cells thick. These additional layers of hepatocytes function to control entry and exit of substances from the sinusoids (Walthall et al. 2005). The thick plates gradually reduce to single cell thickness by PND 14–PND 21. Some morphological appearances of the liver should not be misinterpreted as lesions. The thick irregular hepatic plates of the

788

Catherine A. Picut and Amera K. Remick

liver at birth could look similar to the trabeculae characteristic of hepatocellular carcinomas and should not be interpreted as a dysplasia, let alone neoplasia. Glycogen is very common in neonates at birth, but the vacuolar appearance quickly disappears within 1–2 days of life. Therefore, vacuolation in animals over 2–3 days of age should not be automatically dismissed as normal. The liver is the primary source of red and white blood cells, and extramedullary hematopoiesis (EMH) is prominent for the first 10 days of life. Thereafter, it diminishes as the bone marrow becomes the primary tissue for hematopoiesis. Identifying a deficiency of EMH would be a lesion in rat up to 10 days of age, whereas in older animals, an increase in EMH is often the lesion. Some lesions could conceivably appear in the juvenile rat due to experimental manipulation. Handling rats at very young ages (prior to PND 21) for purposes of daily gavage can be technically difficult. Handling can result in compression of the abdomen. This periodic compression may result in multifocal/focal necrosis of the liver, due presumably to pressure and/or vascular compromise (Fig. 1). Similar liver lesions (necrosis) have been associated with wrapping of the torso in rats (Parker and Gibson 1995). Lesions associated with wrapping/compression have been attributed to hypoxia and are described as a centrilobular coagulative necrosis with inflammatory cell infiltration around biliary tracts, histiocytosis, fibrosis, and granulomatous inflammation. The juvenile liver has reduced drug biotransformation capacity, compared to the adult. This reduced biotransformation capacity can increase or decrease the susceptibility of the animal to toxic effects, depending on if the primary agent is the toxicant or if the metabolic end-product is the toxicant. A well-known example of toxicity associated with reduced drug transformation is gray baby syndrome associated with chloramphenicol administration to

Fig. 1 Liver necrosis associated with handling. This young rat (PND 21) had been handled twice daily in a gavage study, starting at PND 4. These subcapsular zones of necrosis and inflammation were considered secondary to pressure associated with handling, rather than to a test article-related effect. H&E. 5.6×

Pathology of Juvenile Animals

789

infants. The immature GDP glucuronyl transferase system in the infant (or juvenile animal) slows the metabolism of chloramphenicol in infants (Fernandez et al. 2011). This leads to relatively higher and longer exposure to chloramphenicol in young animals. The ashen or gray color to the skin (and blue discoloration of the lips) of affected newborns is due to cyanosis that results from chloramphenicol’s impairment of electron transport and inhibition of cellular respiration. Increased sensitivity to hexachlorophene due to lower glucuronidation in the liver and lower biliary clearance will be covered under the section on brain and is one more example of how relatively deficient liver metabolism in juvenile rats affects distant organs. While there may be lower glucuronyl transferase in immature animals, there is increased activity of sulfation in the immature liver. This leads to decreased susceptibility of the immature liver to acute acetaminophen toxicity when compared to the adult (Penna and Buchanan 1991; Groseclose et al. 2015). Biliary clearance mechanisms in the rat are underdeveloped prior to PND 21, and those drugs that are eliminated by the biliary transport system may maintain higher circulating levels. This occurs with Dabrafenib (DB), a serine/threonine kinase inhibitor. This drug depends on biliary excretion. When DB is given to rats less than 21 days of age, higher levels of DB metabolites accumulate in the kidney resulting in kidney injury in juvenile rats (see VIII. Urinary System: Kidney) (Groseclose et al. 2015). The P450 cytochrome enzymes and the conjugating enzymes are substantially lower in early neonatal life. Those drugs that require CYPs for metabolism might therefore accumulate in juvenile animals. This was the case for antiretroviral therapy nevirapine (NVP), which is a substance that depends on CYP3A4 for biotransformation in the liver (Murphy et  al. 1999). In a juvenile study in rats, NVP resulted in necrosis, lipid deposition, and enzyme alterations of elevated alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) at 4 mg/kg body weight (Adikwu and Bokolo 2017). The toxic effect of any test article that gets metabolized by the liver might also be manifested as a latent effect of increased susceptibility to carcinogens. Any test article that is metabolized by the juvenile liver using cytochrome P450 enzymes might result in CYP isoform imprinting during this formative stage. This imprinting has been reported to occur following prenatal and perinatal exposure to phenobarbital (Downes 2012). Imprinting is tantamount to programming the hepatocytes to overexpress constitutive CYPs over a lifetime resulting in enhanced tumor formation and reduced life expectancy (Agrawal and Shapiro 2005; Gold et al. 1978). Therefore, even if exposure to a test article results in increased liver weight in a juvenile study, there is a possibility that the CYPs were programmed to overexpress in such a way to be a

790

Catherine A. Picut and Amera K. Remick

potential long-­term hazard. As the experience with phenobarbital has shown, ­epigenetic flags that are set in early life can alter the function in the liver for a lifetime. The transgenerational effects of CYP imprinting, or even epigenetic imprinting, are recognized by some regulatory agencies (EPA and European Commission) as toxic effects associated with endocrine disruptors or environmental compounds, but to the authors’ knowledge, imprinting is not yet considered in risk assessment (Kortenkamp et al. 2012; Johri et al. 2008; Anway et al. 2005; EPA 2011). Some findings in the liver of young animals may be considered within the normal range of biological variability. In the juvenile pig, there is a reported incidence of up to 20–30% of 23–24-day-­ old Yorkshire crossbred piglets as having EMH in the liver and more than 50% of animals having mononuclear cell infiltration in the liver (Kumar and Beazley 2017). Whether one would consider EMH or mononuclear cell infiltration in the liver to be a finding or to be a normal constituent of liver at 23–24 days of age is questionable. Certainly, the pathologist must compare the degree of EMH to that in age-matched controls before an increase in incidence or severity is recorded as a finding or lesion.

7  Gastrointestinal System The gastrointestinal (GI) tract of the rodent is highly underdeveloped at birth with very little capacity to metabolize fat and protein. At birth, digestion is carried out by lingual lipase produced by the tongue allowing for initial digestion of milk lipid upon the first suckle. At this early time, there is minimal to no protein metabolism in the stomach, since the stomach has a bland pH with no parietal or chief cells. By PND 21 (weaning), other enzymes along the GI tract develop and this robust physiological development parallels considerable morphologic change and cellular differentiation in all segments of the GI tract. Toxicologists designing gavage studies in juvenile animals should be aware of the evolving pH levels of the stomach of juvenile animals, since pH can affect exposure and/or metabolism of test article. The development of the stomach can be divided in an early bland phase (birth to < PND 14), transitional phase (PND 14–21), and acidic phase (> PND 21). During the bland phase, there is little to no hydrochloric acid (HCl) or pepsinogen production; this is an evolutionary protective design so that protective antibody proteins suckled from the dam are not denatured (Henning 1981). During this bland phase, it is common to find colonies of lactobacilli, enterobacter, and streptococci on the mucosal surface of the stomach and along the entire GI tract. The pH of the gastric lumen drops from about pH 6 to pH 4 during the transitional phase; glucocorticoid and thyroxine are two

Pathology of Juvenile Animals

791

hormones that help direct this pH change. A glucocorticoid surge occurs at around PND 18, and glucocorticoid, together with thyroxine, stimulates gastrin production and formation of gastrin receptors in the stomach. Gastrin in turn stimulates HCl production by the parietal cells and has an overall trophic effect on chief cells (responsible for zymogen production). The collective actions of glucocorticoid, thyroxine, and gastrin therefore lead to the lowering of pH through this transitional phase up to PND 21. Knowing that the maturation of the stomach is tied into an initial glucocorticoid surge helps clarify why any stress in a young animal (prior to the expected normal glucocorticoid surge) could result in a premature glucocorticoid surge, premature gastrin production, and precocious development of the stomach. After PND 21, the stomach is in the acidic phase, with dramatic increases in HCl and pepsinogen production. By PND 40, adult levels of enzymes and pH (approximately pH 2) are attained (Walthall et al. 2005). For comparison, in humans at birth the pH is neutral (pH 6–8), then falls to approximately 1–3 within the first 24 hours after birth, and later gradually returns to neutrality by day 10. The gastric pH slowly declines again to reach adult values of pH 2–3 by 3 years of age (Fernandez et al. 2011). Microvilli in the stomach, vacuolation, and hyperplasia are morphologic features of the normal developing GI tract. Transient villi in the stomach and colon are normal in young animals (birth to about PND 7), and these villus structures provide necessary increased surface area in the neonatal animal (Fig. 2). When identifying the site of origin of the tissue section, knowing that villi can be present in the stomach and colon will avoid confusion. Vacuolation of the small intestine is normal in rats less than 21 days of age, and must not be mistaken for toxic vacuolation, such as that associated with phospholipidosis. The normal vacuoles of the small intestine in young rats take on varied appearance depending on whether the

Fig. 2 Normal stomach at PND 7. This is stomach with normal microvilli to increase surface area. The presence of these villi should not cause confusion as to tissue type. H&E. 26×

792

Catherine A. Picut and Amera K. Remick

Fig. 3 Normal ileum at PND 7. This image of normal ileum from a PND 7 rat shows the vacuolated appearance of the small intestine. These vacuoles contain proteinaceous eosinophilic material, and the clear space around the protein likely represents intracellular digestion of proteins. This finding should not be interpreted as a toxic effect. H&E. 51×

molecule being absorbed is protein or lipid. Large intracytoplasmic protein droplets are normally present in the small intestine (duodenum, jejunum, and ileum) for the first 2 postnatal weeks (Fig. 3). The droplets are eosinophilic and represent both selective and nonselective receptor-mediated pinocytosis of proteins. Normal intracellular digestion of these proteins in preweaned rats may impart a clear vacuolated appearance around the droplets. Intracellular digestion of proteins is physiologically important because there is lack of protein metabolism within the lumen of the bland stomach during these first 2 weeks. Other vacuoles can be entirely clear and represent fat absorption. Lipid absorption by the small intestinal epithelial cells is necessary since the young animal has very limited capacity to digest fat—other than via lingual lipase. There are no bile salts at this early time to handle lipid metabolism. At PND 21, most sections of GI tract are in a state of rapid proliferation of epithelial cells, and this physiologic hyperplasia imparts a deep basophilic appearance to the epithelial cells, and a high mitotic rate is apparent. This physiologic hyperplasia should be expected and not confused with test article-related hyperplasia. Spontaneous lesions of the gastrointestinal tract have been identified in juvenile pigs. Juvenile pigs often serve as the most suitable non-primate animal model for human pediatric nonclinical research because of a similar physiologic gastrointestinal transit time and drug metabolism (Kumar and Beazley 2017). In 23–24-day-old Yorkshire piglets, some of the findings reported include bacterial colonies in the nonglandular stomach in greater than 33% of animals, hyperkeratosis of the nonglandular stomach in over 40% of animals, acute inflammation in the nonglandular stomach in over 22% of males, and acute inflammation of the large intestine in over 60% of males (Kumar and Beazley 2017).

Pathology of Juvenile Animals

793

Fig. 4 Coccidia in the small intestine of dog. Coccidiosis may be seen as a spontaneous finding in young beagle dogs. Note the protozoan parasite in the epithelial cells of the small intestine (arrows). H&E. 40×. (Image courtesy of Dr. George A. Parker)

Coccidia protozoa in the intestines are most commonly encountered as an incidental finding in young dogs housed in close proximity (Fig. 4).

8  Urinary System: Kidney The developing kidney is sensitive to xenobiotics during the postnatal phases of growth in the juvenile rat. Non-specific findings such as dysplasia, polycystic kidney, hydronephrosis, dilated pelvices and tubules, mineralization, tubular basophilia, and epithelial hypertrophy/hyperplasia are typical non-specific lesions associated with interference of test article on morphologic and physicologic development of the nephron in the rat (Figs.  5, 6, 7, and 8). During this early postnatal development, there is ongoing differentiation and elongation of tubules, maturation of anionic and cationic transporters that control acid-base and Ca-P04 balance, production of metabolizing enzymes, diminished concentrating ability of the kidney, and lower renal blood flow and glomerular filtration rate (GFR). Due to these pharmacokinetic, hemodynamic, and morphologic “immaturities,” the lesions in young rats poorly predict lesions in other species and may poorly predict lesions in the adult rat, and vice versa. The profile of lesion incidence in juveniles is further complicated by the fact that tubule dilatation, renal cysts, hydronephrosis, and even infarcts can be spontaneous background findings. To be sure, interpreting kidney findings in young rats can be extraordinarily difficult, and the

794

Catherine A. Picut and Amera K. Remick

Fig. 5 Common toxic effects in juvenile kidney (tubular dilatation and hypertrophy/hyperplasia). Dilatation of tubules, tubular epithelial hyperplasia, and epithelial hypertrophy are toxic effects that may be seen in the cortex of juvenile rats. This rat was exposed to an agent from PND 7 to 28 during the time of significant postnatal development. H&E. 20×

Fig. 6 Common toxic effect in juvenile kidney (tubular basophilia). Image (a) is from a PND 28 rat with the finding of “tubule basophilia” in the cortex. This PND 28 rat had been exposed to a toxicant from PND 7 to PND 28, and small clusters of basophilic tubules (arrows) were present in the cortex. Image (b) image represents the normal appearance of a kidney from a PND 13 rat (b). In this PND 13 rat, all tubules are still relatively undifferentiated and have a basophilic appearance similar to that in the PND 28 rat. While difficult to be sure of the pathogenesis of basophilic tubules in rats over age PND 21, the basophilic tubules (a) may represent foci of delayed/abnormal development. H&E. 31×

­on-­ n specific nature of the lesions and the lack of translation between species and ages make it difficult to identify a mechanism of action. Nephron formation is “complete” in the rat at PND 10 (Brown et al. 2016). “Complete nephron formation” means that all nephrons are in stage 3 or 4 (out of a total of four stages), with nephrons having recognizable glomerular tufts and tubules. The kidney appears morphologically “adult-like” by PND 21 under the light microscope, yet tubule elongation and nephrogenesis are not fully complete until PND 28 in rats. The mouse, monkey, and human

Pathology of Juvenile Animals

795

Fig. 7 Renal mineralization associated with soy-free diet. This 12-week-old female rat was exposed to a soy-deficient diet indirectly by lactation (through diet of dam) and then directly through feed until euthanasia at 12 weeks of age. The diet-induced renal mineralization is pronounced at the cortico-medullary junction. H&E. 1×

Fig. 8 Renal pelvic dilatation. In this PND 23 rat, gavaged twice daily from PND 7 to PND 23, there is renal pelvic dilatation. Renal pelvic dilatation may also be a normal background finding in juvenile rats. Empirical evidence suggests the incidence and severity of this change may be exacerbated by abdominal pressure associated with handling. H&E. 1.09×

796

Catherine A. Picut and Amera K. Remick

develop earlier than the rat; nephrogenesis is complete by birth in the mouse, monkey, and human (Fouser and Avner 1993). Glomerular tufts continue to mature, and tubules elongate through 1 year of age in human infants and up to 5 months of age in monkeys (Zoetis and Hurtt 2003). Functional maturation of the kidney in general lags behind morphologic maturation in the kidney. Even though the major morphologic changes of nephrogenesis and tubule differentiation are complete by birth in mice and human, urine-concentrating ability matures at postnatal timepoints in mice (PND 21), rats (PND 11), rabbits (PNW 5), and human (1 year of age) (Frazier 2017; Zoetis and Hurtt 2003; Snodgrass 1992). In rats, even though the kidney appears “adult-like” by PND 21 under the light microscope, and even though renal tubule elongation (morphologic change) is complete by PND 28, functional/physiologic maturation with maximum and stable maximum glomerular filtration rate are not obtained until PND 42. Functional maturation in humans occurs at 2  years of age (with maximum GFR) and at about 6–7  months of age in monkeys. The reader is referred to Frazier (2017) for a species comparison of morphologic and functional renal development in rat, mouse, human, monkey, pig, and rabbit (Frazier 2017). The developmental sequence of the kidney will affect, and help predict, the sensitivity windows to certain agents. Ace-inhibitors in the rat can cause renal tubule dysgenesis due to low perfusion when given the last 5  days of gestation and through the first 2 weeks of life (McCausland et  al. 1996). However, ace-inhibitors do not cause this effect in postnatal mice or humans. In animals with completed nephrogenesis by birth, like the mouse and human, susceptibility to ace-inhibitors occurs only during the prenatal time period (Frazier 2017). A couple of non-specific lesions in the juvenile kidney deserve further comment. One such finding difficult to interpret is basophilic tubules. In adult rats, foci of tubule basophilia are generally indicative of a degenerative change, are commonly associated with the onset of chronic progressive nephropathy, yet may also represent regenerative changes in response to tubular insult. However, in juvenile rats, an increase in tubule basophilia, such as associated with ace-inhibitors, may be more consistent with a delay in development rather than a degenerative change (Gomez et al. 1999). Basophilic tubules have also been associated with soy-deficient diets in rats, especially in 12-week-old F1 females that were exposed to such diet from birth (via suckling and then by ingestion) (Picut et  al. 2018). These same young rats develop pronounced renal mineralization at the cortico-medullary junction (Fig.  7) (Picut et  al. 2018). The mineral deposition is most likely related to a Ca-P04 imbalance associated with the casein-based diet (Reeves et  al. 1993). That the young animals on a soy-deficient diet are

Pathology of Juvenile Animals

797

more prone to develop renal lesions (basophilic tubules and mineralization) compared to adult animals suggests that the juveniles are being exposed during a sensitive window of evolving Ca-P04 homeostasis during postnatal development. To be sure, the phosphate transporter proteins undergo significant downregulation through the juvenile stage of development, and phosphate regulation matures only when nephrogenesis is complete (PND 28) (Frazier 2017; Traebert et al. 1999). Dilatory changes in the juvenile rodent kidney also require a few comment. Dilated renal pelvises, dilated tubules, and cyst formation are quite common in juvenile rodents and, in contrast to the adult, may be considered potentially reversible in the juvenile kidney. Dilated pelvices generally result from a deficient ability to concentrate or reabsorb water, but it also may be due to, or exaggerated by, pressure associated with handling during gavage studies (Fig. 8). Dilated tubules and multifocal cysts in the cortex may represent an upstream manifestation of pelvic dilatation or may also be a consequence of direct ischemia/pressure following handling. These dilatory changes (pelvic dilatation, tubule dilatation, renal cysts) can also be caused by obstructions in the ureters or the renal pelvis by crystals or by urothelial hyperplasia, or atony of the ureters, either one of which can represent the primary toxic lesion. Hydronephrosis or dilatation of the renal pelvis in young animals has been linked to exposure to 2,3,7,8-tetrachlorodibenzo-p-­ dioxin (TCDD) (Frazier et al. 2012) and gaba-agonists (Report on Neurontin (Gabapentin) 2009). Juvenile kidneys may be more or less sensitive than their adult counterparts to xenobiotics. The reason for different sensitivities is complex and as mentioned above can represent differences in metabolism and clearance of the drug, differences in fluid dynamics, and variations in maturation of anionic and cationic transporter functions (Fernandez et al. 2011; Frazier et al. 2012). These variations in sensitivities with age do not automatically translate between species. For example, juvenile rats and juvenile rabbits are more sensitive than adult counterparts to cyclosporine toxicity, yet the human child is not more sensitive than the human adult to cyclosporine. The reason why the human child may not be affected could arguably be because the human kidney does not undergo the degree of postnatal tubule development as in the rat or rabbit (Tendron-Franzin et al. 2004). Dabrafenib-associated nephrotoxicity is also age dependent in rats, and young pups exposed during the preweaning period (< PND 21) are more susceptible than adult counterparts (Groseclose et  al. 2015). Dabrafenib is an inhibitor of B-RAF kinase activity that normally mediates cell proliferation, differentiation, and survival (Groseclose et al. 2015; Trinh et al. 2014). Dabrafenib has been developed as a chemotherapeutic agent that inhibits cell

798

Catherine A. Picut and Amera K. Remick

­roliferation or tumor progression. Dabrafenib-induced toxic p lesions in kidneys include intraluminal calcium phosphate deposits, increased incidence of cortical cysts, tubular dilatation, and increased incidence of tubular basophilia—many of the findings that are non-specific and seen in young kidneys. In teasing out the pathogenesis of the various lesions, it was determined that calcium phosphate calculi were formed when high levels of the metabolite carboxy-dabrenafib damaged the tubular epithelium of the collecting ducts causing local disruption of acid-base homeostasis. The calculi resulted in an obstructive nephropathy, which explains the cysts and tubular dilatation. Only juvenile kidneys had the deposits because the otherwise normal biliary elimination of dabrafenib (the major clearance route) was impaired during this preweaning stage (Fattah et al. 2015; Gao et al. 2004). Not only were the preweaned rats more susceptible to dabrafenib because of impaired biliary clearance and enhanced buildup of metabolite in the kidney, but their kidneys were also more vulnerable because there was decreased capacity of transporters to maintain acid-base balance in the collecting ducts (Ingulli and Mak 2014). Teasing out the pathogenesis of DB-induced renal lesions in susceptible juveniles exemplifies the complexities of juvenile pathology in general. While cyclosporine and dabrafenib exemplify situations in which the young animal is more sensitive than the old, there are situations in which the young are less sensitive than adult counterparts. The young rat is less sensitive to mercuric chloride, gentamicin, and cisplatin. The diminished sensitivity of the young rat compared to the old rat in these situations is due to lower renal blood flow in the young rat with decreased perfusion of the cortical nephrons and therefore decreased exposure of the kidney to toxic agent. Knowing that decreased renal toxicity due to gentamicin is connected to the reduced renal blood flow in juvenile rats helps explain why translation of toxic effects between species is not automatic. In humans, pediatric patients have higher renal blood flow more consistent with that of the adult human. After all, renal vasculogenesis is complete by birth in humans, but is not complete until PND 17–19  in rats (Frazier 2017). Therefore, the relative gentamicin resistance of young rats is not translatable to the human pediatric population. Indeed, young children are as sensitive as older individuals to gentamicin. Alpha 2u globulin is a common finding in the renal tubules of male rats; however, alpha 2u globulin is not produced by the liver in substantial amounts until puberty (Vettorazzi et  al. 2013). Therefore, juvenile rats may not be an appropriate animal to use when investigating alpha 2u globulin accumulation. One spontaneous finding in young rats is a renal tumor classified as amphophilic vacuolar renal tubule tumor. This tumor can occur in Sprague Dawley rats as young as 7–10  weeks of age. Females are affected more frequently than males (Crabbs et al. 2013).

Pathology of Juvenile Animals

799

9  Neurologic System: Brain The brain of the juvenile rat at PND 10 is generally considered developmentally equivalent to a human brain at birth. Therefore, the developing rat brain in the first 2 weeks essentially provides a window into the third trimester of human fetal life. In the rat brain, 90% of neurons are already formed by birth; neurons have completed their proliferation and have already migrated into position. However, there are three major exceptions to this prenatal plan. Neurons in the hippocampus, cerebellum, and olfactory bulbs undergo most of their proliferation and migration during postnatal life. These postnatally differentiated neurons form from primary germinal matrix that lines the ventricles and from secondary germinal matrix that lies at the outer subpial surface of the cerebellum during the first 21 days of life. Neuronal development is considered complete by PND 21. In addition to active neuronogenesis during postnatal life, there is concurrent apoptosis of neurons during the first 2 postnatal weeks. In fact, 70% of neurons present at birth are pruned away within the first 14 days of life. Unlike neuronogenesis, which occurs pre- and postnatally, the differentiation of glial cells is limited to postnatal life and is especially prominent in the first 2  weeks of life (Picut et  al. 2016a). Glial cells, unlike neurons, can divide after they migrate into position, and therefore one may expect to see mitotic activity in multiple regions of the brain and spinal cord during early life. Glial proliferation, and in particular astrocyte proliferation, is required for proper synapses to form. Synaptogenesis is complete by PND 28 in the rat (Semple et al. 2013). Myelin formation is also limited to the postnatal time period in rats, and most of the myelin is formed by PND 37 (Downes and Mullins 2014). The blood-brain barrier (BBB) forms by about PND 21; this is a functional barrier based on formation of transporter systems in addition to a physical barrier created by the choroid plexus epithelium. A schematic of the major elements of brain development are depicted in Fig. 9. Cellular changes that occur during normal brain development can create a complex microscopic picture that could be misinterpreted as lesions. For example, the normal pruning process of neurons results in apoptotic neurons and nuclear debris on histologic sections in certain areas of the brain. In the authors’ experience, the superior and inferior colliculi and the habenular nucleus are two sites where apoptotic neurons are quite frequently observed in brain tissue from animals less than 21 days of age. Another normal finding in the brain is vacuolization of the choroid plexus during the first 3 days of life, and this might be related to “leakiness” of the choroid plexus epithelium due to an underdeveloped BBB. Mitotic activity in the brain and spinal cord is unusual in the

800

Catherine A. Picut and Amera K. Remick

Fig. 9 Normal brain development. This schematic shows the general timing of major events in brain development in the rat. Neurons in most areas of the brain, including the cerebral cortex, are formed prior to birth. Those neurons in the cerebellum and hippocampus form predominantly in postnatal life. As opposed to neuronal development, glial proliferation, axon elongation, synapse formation, and myelin formation are strictly postnatal events. The blood-brain barrier (BBB) is considered formed (physically and functionally) by PND 21

adult brain, but is frequently observed in the juvenile brain, and this mitotic activity most assuredly reflects either the rapid proliferation of glial cells in the developing white matter, or proliferation of undifferentiated neurons in the germinal matrix. Thick bands of germinal matrix lining the cerebellum or ventricles may exist up to PND 21, and these should not be interpreted as inflammatory cells. The developing nervous system is especially vulnerable to certain chemicals (Bondy and Campbell 2005; Rodier 1995). It is believed that the accentuated neurodegenerative mechanisms that are normally operative in the immature brain (i.e., normal pruning of the neurons), not to mention an incomplete BBB, increase the brain’s susceptibility to anesthetics, addictive drugs, environmental toxicants, as well as various metabolic stressors such as hypoglycemia, hypoxia, hyperoxia, infection, ischemia, and seizures (Reich et al. 2016). Hexachlorophene is the “poster child” for a chemical that affects the developing brain much more than the adult brain. Human neonates and developing rats, mice, and monkeys are more vulnerable to hexachlorophene neurotoxicity than adult counterparts (Tripier et  al. 1981). Hexachlorophene has an LD50 of 9 mg/kg in suckling mice, but an LD50 of 120 mg/kg in weanlings. This antibacterial product in the commercial product PhisoHex® causes a spongiosis of the white matter of the brain and spinal cord (vacuolar myelinopathy) including the optic nerve. This intramyelinic edema, characterized by wide interlamellar

Pathology of Juvenile Animals

801

space, eventually leads to axonal degeneration and demyelination (Cohen and Stommel 2015). Degeneration of the ganglion cell layer and retinal necrosis follow the changes in the optic tract. The cause for the heightened sensitivity of the young developing brain may be due to underdeveloped metabolic liver enzymes. Hexachlorophene is normally conjugated to glucuronide and excreted in the bile, yet glucuronidation is relatively deficient in young animals. Kainic acid and NMDA (N-methyl-D-aspartate) have been used as positive controls to produce acute neuronal degeneration in juvenile rat brains as well as in adult rat brains. From these positive control studies, it was determined that the juvenile rat brain has greater susceptibility to leukocyte recruitment compared to adult rats, and this is likely related to an incomplete BBB (Bolton and Perry 1998). Therefore, one may expect to see more intense inflammation in the juvenile brain compared to the adult rat following a similar neurotoxic insult. Increased recruitment of neutrophils into the brain is one reason why there is exacerbated acute neuropathology in models of stroke and head trauma in the young animals. This window of susceptibility for a robust inflammatory response in the juvenile brain is receiving considerable attention. The immature brain is not only more sensitive to developing inflammatory lesions, but it is also more sensitive to general anesthetics. When general anesthetics are administered during the period of active synaptogenesis, which is most profound during the first 2 weeks of life in the rat, widespread “pathological” apoptotic cell death can occur (Olney et  al. 2000). Anesthetics that block NMDA receptors (ketamine) or activate GABA A receptors (midazolam) increase neuronal apoptosis in the neonatal rat brain (Anand and Soriano 2004; Ikonomidou et al. 1999). Additional drugs that cause widespread necrosis are ethanol, phencyclidine, nitrous oxide, barbiturates, benzodiazepines, halothane, and propofol. Since the first 2 postnatal weeks in rat pups are equivalent to the perinatal period in human brain development, studying the rat brain allows us to determine potential effects on the human fetus and newborn brain with exposure to these anesthetics and addictive drugs (Anand and Soriano 2004; Ikonomidou et  al. 1999; Karen et al. 2013). The juvenile brain has also been shown to be more sensitive than the adult brain to environmental toxicants such as methylmercury and triethyltin, and early exposure to these toxic environmental compounds result in lifelong aberration in motor and sensory function (Rice and Barone 2000; Barone et al. 1995). The very young rat brain is also more sensitive to hypoxic ischemic injury than the adult counterpart. This is because the glial cells (and white matter components in general) are actively forming in

802

Catherine A. Picut and Amera K. Remick

the early postnatal period in rats and mice. When there is hypoxic ischemic injury to rat at PND 3 during this period of rapid white matter formation, the lesion is a reduction in size of cortical white matter with persistent gliosis at PND 21. Interference with white matter development in the immature rat and mouse brain can also result in hypomyelination and ventriculomegaly (Sizonenko et al. 2003; Follett et al. 2000; Skoff et al. 2001; Liu et al. 2002). In man, hypoxic ischemia is an important cause of periventricular white matter injury in premature births, since white matter development in humans occurs both pre- and postnatally. The hippocampus (gray matter) also shows selective sensitivity to hypoxia compared to the adult brain. All regions of the hippocampus are quite vulnerable to hypoxia from birth to PND 13, and from PND 13 to 21, the CA1 and CA3 regions of the hippocampus are more sensitive than in the adult. After PND 21, the juvenile hippocampus demonstrates the same sensitivity as in the adult brain (Towfighi et al. 1997). Increased sensitivity of the hippocampus to hypoxia in early postnatal life, when compared to other gray matter areas, makes sense considering a substantial number, and perhaps the majority, of hippocampal neurons proliferate and migrate into position during the postnatal period. Young animals also appear to be more sensitive to vigabatrin, an inhibitor of GABA transaminase that results in elevated GABA levels in the brain (Sidhu et al. 1997). Since vigabatrin is eliminated by the kidney, decreased clearance of the drug in young animals is proposed to result in relatively higher exposure in these animals. Not only do the sensitivities differ with age; the nature of the lesions in the brain also differs between young and adult rats, due primarily to differences in the state of myelin formation. In animals dosed PND 7–14 via oral gavage, the lesion consists of subtle neuropil vacuolation in the thalamus, midbrain, pons, and medulla. The vacuoles in the thalamus are due primarily to swollen oligodendrocytes, while those in the midbrain, pons, and medulla are due to intramyelinic edema (Walzer et  al. 2011). Because myelin is in early stages of formation at this early time point, especially in the thalamus, the vacuoles are quite small (Rasmussen et al. 2015). In animals dosed as adults with vigabatrin, the lesion is much more visually apparent, and intramyelinic edema presents as larger vacuoles more consistent with spongiosis (Rasmussen et al. 2015). The different presentations between young rats (small subtle vacuoles and swollen oligodendroglia) and adult rats (large vacuoles with no swollen oligodendroglia) reflect the age difference in oligodendroglia activity and quantity of myelin. The point of this discussion is that a toxic effect on myelin may be similar in both young and adult animals, but the developmental state of myelin formation affects the appearance of the lesion.

Pathology of Juvenile Animals

803

10  Neurologic System: The Developmental Neurotoxicity (DNT) Study Special pathology endpoints in the juvenile brain include those associated with developmental neurotoxicity (DNT) studies; the PND 22 pup brain is the age of brain commonly evaluated in developmental neurotoxicity studies. It is at this time when the brain has a full complement of neurons, the neurons in all sites of the brain have completed migration into final position, and there is no longer an external germinal matrix lining the cerebellum or ventricles. At this time, apoptotic and/or the mitotic activity of the very young brain should no longer complicate the interpretation of the histology. The pathology evaluation associated with DNT studies is considered a special pathology procedure, similar to how the mammary gland whole mount is a special pathology procedure relating to mammary gland tissue. Several regulations and guidance documents govern the conduct of the DNT study (OECD 2007, 2013; EPA 1998). The pathology endpoints in a DNT study are notoriously difficult from a technical standpoint and require significant laboratory technical and professional expertise. The quantitative endpoints at PND 22 include brain weight, brain length and width, and approximately five to eight microscopic (morphometric) brain measurements. Most of the morphometric brain measurements are bilateral and include the sensory and motor cortex of the cerebral cortex, corpus callosum, basal ganglia, hippocampus, and cerebellum. The SOPs for the laboratory should be drafted to follow the “best practices” in the toxicologic pathology industry; these best practices are currently found in two publications (Garman et al. 2016; Bolon et al. 2006). One of the most important recommendations by the “best practices” is to ensure the laboratory perform each technical step in handling the brain tissue (i.e., necropsy, fixation, weighing, gross measurements, trimming, processing, and embedding) in a counterbalanced fashion. Counterbalanced design is a prospective plan to orderly sequence the animals across dose groups so that one dose group is not systematically biased over another dose group. Such systematic bias can occur by personnel change, technique change, time delay, or environmental changes. How this orderly sequencing is performed is up to the laboratory, but the important point is that the plan or sequence must be referenced and detailed in the protocol, SOPs, and pathology report. Generally, the counterbalanced approach to each step of tissue handling will help prevent false positive results. For example, it is well known that duration of fixation time can affect brain weight and gross brain measurements (Acciani et al. 2016). Upon immersion fixation, brain weights can increase up to 50% within the first day of fixation and, thereafter, decrease each day, until the weights statistically stabilize after 6 days (Acciani et  al. 2016). A ­counterbalance design therefore would insist that

804

Catherine A. Picut and Amera K. Remick

trimming of fixed brains be done so that the brains of one group are not all trimmed first and therefore fixed for a shorter time than any other group. The effect of duration of immersion fixation on brain weight and size is not as apparent with perfusion fixation, and this is one reason why best practices recommend that PND 22 brains be perfusion fixed. Following fixation, brain weights and gross brain measurements are obtained. When brain weights are obtained, it is important to calculate both absolute and brain weight relative to body weight. Although absolute brain weights are spared in adult rats experiencing body weight loss, this sparing does not occur with juvenile rat brains. To be sure, the absolute weight of juvenile rat brains decreases when there is weight loss or growth retardation. As has been shown in feed restriction studies in pregnant rats, a 10–20% lower maternal body weight can be associated with up to a 12% drop in brain weight in the F1 pups when they reach PND 60 (Charil et  al. 2010; Carney et  al. 2004; Branes and Altman 1973). In DNT studies, trimming of the fixed brain requires approximately nine cuts using landmarks on the ventral surface of the brain. These nine cuts or slabs of brain are then embedded in paraffin to produce histologic sections. Out of these nine blocks, three blocks are used to produce highly homologous sections not only for qualitative examination but for morphometric measurements. To help achieve homologous sections (sections identical between animals), the trimming step becomes critical. Therefore, the brain is commonly cradled in a brain matrix or mold to assure consistent trimming between animals. Adult female brains are smaller than male brains, and there is more movement of female brains in the adult rat matrix. Therefore, one can expect to encounter more difficulty achieving homologous sections with adult female rat brains. This sexual dichotomy is not as pronounced in juvenile rat brains. After the nine slabs of brain are embedded in paraffin, the next step is microtomy. Even though microtomy is not one of the steps for which “best practices” insist on a counterbalance design, this step can also be subjected to bias due to personnel change or environmental change. Microtomy requires that thin ribbons of tissue be floated in a warm water bath to flatten out the sections. The temperature and duration of flotation can affect the size of the tissue. Therefore, a digital thermometer should be used in the water bath to keep the temperature between 39 and 41 °C. Further, the same technician should microtome all groups, since different technicians may float sections in the bath for different lengths of time. Sections floated for longer periods will tend to expand further compared to those floated for a relatively short period of time. When all nine sections of brain are microtomed and slides are prepared from all nine cuts, the pathologist then scrutinizes closely the slides from three cuts that will be used for morphometry. For

Pathology of Juvenile Animals

805

these three sections, the tissue must be homologous (i.e., identical) for all animals. To ensure that all sections between animals are homologous to one another, the pathologist generally ensures that each section is homologous to a given image in a brain atlas. Paxinos and Watson (1986) is one brain atlas commonly used, but any atlas would suffice (Paxinos and Watson 1986). The pathologist determines if the particular section is first taken at the correct Bregma level by looking for internal landmarks and then determines if the section is skewed in a dorso-ventral or lateral plane. The pathologist should exclude sections based on a predetermined plan of exclusion. For example, one exclusion criteria could be that dorso-ventral or lateral homology differs by no more than 0.3 mm. Another exclusion criterion could be that a section gets excluded if an artifact interferes with both right and left sides of any one bilateral measurement. While it is acceptable to exclude non-­homologous sections from morphometry, it is important that the end number of sections per group is at least 6 to provide sufficient statistical power. This means that in starting with a group number of 10, it may be acceptable to exclude up to four sections at any one level. The homologous sections, once identified for each animal, are then scanned digitally, and the image specialist takes measurements at pre-defined sites using appropriate digital imaging software. The anchor point and termination point for each measurement must be clearly identified in the SOP, the protocol, and/or the pathology report. A collage of the digital images can then be prepared and included in the pathology report. This collage serves as proof to the regulator that homologous sections were indeed obtained and used to arrive at the data. Generally speaking, the microscopic measurements are performed on sections stained with luxol fast blue/cresyl violet (LFB/ CV), which is also known as the Kluver-Barerra stain (Fig.  10). The LFB stains the myelin and provides clear distinction between white and gray matter so that anchor and termination points are clearly identified. However, myelination is not complete in rats until PND 37. Incomplete myelination in juvenile animals at PND 22 undermines the benefit of the LFB/CV. At PND 22, the LFB/ CV stain is often not any more useful than routine hematoxylin & eosin in providing contrast between white and gray matter. After quantitative morphometric measurements are obtained, the pathologist performs qualitative examination of the nine sections of brain, which are stained by both hematoxylin & eosin and with LFB/CV. Microscopic lesions in the brain are generally different in DNT studies than in routine neurotoxicity studies. In DNT studies, the dosing level and regimen is designed to avoid maternal or pup toxicity, but to still interfere with development. Development involves neuronal proliferation, neuronal migration, neuronal differentiation, axonal myelination, and synaptogenesis, generally in

806

Catherine A. Picut and Amera K. Remick

Fig. 10 Morphometric measurements in a developmental neurotoxicity (DNT) study. The three figures from a PND 70 (adult) rat collectively show an example of five bilateral and one unilateral linear morphometric measurements obtained in a DNT study. These same measurements are also typically taken at PND 22 (juvenile) rats. In the top panel (a), the motor and sensory cortex, the caudate putamen, and the corpus callosum are measured. In the middle panel (b), one bilateral measurement (height of the hippocampus) is taken, and in the bottom panel (c), the height of the cerebellum at the level of the seventh cranial nerve (marked with a “7”) is obtained. The anchor and termination points, and the target Bregma level, must be included in the pathology report. Luxol fast blue/ cresyl violet

Pathology of Juvenile Animals

807

that order. Therefore, the lesions seen microscopically when these steps are disrupted are heterotopia (displaced or poorly migrated neurons) or neuronal loss (that results from either failure of adequate proliferation or excessive pruning) (Sharma et  al. 2016; Kaufmann et  al. 2012). These types of lesions persist into adulthood. Microscopic lesions of astrocytosis, microgliosis, necrosis, hemorrhage, or neuronal necrosis that one may see in routine neurotoxicity studies are not expected in DNT studies. It follows that some of the more common special stains used in routine neurotoxicity studies, such as Fluoro-Jade (to identify degenerating neurons), glial fibrillary acidic protein (GFAP) (to identify reactive astrocytes and gliosis), or Iba-1 (to identify microglial proliferation) are not highly useful in DNT studies. There are a couple of neuron stains, like MAP-2 or NeuN, that can be used to help identify abnormal positioning and density of neurons, and in fact, this neuronal stain on thick sections (approximately 40  μm) might increase the likelihood of identifying heterotopic or abnormal positioning of neurons (de Groot et al. 2005). The pathologist has discretion under DNT guidelines to request such thick sections or neuronal stains. More often than not, there will be no observable microscopic findings in DNT studies. The absence of microscopic findings is not unexpected even though there may be statistically significant changes in gross brain weights, gross measurements (length/ width), or in one or more morphometric (microscopic) measurements. DeGroot, in 1995, using methylazoxymethanol acetate (MAM) as a positive control developmental neurotoxicant, reported up to a 30% reduction in the number of pyramidal neurons in the hippocampus (based on stereology), but was unable to appreciate any microscopic lesion in the hippocampus. He concluded that “substantial effects on brain morphology [quantitative measurements] … go unrecognized during slide reading” (de Groot et al. 2005). Certainly, microscopy is simply not the most sensitive pathology endpoint in DNT studies. Qualitative examination includes the assessment of the degree of myelination; this is performed on LFB/CV stained slides. The effect on myelination is most dramatic in the adult animals when myelination is complete rather than at PND 22, when myelination is still forming. In fact, caution should be exercised before demyelination is ever diagnosed in PND 22 animals (Fig. 11). Once the qualitative and quantitative pathology endpoints are obtained, it is up to the toxicologist to use the weight-of-evidence approach to determine whether the test substance is a developmental neurotoxicant. Garman et al. (2016) outline this weight-­ of-­evidence approach. According to this publication, there is clear evidence of developmental neurotoxicity when at least one of the following three criteria is met: (1) there is a dose-related change

808

Catherine A. Picut and Amera K. Remick

Fig. 11 Normal myelination in PND 22 and PND 70 rats. The images of the corpus callosum demonstrate the dramatic absence of myelin in the PND 22 rat brain (a), when compared to the PND 70 rat brain (b). Since myelination is not fully developed until PND 37 in the rat, and proceeds from a caudal to cranial direction in general, it would be difficult to diagnose demyelination in the cranial aspects of the brain in a PND 22 rat. Luxol fast blue/cresyl violet. A at 6×; B at 5×

in the same linear morphometric measurement at PND 70 and at PND 22, especially if PND 70 animals have more pronounced changes than at PND 22 and if both sexes are affected; (2) there is a dose-related change in either histopathology or gross brain measurements (weight, length, or width); or (3) there are doserelated changes in neurobehavioral effects. There are two important take-­home messages. One is that there may be clear evidence of neurotoxicity even without any abnormal pathology endpoints. The reason this occurs is because the neurobehavioral endpoints are designed to detect abnormalities with synaptogenesis, but the pathology endpoints are deficient in detecting such abnormalities in synapse formation. Of the five developmental processes (neuronal proliferation, neuronal migration, neuronal differentiation, myelination of axons, and synaptogenesis), current pathology endpoints in a DNT study may detect all but the synaptogenesis (Fig.  12). The second take-home message from the weight-of-­ evidence approach is that the changes at PND 60–70 (adult) should have more pronounced changes than at PND 22, if indeed the changes are indicative of a developmental neurotoxicant. This exacerbation of effect over time is because any developmental abnormality can be expected to persist and likely become more pronounced as the brain grows larger. If findings at PND 22 are more pronounced than at the adult timepoint, it is quite possible that the changes were either spurious (perhaps representing statistical noise) or due to general growth retardation rather than to neurotoxicity. Recall from the discussion above that maternal or pup toxicity or lower group mean body weights in the dams or pups can affect the size of the brain. There are a few avenues of future work that can be done to improve the pathology endpoints, and to expand the pathology endpoints to detect abnormal synaptogenesis. Using thick sec-

Pathology of Juvenile Animals

809

Fig. 12 Developmental events detected in developmental neurotoxicity (DNT) studies. The pathology endpoints in the current DNT studies are designed to detect abnormalities in four out of the five major developmental processes in the brain, namely, proliferation of neurons, migration of neurons, and differentiation of neurons and myelination of axons. Current pathology endpoints do not detect abnormalities in synaptogenesis. Rather, behavioral testing is designed to detect abnormalities in synapse formation

tions, confocal microscopy, multiplexed immunohistochemistry, and image analysis, excitatory or inhibitory synapses could be quantified by identifying sites of co-localization of pre- and post-­synaptic proteins (i.e., a synapse). V-glut and PSD-95 represent two pre- and post-synaptic proteins, respectively, the co-­ localization of which would identify an excitatory synapse, and V-gat and gephyrin represent pre- and post-synaptic proteins, respectively, to help identify an inhibitory synapse (McLeod et al. 2017). The toxicologist should be aware of the best practices used in obtaining the pathology endpoints in DNT studies. The toxicologist can take an active role in ensuring the validity of the pathology endpoints by ensuring that the pathology laboratory has Standard Operating Procedures (SOPs) in place that follow the “best practice” guidelines, ensuring the protocol and/or report includes detailed procedures for following a counterbalanced design method for all histology steps through the embedding procedure, and ensuring that the pathology report includes (1) exquisite detail about taking morphometric measurements including the Bregma level, anchor points, and termination points, (2) justification for excluding non-homologous sections from morphometry, (3) an affirmative diagnosis for all sub-anatomic sites of the brain evaluated, and (4) a collage of images of homologous sections used to take morphometric measurements. With such thorough reporting, the regulators have confidence in the results.

810

Catherine A. Picut and Amera K. Remick

11  Respiratory System: Lung The lung of the newborn rat is in the saccular stage of development, and this saccular stage corresponds to prenatal timepoints of human lung. Like the situation with the brain, the newborn and early postnatal lung is a window into prenatal human life. The saccule, as opposed to the alveolus, is characterized by relatively large airspace lined by a thickened smooth inter-airway wall that contains two capillaries, and a relatively large amount of condensed mesenchymal stroma. This saccular stage lasts up through PND 4, when the lung enters the bulk alveolarization stage during which most of the saccular walls are transformed into thin (mature-­ looking) alveolar walls by PND 14. Thereafter, there is continued alveolarization up to PND 21. The very young rat in the saccular stage provides a model to study oxygen-induced dysplasia in the preterm human. Preterm humans are routinely supplemented with 95% oxygen, but this oxygen therapy also results in a failure of normal alveolarization. Providing 95% oxygen to rat pups from birth to PND 15 also results in dysplastic/abnormal formation of alveoli, affording an opportunity to study therapeutic agents that can prevent this side effect (Picut et al. 2017b). The dysplastic alveoli in the rat model look like saccules in that the air spaces are relatively enlarged with a smooth-contoured thick wall and reduced density of septae (Fig. 13). This is referred to as alveolar simplification. To properly detect this change, the methodology for procuring and fixing the lungs is important. Infusion with liquid formalin at a pressure of 25–30 mm Hg for 30 minutes is important to adequately expand the lungs for histologic processing. The young immature lungs are less elasticized or expansile, compared to the adult counterpart, and therefore the full 30 minutes for infusion is important.

Fig. 13 Alveolar simplification associated with 95% oxygen. The alveolar simplification (or alveolar dysplasia) in a PND 15 rat exposed to 95% oxygen (a) arguably looks similar to the saccules present in the normal lung of a PND 1 rat (b). In both instances, the alveolar walls are thickened and hypercellular and the walls of the airspaces are smooth with few septae. H&E. 20×

Pathology of Juvenile Animals

811

In the same way that lesions affecting development of the mammary gland may have latent effects later in life, lesions in the juvenile lung may have delayed effects as well. In mice, mutations affecting lung alveologenesis during early postnatal life can lead to subsequent alveolar enlargement (Chen et  al. 2005), and these abnormalities might render the lung more susceptible to chronic obstructive pulmonary disease (COPD) (Boucherat et al. 2016). Spontaneous findings in juvenile laboratory animals should be recognized and not confused with a test article-related effect. Spontaneous background findings have been published and with regard to the lungs of day 23–24 young Yorkshire pigs include pleural adhesions, inflammation, and/or fibrosis in over 40% of untreated control animals (Kumar and Beazley 2017).

12  Immune System: General The developing immune system of children has been identified as a likely target for adverse effects of chemicals (Richter-Reichhelm et  al. 2002; Landrigan et  al. 2004) and medicine (Dietert et  al. 2000; Hurtt et al. 2004). PND 4–28 is a typical dosing regimen for rats on a pediatric study to test the safety of pediatric medicines intended for babies and infants. This exposure period comprises the critical period of postnatal immune maturation in the rat. Damage to the lymphoid organs during this period has the unique potential to result in a lifelong impairment of the immune function. A lot of work has been sparked by the finding that cyclosporine influences the developing immune system in rats. Cyclosporine exposures in utero and postnatally result in morphological and functional abnormalities of the thymus and spleen (Holladay and Smialowicz 2000). It causes decreased size of the T-cell areas of most lymphoid organs, including the spleen (i.e., the periarteriolar lymphoid sheaths), thymic medullary regions, paracortex of peripheral lymph nodes, and interfollicular zones of the GALT (Allais et al. 2009). Rodents lag significantly behind the human and nonhuman primate (NHP) when it comes to morphologic and functional maturation of the immune system. T and B cells form prenatally in humans and NHPs, but these cells primarily form in the postnatal period of rodents(Weinstock et al. 2010). For purposes of this section, the immune system organs include the thymus, spleen, peripheral lymph nodes (mesenteric, mandibular, and axillary), and mucosal associated lymphoid tissue (MALT), including that which is bronchus-associated (BALT), nasal-associated (NALT), and gastrointestinal-­associated (GALT). The thymus is a primary lymphoid organ that develops regardless of the degree of environmental stimulation. The other lymphoid organs (peripheral lymph nodes, spleen, and mucosal associated lymphoid tissue) develop

812

Catherine A. Picut and Amera K. Remick

follicles and germinal centers (i.e., B-cell areas) in response to antigenic stimulation. In rats during postnatal life, there is a gradual increase in cellularity and size of all lymphoid organs beginning at PND 10 and concluding at PND 42, when these immune organs are all mature and morphologically similar to that of the adult. In general, the first sub-anatomic sites in any lymphoid organ that develop are T-cell areas, followed by B-cell areas. It makes sense, therefore, that the thymus reaches morphologic maturity first (by PND 14) because it is entirely T cells that develop independent of antigenic stimulation. The next immune organ to reach maturity is the mesenteric lymph node (at PND 14–21), then BALT, mandibular and axillary nodes (at PND 28), then NALT (at PND 35), and then spleen (at PND 42) (Parker 2016b; Parker and Papenfuss 2016). The splenic B lymphocyte follicles are the last structures to appear in the development process, even though the T-cell areas of the spleen (i.e., periarteriolar lymphoid sheaths) are programmed to develop earlier at about PND 28. Even among the secondary lymphoid organs in control animals, there is considerable morphologic variation in the size of germinal centers. BALT and axillary lymph nodes rarely have germinal centers in the control resting laboratory rat even at the adult age (van der Brugge-Gamelkoorn and Sminia 1985). Whether the morphologic maturation of the various immune organs parallels or predicts functional maturation is not very clear. Within those secondary lymphoid organs that depend on antigenic stimulation (i.e., peripheral lymph nodes, mucosal associated lymphoid tissue [BALT, GALT, and NALT], and spleen), follicles and/or germinal centers might appear relatively small and quiescent in a relatively “sterile” laboratory environment, even though they are capable of responding upon confronting antigen. The first month of age in rats, and the first year in humans is considered a period of perinatal immunodeficiency, and immune responses should not be evaluated (morphologically or functionally) until this immunodeficient period is over (Landreth 2002). This is consistent with current practices in developmental immunotoxicology. Immune function tests are generally not conducted until the first filial generation (F1) animals reach PND 56, at which time the T-cell dependent antibody response (TDAR) is performed (Dietert 2006; OECD 2011). Other immune function endpoints are not assessed until PND 90, including phenotyping for T cell, B cell, NK cells, histopathology, hematology, and organ weights. As with most organ systems, the functional developmental of the rat immune system (in addition to the morphologic development) lags relative to that of the human and NHP. Primates are born capable of producing a humoral immune response, but in rodents, a humoral immune response is detectable only after PND 20 (Holladay and Smialowicz 2000). Humans are considered to

Pathology of Juvenile Animals

813

have a fully mature functional immune system by 12 years of age (adolescence), and rats have a mature immune system (at least morphologically) by the same relative age (PND 42). Although significant similarities of immune system function are shared among mammals, important differences highlight the limitations in ­automatically extrapolating results in rodents to the human condition (Weinstock et al. 2010).

13  Immune System: Thymus The thymus is well-developed at birth, and this is not surprising, since T-cell areas develop prior to B-cell areas. The thymus is the immune organ of choice to pick up a T-cell toxicant in a young neonatal and infantile rat. The architecture of the thymus is adult-­like by PND 7, but the cellularity of the cortex maximized by PND 14.

14  Immune System: Spleen The spleen at birth consists of a uniform sheet of cells with no distinction between white and red pulp, except for a few periarteriolar aggregates of mononuclear cells. There are no lymphoid follicles or germinal centers at this time. At PND 7, recognizable periarteriolar lymphoid sheaths and marginal zones can be recognized, and these substructures reach normal adult size by PND 28. By PND 35, there are small “immature” follicles, and by PND 42 the spleen can be considered histologically mature with well-developed follicles, marginal zones, and periarteriolar lymphoid sheaths. Because follicles are inapparent during the first 4–5  weeks, the juvenile spleen is not a good organ to detect B-cell toxicants. Most importantly, pathologists should use caution to diagnose depletion of white pulp in the spleen of animals less than 4 weeks of age. Extramedullary hematopoiesis is common and robust from PND 0 to 14 and thereafter reduces to a low sustainable level in the red pulp of the spleen. Accessory spleen is reported as a development variation and can be seen in up to 1% of litters of Cr1:CD(SD) rats.

15  Immune System: Lymph Nodes Lymph nodes in an adult rat provide an opportunity to examine both B-cell effects (follicles and germinal centers) and T-cell effects (paracortical areas). Prior to PND 14, however, the peripheral lymph nodes lack a distinct paracortex or follicle zone. For that reason, it would be very hard, if not impossible, to detect an immunologically toxic agent on the lymph nodes in animals less that

814

Catherine A. Picut and Amera K. Remick

Fig. 14 Mandibular lymph node development. These images are from the mandibular lymph node of a normal PND 14 rat (a) and a PND 28 rat (b). In the PND 14 rat (top panel), there are no observed germinal centers to evaluate for potential B-cell toxicity. By PND 28, however, germinal centers and follicles begin to develop, and only after this stage can B-cell toxicity be evaluated histologically. H&E. 15×

PND 14 (Fig. 14). Of the three peripheral lymph nodes routinely examined in preclinical studies (mesenteric, mandibular, and axillary), the mesenteric nodes develop earliest, followed by the mandibular and axillary nodes.

16  Immune System: Mucosal Associated Lymphoid Tissue BALT, GALT, and NALT develop in response to antigen influx and as such are secondary immune organs. In the rat, BALT is relatively large compared to that of humans or mice. BALT cellularity reaches adult levels by PND 28, and the size of BALT is affected by the hygienic environment of the rodent facility. Regardless of the size of BALT, germinal centers or follicles are not a morphologic feature of BALT in control animals at any stage of maturation. GALT reaches adult cellularity by PND 28, at the same time as BALT, and GALT normally will have germinal centers.

17  Male Reproductive Tract: Testes Evaluating the testes in juvenile males is difficult and complex, not only because there are different populations of germ cells compared to the adult but also because the tubules are dynamically expanding in diameter and the 14 stages of tubules are not yet established. The picture is further complicated by the fact that there are ongoing degenerative and proliferative changes in the normal developing male testes and these must be differentiated from the abnormal degenerative and proliferative changes that can accompany a toxic effect. Under normal circumstances, the male testis has significant apoptosis of germ cells during the first wave of spermatogenesis, and this occurs around PND 12–15. Many of the lesions associated with low testosterone levels in older animals can

Pathology of Juvenile Animals

815

be seen in the juvenile rat at PND 21–32, which is not surprising given the relatively low physiological level of testosterone in juveniles. During this time, there is apoptosis of pachytenes especially in stage VII and VIII tubules. Many lesions associated with Sertoli cell toxicity can be seen normally in the young animals, because Sertoli cells are immature and do not have their full complement of functions. Changes that can be attributed to immature Sertoli cells include sloughing of germ cells, enlarged residual bodies, disorganization of the germ cells, and a motheaten irregular wall of seminiferous epithelium. Sloughing of germ cells results in cell debris in the epididymis, and this cell debris can be quite prominent during the juvenile period (Picut and Remick 2017). Identifying with certainty test article-related effects in the testis and epididymis is not straightforward at any age, and this task is more daunting in young animals. The blood-testis barrier (BTB) forms around PND 18, and the developing germ cells are exposed to test article prior to PND 18. While this early time point should be an ideal time to evaluate a toxic effect on germ cells, this is not the case. There are simply few to no germ cells present to interact with the toxic agent prior to PND 18. The germ cells (spermatocytes and spermatids) don’t form until the BTB is in place to protect them (Picut and Remick 2017). While evaluating the testes in young animals is through a lens of noisy normal background degenerative and proliferative changes on one hand, and a lack of a susceptible germ cell population on the other hand, it does provide some unique opportunities. Contrary to conventional wisdom, evaluating immature testes can provide a wealth of information not otherwise obtained from sexually mature animals. The immature tubule gives unusual clarity to the mode of action. This is because the neonatal and infantile periods are times when the testis is uncomplicated by endocrine feedback loops, there are relatively little confounding stress effects on gonadotropins, there are a large number of uniform-appearing tubules (since the 14 stages have not yet developed), and there are highly visible, mitotically active, and susceptible spermatogonia and Sertoli cell populations (Whitney 2012; Picut et  al. 2015a). Therefore, evaluating testes at the neonatal and infantile stages may actually provide an ideal opportunity to identify a toxic effect that could be unique to the early postnatal time period, and also to help determine the target cell that led to a non-specific testicular lesion in an adult testis. Studies performed during a critical window (PND 4–15) have helped define testicular toxicants and their modes of action (Whitney 2012). N-methylthioltetrazole, a toxic agent in cefamandole (a β-lactam antibiotic), inhibits aldehyde dehydrogenase and interferes with proliferation of spermatogonia. The effect was apparent in a targeted study from PND 4 to 13 (Whitney 2012; Hoover et al. 1989). When the same compound

816

Catherine A. Picut and Amera K. Remick

was administered over a wider time period (PND 6–36), the degenerative findings were non-specific and did not clearly reveal the mode of action. Therefore, this example demonstrates the importance of designing targeted short-term studies during windows of susceptibility to toxicants (Hoover et al. 1989). Unique lesions can also be seen during the neonatal and infantile period. Test articles that interfere with tyrosine kinase receptor activity can inhibit migration of gonocytes to the basement membrane during the first week of postnatal development and prevent their transformation into spermatogonia (Whitney 2012). Imatinib mesylate (Gleevec®), a tyrosine kinase receptor inhibitor against c-kit and platelet-derived growth factor receptor (PDGFR), when given for 3 days during the first postnatal week (PND 5–7), led to increased persistence of central gonocytes in tubules (Whitney 2012; Nurmio et al. 2007). Endocrine disruptors given during the critical window (PND 4–15) also have effects that would arguably not be apparent had the testis been allowed to mature. By giving estradiol to neonatal rats, it was revealed that estradiol inhibited the maturation of the Sertoli cells. Therefore, the estradiol administration resulted in a pseudostratified appearance of immature Sertoli cells with a paucity of spermatocytes by PND 22 (Gaytan et  al. 1986). When estrogenic substances are given during the neonatal period, there is alteration of estrogen receptor expression in the testis, which will influence the ability of Leydig cells to function, and which will delay the eventual onset and progression of puberty (Sharpe et al. 2003). This correlates well with Gaytan’s report that estrogens inhibit the maturation of Sertoli cells (Gaytan et al. 1986). While estrogens inhibit maturation of Sertoli cells, other hormones have their effect on the Leydig cell population. Thyroid hormone is required for Leydig cells to mature. Therefore, neonatal hypothyroidism will arrest postnatal Leydig cell maturation yet, perhaps counterintuitively, permit overzealous proliferation of mesenchymal precursor cells leading eventually to hyperplasia of “immature” Leydig cells (Mendis-Handagama and Ariyaratne 2004). Polychlorinated biphenyls are one toxicant that can cause a hypothyroid state in neonatal rats and is associated with increased number of Leydig cells, reduced size of Leydig cells, and decreased steroidogenic function of Leydig cells (Mendis-Handagama and Ariyaratne 2004; Kim et al. 2001). Just like there are latent or delayed effects associated with toxicity in the lung, mammary gland, and liver, prenatal exposure to di(n-butyl)phthalate (DBP) induces latent morphological and biochemical changes in the testes of post-pubertal rats. When rats are administered 100 mg/kg/day DBP GD 12–21, an increased number of Sertoli cells in the postnatal testis do not become apparent until postnatal weeks 14 and 17. Other studies show that in utero exposure to higher doses of DBP (500  mg/kg/day) resulted in

Pathology of Juvenile Animals

817

decreased Sertoli cell numbers in the perinatal period (Okayama et  al. 2017). As discussed under testosterone and its effect on mammary gland development, the timing and dosage of endocrine disruptors can cause seemingly opposite histologic findings. The readers are referred to Picut et  al. (2017) for a species comparative review article that provides morphological and endocrinological timepoints during development of the male reproductive tract in laboratory animals and in humans (Picut et al. 2017a). Testicular weight is spared in sexually mature animals in the face of weight loss. In adult rats experiencing loss of body weight, absolute testicular weight stays constant, and relative testicular weight (testes weight relative to final body weight) may increase. However, in the authors’ experience, this “sparing” of testicular weight does not hold true in immature animals. In immature animals, the testes weight is closely linked to body weight in the rat and the dog (Picut et al. 2015a; James et al. 1979), and therefore, it is not unreasonable to expect a lower absolute testes weight with a loss of body weight. However, there are no publications that support this lack of sparing in immature rats. In young Sprague Dawley rats with a restricted diet and significant body weight loss, only negligible effects on testicular weight were reported (Rehm et al. 2008). The immature testis might show increased sensitivity to test agents when compared to adults. It has been shown that the immature testis in rats is more sensitive to cyclophosphamide than the adult testis, and this is likely due to greater degree of drug penetration into the seminiferous tubules of young animals with an immature blood-testis barrier compared to older animals (Comereski et al. 1987). Spontaneous background findings are common in dogs, monkeys, and minipigs. In beagle dogs, bilateral tubular atrophy/ hypoplasia can be a spontaneous occurrence in up to 87% of dogs 6–7  months of age (Rehm 2000; Goedken et  al. 2008). This is characterized by triangular clusters of Sertoli-only tubules, admixed with some tubules having degenerative changes (Goedken et  al. 2008). Testicular hypoplasia is a spontaneous finding in minipigs and appears similar to testicular hypoplasia/atrophy of dogs. The lesion is generally a combination of atrophic (Sertoli-only tubules) admixed with degenerative-appearing tubules (Bode et  al. 2010; Thuilliez et al. 2014). These mixed-type lesions provide a level of complication to the pathologist in interpreting the immature testis. This change can be seen in 24% of Gottingen pigs from North America and 37.3% from Europe (Thuilliez et al. 2014). In monkeys, perhaps the most commonly encountered spontaneous finding is fibrous hypoplasia of the testis in juvenile cynomolgus monkeys. This entity is a unilateral or bilateral background lesion in cynomolgus monkeys, especially those imported from

818

Catherine A. Picut and Amera K. Remick

Indo-China (Creasy 2012). Fibrous hypoplasia (or increased stromal collagen) is encountered with increased incidence and severity in sexually immature monkeys (approaching 50% incidence with 12.5% having moderate to marked severity), when compared to peripubertal monkeys (incidence 29.4% with 5.9% being moderate to marked severity). Peripubertal monkeys have a higher incidence and severity than that of sexually mature monkeys (incidence 10.7% with 0% incidence of moderate to marked severity) (Coe et  al. 2017). The increased stromal collagen is commonly associated with another spontaneous finding in the young cynomolgus testes, that of seminiferous tubule dilatation (Fig. 15) (CVemireddi and Creasy 2017). In the pig, one must be careful when evaluating interstitial cells. The numbers and size of the Leydig cells are exceptionally large in the pig. Pigs have two morphologically distinct Leydig cell populations: relatively large inter-tubular Leydig cells and a peritubular population of smaller Leydig cells. Therefore, one should expect to see both small and large Leydig cells, and not confuse this disparity with a lesion, such as Leydig cell hyperplasia (to account for the large Leydig cells) or atrophy (to account for a smaller population of Leydig cells).

Fig. 15 Background changes in testis of cynomolgus monkey testis (dilatation of tubules and fibrous hypoplasia). In this sexually immature cynomolgus monkey, there are two background findings. One is dilation of seminiferous tubules (a, 3×), and the other is fibrous hypoplasia (b, 2×). In (a), note the clusters of dilated empty tubules. There is arguably also increased collagen in the stroma, suggestive of a mild degree of fibrous hypoplasia. In (b), note the more severe fibrous hypoplasia, characterized by expansive amount of dense eosinophilic collagen that permeates and replaces seminiferous tubules. Panel (c) represents a normal testis from a sexually immature cynomolgus monkey. H&E

Pathology of Juvenile Animals

819

18  Integumentary System: Mammary Gland The mammary glands of the juvenile rat are quite useful to detect endocrine disruption. Due to concern that early exposure to environmental factors can cause breast cancer later in life, and the relatively recent finding of early breast development and puberty in girls, the use of mammary gland tissue as an endpoint in rodent toxicity testing has increased (OECD 2018; Fenton 2009; NTP 2011). More specifically, National Toxicology Program (NTP) carcinogenicity bioassays have been modified to include early life or multigenerational exposures, along with special morphologic evaluation of the young mammary gland (OECD 2018; NTP 2011). The mammary gland of the young rat is evaluated for qualitative and quantitative changes that are associated with prenatal and/or postnatal exposure to chemical compounds. These changes detected at a young age then serve as evidence that the test compound is an endocrine disruptor, but more importantly they can serve as an indication of increased risk for mammary gland cancer later in life. There is data linking early exposures to dioxin, atrazine, dimethylbenz-α-anthracene (DMBA), N-nitroso-N-­ methylurea (NMU), and perfluorooctanoic acid, with mammary gland tumors later in life (Fenton 2006; Macon and Fenton 2013). In addition to cancer, other latent effects of early life exposure to toxicants include insufficient lactation during pregnancy/lactation and dilated ducts (Rudel et al. 2011). The mammary glands of adult rats extend from the cervical to the inguinal regions and are composed of lobules of branched ducts with terminal alveoli primarily oriented around six pairs of ventrolateral nipples. The mammary gland of males and females exhibit sexual dichotomy microscopically, and this becomes apparent after PND 35. Male rats have a lobuloalveolar structure, a low number of ducts, and plump vacuolated and often apoptotic epithelium, while the females have a tubuloalveolar structure, a high number of ducts, non-vacuolated epithelium, and rare apoptotic cells (Fig. 16). The structure and sexual dichotomy of the mammary gland are reviewed (Lucas et al. 2007). In young immature male and female rats, the alveolus does not yet exist and, in its place, is the relatively undifferentiated terminal end bud (TEB), which is a highly proliferative structure (>100 μm wide). Although male and female mammary glands are morphologically similar microscopically until about PND 35, there is physiologic differentiation between males and females as early as PND 23. In females, starting at about PND 23, there is an increase in mammary gland growth, in concert with estrogen and progesterone secretion by the ovary. At puberty (around PND 35) in the females, the estrogen surge associated with each proestrus and estrus in each cycle promotes continual ductule branching and transformation of TEBs

820

Catherine A. Picut and Amera K. Remick

Fig. 16 Sexual dichotomy of normal mammary gland. There are morphologic differences between the male rat mammary gland at PND 42 (a) and the female rat mammary gland at PND 42 (b). The male gland is comprised of vacuolated plump epithelial cells forming a lobular structure, while the female gland is comprised exclusively of open ductular structures lined by cuboidal non-vacuolated epithelial cells. H&E. 45×

into alveolar buds and progressively into alveoli (Masso-Welch et  al. 2000). In contrast, the male mammary gland stays as a quiescent-­ appearing, non-proliferative tubular structure until around PND 35, at which time increasing circulating testosterone sets in motion a dramatic morphologic change to a lobuloalveolar structure. The reason the mammary gland of juvenile rats provides a good opportunity to study the effects of endocrine disruptors is because of the terminal end buds (TEBs) in immature glands. These small proliferative undifferentiated structures are most sensitive to the effects of endocrine disruptors, and the window of susceptibility would be during the time when the numbers and/or density of TEBs are maximal (Teitelbaum et al. 2015). Other windows of susceptibility include the prenatal period (GD 15–19), and it is quite common to examine juvenile mammary gland tissue after the test article was given only during gestation (Macon and Fenton 2013; Filgo et al. 2016). Routine histologic assessment is the mainstay for examining mammary gland tissue in nonclinical safety studies. The best way to examine mammary gland tissue is by taking longitudinal sections of the fourth and fifth inguinal mammary glands. Sometimes the glands are placed on fiberboard to fix them flat, prior to processing them and cutting sections at 5 μm thick. The whole mount technique improves the opportunity to examine mammary gland tissue, because it provides quantitative endpoints. With whole mounts, the entire fourth and fifth gland are dissected and mounted onto a slide. Because the entire gland(s) are represented, objective quantitative and/or semi-quantitative assessment of TEBs, alveolar buds, ducts, branching density, convergence of the two glands, and overall growth of epithelium is possible (Filgo et al. 2016; Osborne et al. 2015). Typically, the mammary gland is evaluated at set time points during the immature stages, namely, PND 3, PND 22–28, PND 33, and PND 40–45 (Davis and Fenton 2013). The whole

Pathology of Juvenile Animals

821

mount technique for the juvenile rat mammary gland tissue is detailed (Davis and Fenton 2013). The literature cites many examples of compounds with effects of endocrine disruptor on mammary gland tissue in the juvenile rat. Genistein and methoxychlor (a pesticide that binds to estrogen receptor) are two of these (Bulger and Kupfer 1983; You et  al. 2002). Following prenatal and postnatal exposure to these endocrine disruptors, the total glandular area and the numbers of branch points, lateral buds, and terminal end buds were increased in male rats at PND 22 when compared to non-exposed male rats (You et al. 2002). TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) given GD 15–18 has been shown to delay mammary gland development in male and female rats. TCDD-exposed males contained fewer terminal branches at PND 33, stunted epithelial outgrowth, fewer branches, and decreased bud development, and these findings were gone by PND 70 (Fig. 17). The females exposed to TCDD had fewer TEBs, fewer branches at PND 21, and lack of convergence of the fourth and fifth glands (Filgo et al. 2016). Intraductal hyperplasia is a unique lesion in the peripubertal rat following prenatal exposure to endocrine disruptors early in life. Pre- and perinatal exposure to bisphenol A (i.e., during the fetal, neonatal and infantile periods) results in intraductal hyperplasia by PND 50–90 (Macon and Fenton 2013; Markey et al. 2001; Acevedo et al. 2013). Agents that cause precocious or delayed development of the mammary gland (i.e., estrogen agonists; bisphenol A; TCDD) can render the gland susceptible to late-in-life cancer. It may seem counterintuitive that delayed or stunted growth would render the mammary gland more susceptible to cancer. However, delayed development of rodent mammary glands often means there are fewer carcinogen-sensitive TEBs, but these few TEBs persist and experience prolonged exposure to potential environmental carcinogens (Macon and Fenton 2013). Timing of the insult to the mammary gland is important in lesion development, and metabolism of the compound will determine the susceptible window of time. Prepubertal administration of N-nitroso-N-methylurea (NMU) is most effective in enhancing mammary cancer development, whereas post-pubertal exposure to 12-dimethlybenzyl-α-anthracene (DMBA) is most effective in this regard. In the case of NMU, the prepubertal age susceptibility pattern is attributed to a deficiency of DNA repair enzymes in the immature gland that permit the induction of H-ras mutation in epithelial cells. In the case of DMBA, the post-pubertal age susceptibility is due to the development of enzymes required to metabolize a chemical, such as DMBA, to its carcinogenic form (Davis and Fenton 2013).

822

Catherine A. Picut and Amera K. Remick

Fig. 17 TCDD effect on mammary gland development in PND 21 rat. This panel of images is from the fourth mammary gland of PND 21 female Harlan Sprague Dawley (HSD) rats. The top panel shows the normal mammary gland in whole mount preparation (a) and by routine microscopy (b). In (a), the normal arborization and branching pattern of ducts leading to slightly bulbous terminal end buds can be seen, and the mammary gland is closely approximated to the dark staining inguinal lymph node. In (b), the normal tubuloalveolar duct leading into a densely cellular terminal end bud (TEB) is depicted. The bottom panel shows the appearance of the mammary gland after prenatal exposure to 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). The whole mount preparation (c) demonstrates stunted mammary gland growth characterized by reduced size of the gland, and reduced number and branching of ducts, when compared to the normal gland in (a). Microscopically, the gland is comprised of tubuloalveolar ducts without TEBs (d). (Reprinted with permission from Sage Publishing Co., 2016)

The nature and timing of lesion development gets further complicated by the fact that the same compound will produce different lesions depending upon the timing of exposure. For example, exposure to estrogenic compounds through the perinatal period (when TEBs are first developing) will result in hyperplasia of TEBs, while exposure to the same estrogenic compound during the peripubertal periods (when ducts are primarily branching and elongating) is associated with ductal hyperplasia. The timing of testosterone administration shows this same complicated effect when administered to juvenile animals. Testosterone, an endocrine disruptor that results in delayed development of the mammary gland, has been associated with seemingly opposite effects in different publications, namely, ductal hyperplasia, alveolar hypoplasia, reduced apoptosis in TEBS, increased or decreased number of ter-

Pathology of Juvenile Animals

823

minal ducts or lobules, and accelerated alveolar differentiation. The histologic findings apparently depend on the timing and length of exposure to this and all endocrine disruptors. Strain differences in mammary gland development may account for differences in reported findings. Caution should be exercised before extrapolating information from one strain of rat to another (Filgo et al. 2016; Stanko et al. 2016). Harlan Sprague Dawley rats consistently exhibited more well-developed glands with greater branching and greater mass compared to Charles River Sprague Dawley and Charles River Long Evens rats at multiple time points (PND 25, 33, and 45). The glands of Harlan Sprague Dawley rats are also quicker to show convergence of the fourth and fifth inguinal glands, and 100% of animals have convergence of these two glands by PND 33 (Filgo et al. 2016; Stanko et al. 2016). Other strain differences associated with the number and density of TEBs suggest that the Charles River strains (Sprague Dawley and Long Evans) of rats might be most sensitive to carcinogenic effects at PND 33 (Stanko et al. 2016), while other reports suggest that maximum number/density of TEBs occurs at PND 20–21 (Mandrup et  al. 2012; Russo and Russo 1996). Despite these strain differences, no one strain of rat has been determined yet to be more closely related to the human condition over any other strain. Mammary gland development in rodents can also be affected by prolactin disruption. Prolactin is a hormone that becomes more important in the growth of the mammary gland after puberty. In knockout mice lacking the prolactin receptor, mammary gland development is normal until puberty, but subsequently the ducts branch less frequently and the TEBs persist (Brisken et al. 1999). These knockout mice with persistent TEBs can now be used to further investigate any relationship between prolactin and breast cancer. In studies with dopamine antagonists (which result in increased prolactin levels) administered to rats starting at 6–7 weeks of age (peripubertal), lobuloalveolar hyperplasia with increased secretion due to higher prolactin levels has been found in the female rats (Fig. 18). RANK-RANKL (receptor activator of nuclear factor kappa-B ligand) system is best known for its effect on mediating osteoclast formation, function, and survival (Teitelbaum and Ross 2003). There are a number of therapies that inhibit RANK-RANKL and their pharmaceutical action is to inhibit bone resorption associated with osteoporosis. But RANK-RANKL is also important in lactation of the mammary gland. Lactational mammary gland development is absent in RANK knockout mice (Fata et  al. 2000). Therefore, it is not surprising that therapies that inhibit RANK-­ RANKL (and are intended to treat osteoporosis) have a side effect on the lactating nonhuman primate and consequently a non-­specific and indirect effect on the juvenile monkey. For example, the RANKRANKL-inhibiting therapy, denosumab, has been associated with

824

Catherine A. Picut and Amera K. Remick

Fig. 18 Prolactin-induced mammary gland hyperplasia (post-pubertal). Mammary gland hyperplasia of a post-pubertal female rat exposed to a dopamine antagonist starting at puberty (6 weeks of age) to 4.5 months of age. Dopamine antagonists cause a relatively high level of prolactin, which has an effect primarily on the post-pubertal mammary gland. Notice the densely packed lobuloalveolar glands, lined by plump vacuolated epithelial cells and containing basophilic glandular secretion. H&E 32×

poor mammary gland development in lactating monkeys, resulting in suckling young monkeys with reduced body weight gain and decreased neonatal growth (Fata et al. 2000). The unique feature of the developing mammary gland is the TEB, and this structure must not be confused with hyperplasia. TEBs appear as a bulbous cluster of immature epithelial cells in the mammary gland, are >100 μm wide, are present by PND 14, and are most prevalent in the rat from PND 21 to 28 (Fig. 19). It is also common to find lymphocytic and eosinophilic infiltrates accompanying ductule morphogenesis, and these inflammatory cells are normal and should not be diagnosed as inflammation (Davis and Fenton 2013).

19  Integumentary System: Skin The skin is a standard tissue examined in routine 28-day safety studies and likewise is commonly examined in juvenile toxicity studies. The skin is comprised of keratinized surface epithelium, hair follicles, and dermis with underlying panniculus carnosus muscle. There are some unique features of the skin in juvenile rodents. The surface epithelium of the skin is thick with abundant keratin in the neonatal and infant rat (up to PND 7), and this epithelium thins to normal adult levels by PND 14, while the amount of keratin decreases to normal adult levels by PND 21 (Fig. 20). This is true not only in the rat but also in the mouse; the skin of neonatal mice is three to four cells layers thick and reduces to one to two cell layers with age. Hair follicles undergo active morphogenesis in the

Pathology of Juvenile Animals

825

Fig. 19 Terminal end buds in juvenile mammary gland. Terminal end buds (TEBs) in a PND 28 male rat mammary gland. TEBs appear as solid structures of proliferative cells. These TEBs are a normal structure and eventually develop into the alveolus of the mammary gland. H&E. 40×

Fig. 20 Normal skin development. The skin of a male rat at PND 1 (a), PND 14 (b), and PND 21 (c) is shown. The epithelium is relatively thick (up to five to six cell layers thick) with dense abundant keratin within the first 7 days of life (a). The epithelium thins to normal adult thickness of two to three cell layers by PND 14, at which time the keratin is less abundant and loosely arranged (b). By PND 21 (c), the keratin diminishes in quantity, and the skin is morphologically similar to that of an adult rat. H&E. 30×

first few days at birth, at which time the nine stages of hair follicle development can be examined. When the follicles reach the “adult” ninth stage of follicle growth, these mature follicles begin to cycle through three mature phases: anagen (growth), catagen (regression), and telogen (resting). In young animals, the mature follicles can generally be found in the same stage over large regional areas

826

Catherine A. Picut and Amera K. Remick

called zones. This synchronization into zones during the first two follicle cycles does not occur in the human (Porter 2003). Some features of the juvenile skin can be misinterpreted as lesions. For example, the thickness of the epithelium during the first week of life should not be confused with acanthosis or hyperkeratosis. Rats up to PND 10 may appear to have dandruff or flaking of the skin as a gross pathology observation. This flaking is a normal consequence of hair follicle development and shedding of the relatively hyperkeratotic epithelium. The uniform and predictable mature follicle phase provides opportunity for investigators to design a study targeted to a specific follicle stage. The location and timing of these phases have been published for the rat and the mouse. For example, in the rat, anagen follicles predominate at PND 14 in rats and at PND 12 in mice and are especially prominent on the ventral surface; catagen follicles predominate up to PND 21–28 in the rat, and up to PND 17 in the mouse and are especially prominent on the dorsal surface (Muller-Rover et al. 2001; Staska 2016). There are major differences between mouse and human skin that limit the translational information from adult mouse to human skin (Sundberg et  al. 2005), including the fact that the predominant cycle phase in the adult mouse is telogen. The juvenile rat or mouse may therefore provide a better source of anagen hair follicles. For more detailed information on the stages/phases of follicle development and cycling in the mouse, and determining the proliferation rate and cell death rates, the reader is referred to Sundberg et al. (2005). Although likely included in most juvenile toxicity studies, specific lesions in the juvenile skin are sparsely reported in the literature. The authors found a limited number of publications describing toxic effects of chemicals on the skin or hair follicles of juvenile laboratory animals in nonclinical safety studies. Clomiphene citrate results in disorganization of the basal cell layers, and hyperplasia, hyperkeratosis, and irregular hypertrophy of epithelium in newborn rats (Deveci et al. 2000); anastrozole (a nonsteroidal aromatase inhibitor) results in increased keratinization and hypertrophic epidermal cells in neonatal rats (Akcali et al. 2007); and tamoxifen results in epidermal atrophy and dermal fibrosis (Inaloz et  al. 2002). While there has been recent explosion in the variety and availability of transgenic mouse models and spontaneous mutation mouse models for a variety of allergic, autoimmune, proliferative, and inflammatory human skin conditions, most of the investigative studies involve mature animals, because often it takes time for these skin conditions to develop in transgenic mice (Sundberg et  al. 2016; Raghavan et al. 2000; Sengupta et al. 2010). Spontaneous diseases do affect the skin of young laboratory animals. Histiocytoma is one of the few neoplasms that is observed in routine toxicology studies in beagle dogs and 50% of histiocyto-

Pathology of Juvenile Animals

827

Fig. 21 Demodex in skin of dog. This image shows the presence of Demodex mange mites in the hair follicles of the skin of a young beagle dog. H&E. 10×. (Image courtesy of Dr. George A. Parker)

mas arise in dogs less than 2 years of age. This round cell tumor, composed of often bizarre histiocytic-like cells with a high mitotic rate, is commonly found in the dermis on the head and regresses with time. The hair follicles of young dogs are commonly infested with Demodex mites resulting in granulomatous perifolliculitis, especially if the young dogs are immunosuppressed or stressed by experimental manipulation (Fig. 21). Malignant spontaneously regressing melanomas have been described in Sinclair minipigs (Oxenhandler et  al. 1979; Pathak et al. 2000). These tumors appear from birth and culminate in skin depigmentation after tumor regression.

20  Endocrine System: Thyroid Gland The thyroid gland is routinely examined in young peripubertal animals, since histopathology of the thyroid gland is one of the pathology endpoints in the pubertal development and thyroid function assay. In this assay, females are dosed PND 21–43 and males at PND 21–53 (EDSP 2009). These assays are the only in vivo mammalian assays designed to assess the status of the hypothalamic-­ pituitary-­ gonadal (HPG) and hypothalamic-pituitary-thyroid (HPT) axes. Thyrotoxicants, which generally cause hypothyroidism, result in potential endpoint changes including decreased circulating thyroid hormone (T4), increased thyroid-stimulating hormone (TSH), increased thyroid weight, and increased liver weight or enzyme profile. The increased thyroid weight is generally

828

Catherine A. Picut and Amera K. Remick

associated with microscopic findings of increased height of follicular epithelium, with a corresponding decrease in the amount of colloid, when compared to controls. Thyroid weights are less sensitive than histopathology, and weights generally do not increase upon exposure to a thyrotoxicant for at least 2–4 weeks. Any increased weight associated with higher follicular height may be partially offset by decreased colloid. There could conceivably be a paradoxical decrease in thyroid gland weight, since a reduction in colloid may occur at an earlier time before the follicular cells enlarge (Capen 1997). Therefore, in shorter term studies (less than 28 days) one cannot rely on thyroid weight alone to identify a thyrotoxicant. Histological examination of the thyroid gland is considered the most reliable indicator of thyrotoxicosis. Microscopic evaluation requires a semi-quantitative subjective determination of the height of the follicular epithelial cells and the amount of colloid. The guidelines for the pubertal development and thyroid function assay include a five-point grading scale to evaluate these two parameters. A normal resting thyroid gland of a female is generally C5/ F1, meaning there is maximum amount of colloid (grade C5 on a scale of C1–5) and the follicular cells are flattened to low cuboidal (grade F1 on a scale of F1–5). The grading scales progress in ­opposite directions, and as the follicular height increases (up to F5), the colloid content generally decreases (down to C1). The guidelines attempt to harmonize the grading between laboratories by providing photomicrographs that illustrate the grading scales (Fig. 22) (EDSP 2009). There is substantial variation between different regions of the same section of thyroid gland, and the pathologist should examine the entire section, develop a subjective impression, and try to avoid those expanded follicles with flattened epithelium normally present on the surface of the thyroid gland. In addition to examining microscopically the thyroid gland of peripubertal rats (at PND 43 or 53), sometimes the pathologist may be asked to evaluate the thyroid gland of early postnatal rats, such as on targeted juvenile studies designed to detect potential thyrotoxicants. Knowing normal thyroid morphologic development is important if this is the case. Epithelial height is maximum at gestation day (GD) 18, dramatically declines through PND 4, and then temporarily surges slightly between PND 40 and 60. This surge undoubtedly is due to a surge in steroid hormones, since it is well known that androgens cause heightened sensitivity of the follicular cells to TSH (Banu et al. 2002; Keane et al. 2015). Colloid is first observable at GD 18, increases up to PND 4 (at which time it accounts for 20–30% of the tissue), and slowly increases to maximal level at PND 20 (at which time the colloid accounts for approximately 50% of the glandular mass) (Fig.  23) (Picut et  al. 2016b). Because of the dynamic changes in thyroid morphology from GD 20 to PND 40, grading follicular height and amount of

Pathology of Juvenile Animals

829

Fig. 22 Grading thyroid glands in pubertal development and thyroid function assay. These five images of a thyroid gland at PND 43 in a female rat show the grading scales for follicular height (F1–F5) and for the amount of colloid (C1–C5), as used in the pubertal development and thyroid function assay. Exposure to thyrotoxicants in general causes the follicular epithelial height to increase and the amount of colloid to decrease. These images are for exemplary purposes only. (A) F1/C5; (B) F2/C4; (C) F3/C3; (D) F4/C2; (E) F5/C1. H&E. 30×

colloid (as is done in the pubertal assay) is not quite as objective as it is in the peripubertal animals. In animals less than PND 40, the pathologist needs to determine an appropriate grading scale and cannot automatically rely on the scale used for the pubertal assay (Fig. 24). There is sexual dimorphism in the thyroid gland, which becomes apparent microscopically at around PND 42 (Walling et al. 2016). It is accepted that male thyroid glands (in the adult) have plumper more vacuolated epithelium and lesser amounts of colloid. In fact, the normal grading scale for male thyroid gland, if one was using the guidelines for the pubertal assay, is C4/F2, as opposed to C5/F1 in adult females. While the epithelial cells are plumper and more robust in the males, Picut et al. showed that

830

Catherine A. Picut and Amera K. Remick

Fig. 23 Changes in colloid fraction in developing thyroid gland. This series of normal thyroid glands from rats show the progressive changes in the amount of colloid (colloid fraction) at GD 20, PND 4, and PND 30. The colloid (labeled green) increases abruptly as the thyroid gland matures from one day prior to birth (a, GD 20) to PND 4 (b), and thereafter increases slowly to PND 30 (c). The variations in appearance of the thyroid gland with age underscore the importance of having age-matched controls when evaluating any test article-related effect on the thyroid gland. H&E. 30×

Fig. 24 Grading thyroid glands in PND 13 pups. The grading scale used in the pubertal and thyroid function assay (pubertal assay) has limited us in infantile animals less than PND 21. In these young animals, the thyroid gland normally has a high epithelial height and relatively little colloid, and changes upon exposure to thyrotoxicants may be subtle and limited This panel shows an example of a normal thyroid gland from a PND 13 pup (a), and a thyroid gland with increase in follicular epithelial height and decreased colloid upon exposure to a thyrotoxicant (b). The pathologist must devise his/her own grading scale when evaluating thyroid glands in animals less than PND 21 and cannot rely on grading scales published for use in the pubertal assay. H&E. 30×

the actual height of these follicular cells are generally taller in females than in males when measured by image analysis in control rats at PND 30 and again at PND 50. The taller cells in peripubertal/young adult females determined by image analysis on one hand, and the generally accepted “larger” follicular cells of males microscopically, underscores the “subjective” nature of the microscopic evaluation of follicular height, in general. Pathologist perception of follicular height apparently takes into account additional features including the amount of colloid, and alterations of other parameters such as width of the cell, cytoplasmic appearance of the cells, and size of the nucleus. Routine use of image analysis,

Pathology of Juvenile Animals

831

Table 3 Age of thyroid hormone measurements according to selected agency guidelines Age of rat Guideline

GD 20

PND 4

PND 13

PND 21–22

OECD 443 Extended One- Generation Reproductive Toxicity Test



T4



T4, TSH

OECD 421 Reproduction/Developmental – Toxicity Screening Tests

T4

T4

T4

EPA Guidance for Thyroid Assays (2005)

T3, T4, TSH



T3, T4, TSH

T3, T4, TSH

GD gestational day, PND postnatal day, T3 triiodothyronine, T4 thyroxine, TSH thyroid-stimulating hormone

though relatively time consuming, could eliminate any subjectiveness of thyroid evaluation. Hormone analysis is an important aspect of evaluating the function of the thyroid gland in juvenile animals. Though this topic generally falls under clinical pathology, the topic will be discussed in this subsection. OECD and the EPA have developed guidelines that request the evaluation of serum T3 (triiodothyronine), T4 (thyroxine), and/or TSH (thyroid-stimulating hormone) at gestational age 20 (GD 20), PND 4, PND 13, and/or PND 21 (see Table  3) (OECD 2018, 2016; EPA 2005). Since sufficient quantity of serum is difficult in these young animals, especially at GD 20, PND 4, and PND 13, pooling of blood from multiple animals is generally acceptable. In order to avoid fluctuations due to circadian rhythms or stress, blood should be collected at the same time of the day, preferably the morning hours during basal values, and appropriate steps should be taken to minimize stress. At least one laboratory has developed and validated a T3 and T4 assay on pooled blood in GD 20 fetal rats. This assay is based on liquid chromatography/mass spectroscopy (LC-MS/MS) and can detect serum T3 and T4 levels down to 5 and 125 pg/ml, respectively, which is of necessary and heightened sensitivity required for rat fetal concentrations. Based on a single 50 μl sample, serum T3 and T4 (by LC-MS/MS) and TSH (by radioimmunoassay) can be measured (Coder and Groeber 2018; Groeber et  al. 2018). This heightened capability will undoubtedly bolster further investigation into early detection and identification of thyrotoxicants.

21  Skeletal System: Growth Plate The actively growing epiphyseal plate (growth plate) is a dynamic structure and can be an excellent place to identify toxic effects in a relatively short period of time.

832

Catherine A. Picut and Amera K. Remick

In the growth plate, the number of proliferating chondrocytes and their division rate are key factors in controlling longitudinal bone growth. Changes in the appearance of the developing growth plate can result from direct or indirect action of test articles and may be non-specific or specific. A non-specific effect on the growth plate may occur as an indirect effect that test articles may have on food consumption. It has been shown that rats with dietary restriction may have non-specific decreased chondrocyte proliferation in the proliferative zone with decreased transformation of these proliferative chondrocytes into hypertrophic chondrocytes (Svensson et al. 1985; Heinrichs et al. 1997). This non-specific retardation of growth manifests as decreased tibia length, which is one non-­ microscopic endpoint measured at weaning. When this occurs, one might see loss or paucity of trabecular and cortical bone microscopically in the growth-retarded juvenile rat (Allais et al. 2009). Other indications of growth retardation (e.g., delayed incisor eruption or eye opening) may help confirm that these long bone changes are non-specific effects secondary to growth retardation. The effect of methylphenidate on bone growth underscores the importance of identifying non-specific effects on the bone parameters rather than attributing them to specific toxicity. Methylphenidate, a common treatment for attention deficit-­ hyperactivity syndrome (ADHD), has effects on growth and endocrine function in young rats (Greeley Jr. and Kizer 1980); and children and teenagers taking stimulant drugs to treat ADHD have lower bone density than peers. It has recently been shown that the change in height and/or bone density in children and teenagers is secondary to changes in energy balance due to stimulant-related appetite suppression, rather than to any specific toxic effect on the growth plate itself (Poulton et al. 2016). While decreased growth of long bones is a non-specific effect of growth retardation, decreased growth of long bones can also be due to specific toxic effects on proliferating chondrocytes. Antimitotic drugs may directly damage chondrocytes in the proliferating zone. Some agents interfere with vascularization of the growth plate (which is required for endochondral ossification), leading to morphologic defects in the growth plate. Vascular endothelial growth factor (VEGF) is produced by hypertrophic chondrocytes and is required to promote vascular ingrowth into the growth plate. VEGF inhibitors are commonly developed as anti-­ cancer therapies, and these VEGF inhibitors result in disrupted angiogenesis and thickened epiphyseal growth plates with expansion of the hypertrophic zone (Ytrehus et  al. 2007). While this lesion may occur at any age, the effect on the growth plate of the juvenile animal occurs more quickly and more dramatically (Fossey et  al. 2016). Some agents that have antiangiogenic properties include monoclonal antibodies and small molecule inhibitors of

Pathology of Juvenile Animals

833

VEGF, inhibitors of matrix metalloproteinases, inhibitors of TGFβ signaling (Petersen et  al. 2008), and tyrosine kinase inhibitors (Patyna et al. 2008). Methotrexate (MTX) is the most commonly used antimetabolite in pediatric cancer treatment and it causes bone growth defects in children. While it targets the growth plate to a certain degree, it also damages the bone itself (Swierkot et al. 2015). Microscopically, the lesion at the growth plate in young rats exposed to methotrexate is a thinning of the growth plate, an increase in the hypertrophic zone, and increased number of fat cells in the marrow cavity. Due to the growth plate dysfunction, there is decrease in primary spongiosa in adjacent metaphyseal bone (Fan et al. 2011). In 4-week-old Wistar rats, doxorubicin was administered for 9 weeks (into adulthood), and this produced a significant decrease in height of the proliferating zone of the growth plate along with loss of bone marrow cellularity in the metaphysis (van Leeuwen et al. 2003). Diphenylhydantoin or sodium valproate treatment of young rats for 6–7 weeks also showed reduced chondrocyte numbers in the femoral growth plate as well as a reduction in cartilage thickness at the ends of long bones and in the mandible, suggesting these drugs interfere with regulation of chondrocyte proliferation and matrix synthesis (Robinson et al. 1988). While the well-known effects of bisphosphonate are due to reduced bone remodeling, the effects of bisphosphonates have also been seen in the growth plate. In juvenile 12-week-old rabbits, bisphosphonates caused disrupted chondrocytes and chondrocytes failed to align with a reduction in proliferation and/or increased apoptosis (Smith et al. 2005). In young rats, one toxic lesion may be premature closure of the growth plate, and this appears as a replacement of cartilage in the growth plate with mineralized bone. For example, administration of anticoagulants to young rats causes physeal hemorrhage and then premature closure (Hahnel et  al. 1978). This lesion is similar to fetal warfarin syndrome in humans. Unlike in humans, the rat physis does not normally close (or ossify) until old age (about 2 years of age), and therefore, it is not necessary to use a juvenile rat to identify premature closure of the growth plate. In fact, premature ossification of the growth plate was detected in rats treated with warfarin at 8  months of age (Price et  al. 1982). Nevertheless, the juvenile growth plate even in rats is more dynamic and active, and toxic effects would be more apparent if performed in young dynamic tissue. Spontaneous incidence of focal degenerative findings in young physeal growth plates may complicate nonclinical toxicity studies. Young beagle dogs and cynomolgus monkeys may have focal necrosis, fracture, and degenerative changes at the femoral growth plate (Yamasaki 1995; Chamanza et al. 2010). Focal collections or

834

Catherine A. Picut and Amera K. Remick

islands of chondrocytes have been reported in the femoral ­metaphysis in young rats (6–12 weeks of age), and these have been published as focal chondrocyte dysplasia (Shigami et  al. 2016). These foci probably represent remnants of the hyaline cartilage present at the primary ossification center in the shaft of the long bones. They may persist more commonly in rodents due to the minimal bone remodeling that occurs in these species. One finding that should not be interpreted as a lesion is the artifactual separation of the epiphysis and physis from the metaphysis in young animals. This may occur even with routine handling and should be distinguished from separation that can occur due to a treatment-related decrease in primary spongiosa and/or ossification.

22  Skeletal System: Bone Despite its apparent inactivity when examined microscopically, bone represents a dynamic remodeling process that exists in a balance between resorption and formation. Remodeling is performed by the basic multicellular unit (BMU) of bone which consists of osteoclasts, osteoblasts, and matrix. Matrix consists of the unmineralized osteoid and mineralized bone, and the two are separated by a resorption or a cement line. Since many potential therapeutics are directed to tip the balance in favor of formation (e.g., to treat menopausal osteoporosis), a good animal model for bone remodeling is necessary. However, bone remodeling in long bones of rodents is minimal. The minimal amount of bone remodeling that does occur happens not by intracortical remodeling of Haversian systems as in humans, but by a combination of periosteal bone resorption and endosteal intramembranous bone formation on secondary spongiosa. This endosteal formation occurs in the metaphysis, close to the growth plate, in the area of bone referred to as the cut-back zone. Surprisingly little work is published on the bone of the juvenile laboratory animals. The animal model most typically used for therapies directed at bone remodeling is the ovariectomized adult female mouse, since the low estrogen in this model will activate an otherwise inactive bone remodeling process (Thompson et  al. 1995). Using the juvenile rat at a time of fast growth (around PND 21) may arguably be advantageous. Only a short a 7-day treatment with bisphosphonates (compounds that inhibit RANK-­ RANKL) was necessary to yield bones that demonstrated large increases in metaphyseal density. The relatively small body weights of these animals means less test compound is needed and there is increased ability to detect changes in a short period of time (McHugh et al. 2003). Weanling rats have been successfully used to study the effects of chronic alcohol consumption and binge

Pathology of Juvenile Animals

835

drinking on the growing bone (Sampson et al. 1999; Sampson and Spears 1999). The expected microscopic effects of bisphosphonates are to find widened osteoid seams and increased bone in the metaphysis, both of which indicate the bone remodeling process is tipped in favor of bone formation. With this increased bone formation, the edges of the bony trabeculae are commonly undulating with enlarged osteoclasts with increased numbers of nuclei and occasionally apoptosis of osteoclasts. With the impaired osteoclastic activity resulting from bisphosphonates, one may also notice delayed or inhibited molar tooth eruption in young rats (Grier and Wise 1998). In young rabbits treated with bisphosphonates, such as manitronate, one can find characteristic zebra stripes (parallel radiodense lines) in the tibia on radiographic examination (Smith et al. 2005). These radiodense lines represent sclerotic bands where bone fails to resorb (Chakraborty et al. 2017). Some other effects of bisphosphonates that can be seen in sections of bone include decreased marrow space, decreased marrow hematopoietic tissue, and increased extramedullary hematopoiesis. This was present in cynomolgus monkey infants exposed to denosumab during gestation (Bussiere et al. 2013; Boyce et al. 2014). Although typically the femoro-tibial joint and the sternum are examined in adult and juvenile animals, it may be necessary to look beyond these areas of the skeleton in juvenile animals if one expects an effect on the developing skeleton. Malocclusions of incisors have been associated with skull abnormalities associated with reduced fetal testosterone production associated with fetal exposure to dipentyl phthalates (Gray et al. 2016). These lesions (skull abnormalities and dental malocclusion) are lesions seen in the postnatal rat, even though they arose from prenatal toxicity. There are some spontaneous minor skeletal malformations of the bone in laboratory animals that could affect the interpretation of juvenile animal studies. Some minor malformations affecting 1–5% of mice include curly tail, sternebral asymmetry, unossified phalanges, reduced ossification of the 13th rib, and bent ribs. A common skeletal variant that may affect up to 35% of rodents include supernumerary ribs and wavy ribs (Stump et al. 2012).

23  Skeletal System: Articular Cartilage There are a few well-known toxic effects on the articular cartilage. Quinolone-induced changes in the articular cartilage are well known and are seen in pigs, dogs, horses, cattle, cats, and rats. Young animals, especially beagle dogs, are highly sensitive to quinolone carboxylic acid compounds and analogs such as cinoxacin, nalidixic, pipemidic, norfloxacin, and ciprofloxacin. They develop degenerative lesions in the articular cartilage, especially the distal

836

Catherine A. Picut and Amera K. Remick

femur, and these degenerative changes are referred to as osteochondrosis (OCD) (Kato and Onodera 1988; Stahlmann et  al. 1997). The cartilage develops bullae with detachment of the articular cartilage, synovial fluid becomes tinged with blood, and histologically there is loss of matrix and cavitation with focal necrosis of subchondral bone. Reactive synovial changes are prominent. The pathogenesis of these quinolone compounds involves a toxic action on the cartilage or on the metabolism of the chondrocyte in young animals. This compound is proposed to cause mitochondrial dysfunction in the chondrocyte resulting in decreased synthesis of proteoglycans and collagen (Kato et al. 1995). These lesions may be seen acutely, as soon as 2  days following subcutaneous injections in 24-day-old rats (Pfister et al. 2007). The young age of the animal is critical in order for this lesion to develop; the OCD lesion formed only when drug treatment began at 4 weeks of age but not at 8 weeks of age (Kato and Onodera 1988). As similar changes can be induced by magnesium deficiency, it has been suggested that cartilage damage may be related to effects of magnesium (Gough et al. 1979, 1996; Forster et al. 1996). Quinolone antibacterial agents also can lead to lesions in the tendons. Perfloxacin and ofloxacin induced edema and mononuclear infiltration in the inner sheath of inner Achilles tendons in rats and only when administered to 4-week-old rats (Kato et al. 1995).

24  Special Sense Organs: Eye The eye is poorly developed at birth in rats, and the animal is born with fused eyelids. Eyelids open at around PND 12–14. The cornea is composed of a surface epithelium lined by Bowman’s membrane, a middle matrix of type I collagen, and a deep Descemet’s membrane. The cornea is well-formed by the time the eyelid opens at PND 12–14, even though from PND 14 to 21 the corneal epithelium continues to proliferate. The lens undergoes considerably postnatal development in size and shape, starting as a flattened disc that becomes a sphere to occupy 2/3 of the internal volume of the eye by PND 16. The retina undergoes most of its development in the postnatal period. At birth, the ganglion cells have differentiated and are in position, but there is little to no development of other cell layers. After birth, the other cell types (amacrine, Muller, rods, cones, and horizontal cells) develop from the neuroblast layer and migrate into position. By PND 21, the cell layers of the retina reach adult proportion. A delay in eye opening is a non-specific measure of poor health and is used as a developmental landmark. Other physical developmental landmarks in rats include pinna detachment, tooth eruption, vaginal opening, and preputial separation. Effects on the timing of eye opening (or other physical landmarks) may be

Pathology of Juvenile Animals

837

­ on-­specific due to effects on body weight gain rather than to n direct toxic effects on the eye. Some windows of susceptibility have been identified in the structures of the rat eye. Although the cornea is morphologically developed by the time the eyelid opens, it has a window of susceptibility to toxic compounds from PND 16 to 21. This window of susceptibility has been noted with the combined exposure to ketamine hydrochloride and xylazine. This combination administered to rat pups between the second and third weeks of life produces a superficial stromal calcium deposition. Either anesthetic alone does not incite this change (Guillet et al. 1988). Corneal opacities have also been reported in the young eye following exposure of the animals to antidepressants (Vonvoigtlander et  al. 1982), capsaicin (Shimizu et al. 1984), morphine sulfate (Fabian et al. 1967), and 1-alpha-acetylmethadol (Roerig et al. 1980). The greater susceptibility of juvenile animals to centrally acting drugs suggests that the effect is mediated through the evolving innervation, which may be precipitated by increased light exposure when the eyelids open (Guillet et  al. 1988). The pathogenesis of lesion development is still not clear. The juvenile lens also shows heightened sensitivity to develop cataracts to agents when compared to the adult lens. Kalydeco (Ivacaftor) was approved in 2012 for patients with cystic fibrosis from 6 years of age and older. However, cataracts were reported in juvenile rats treated from PND 7 to 35 at dosages >10 mg/kg/day (Baldrick 2010, 2013). Another lesion affecting the lens of the juvenile rat is retrolental fibroplasia which can be seen with hyper-­ oxygen therapy in the young rat (Ashton et al. 1953). Antimitotic agents, such as paclitaxel, can cause cataracts and dysplasia of the lens and retina in young animals when given during the first 2 postnatal weeks. This is the time corresponding to a period of rapid development of the lens and retina (Fig. 25). As in rats, dogs are also born with fused eyelids with eyelid opening occurring approximately 2 weeks postnatally, and development of the anterior chamber and retina continuing postnatally. Anterior chamber elements, such as the uveal and corneoscleral trabecular meshworks, continue to develop until they reach adult maturation around 8 weeks of age (Cook 2013). Retinal development in the dog at birth is equivalent to 3–4 months of gestation in humans (Cook 2013). Retinal development and differentiation proceeds from inner to outer retinal layers and from the central to the peripheral retina, and the retinal layers are developed between 16 and 40 days of age (Cook 2013). The canine retina is generally considered histologically and functionally mature by 6  weeks of age (DeLahunta and Glass 2009). Exposure to toxicants prior to complete development and differentiation of the retina can lead to disruption of the normal retinal architecture and apparent vacuolation of the retina (Fig. 26). Retinal toxicity occurring during this

838

Catherine A. Picut and Amera K. Remick

Fig. 25 Lenticular and retinal dysplasia in PND 21 rat. An antimitotic agent that disrupts microtubules given to a rat for the first 3  weeks of life results in numerous malformation of the eye. The lens is poorly formed and the retina is thickened with loss of normal architecture. These eyes were also severely microophthalamic. H&E. 5.4×

Fig. 26 Retinal toxicity (vacuolation) in a juvenile beagle dog. (a) Normal retina from a postnatal day (PND) 35 beagle dog from a control group for age-matched comparison. (b) Vacuolated retina from a PND 35 beagle dog treated with an unspecified orally dose test article from PND 7 to 35. There is vacuolation of the ganglion cell and optic fiber layers of the retina as well as of the inner nuclear layer of the retina in the treated juvenile animal. Toxicity studies on the same test article conducted in mature dogs did not reveal any retina toxicity; the retinal finding was limited to studies with dosing prior to complete retinal maturation. H&E. 20×

sensitive period of development will not be observed if exposure occurs post-retinal maturation.

25  Clinical Pathology There is limited published information on clinical pathology of the juvenile rat: Few reports provide complete clinical pathology profiles for the neonatal or developing young rat. There are however a few generalities that can be gleaned from publications. Pups have physiological anemia in the early postnatal period. Compared to

Pathology of Juvenile Animals

839

adults, the neonate is reported to have relatively low hemoglobin concentration, low red blood cell counts, low packed cell volume, low mean corpuscular hemoglobin concentration (MCHC), higher reticulocytes, higher mean corpuscular volume (MCV), and lower white blood cell (WBC) count. Activated partial thromboplastin time (APTT) and prothrombin time (PT) are relatively prolonged in the neonate (FDA 2006). In dogs, similar hematology profiles are observed. Young dogs have higher reticulocyte counts at birth and higher MCV during the first month of life, and the hematology values fall within the normal adult range by 3 months of age. Regarding serum chemistry parameters, specific patterns of changes are revealed. In rats and dogs, cholesterol, bilirubin, and blood urea nitrogen (BUN) decrease with age. Alanine aminotransferase (ALT) increases with age. In rats, ALT increases 300% from birth to 5  weeks of age (Beck et  al. 2012). Serum alkaline phosphatase (ALP) generally decreases with age as the animal approaches maturity. ALP profiles can be difficult to interpret, however, because ALP has sources from the intestinal tract and bone. Therefore, while a decrease in serum ALP may reflect normal reduction in bone ALP as the animal reaches maturity, it also may indicate pathological reduction in the intestinal component if there is reduced food consumption. Interpretation of ALP levels, therefore, may be complex especially if the test article impedes bone growth and maturation (of bone) or influences food consumption. In monkeys, reference hematologic and biochemical parameters are published for sexually immature/juvenile animals (ages 19–28  months) vs peripubertal to sexually mature animals (ages 39–72  months). The publications reveal that there are few age-­ related changes after 19–20 months of age, except that the younger cohort of animals had slightly higher serum triglycerides, lower alanine aminotransferase, and lower MCV when compared to adult counterparts (Choi et  al. 2016). No publication could be found that addresses the clinical pathology profiles of neonatal to infant monkeys. Clinical pathology endpoints in a juvenile study are limited by the technical difficulty for obtaining adequate samples for analysis particularly in the case of rodents. Juvenile rats are simply too small to undergo repeated blood sampling by conventional methods that collect 200–300 μl of blood at each time point. Capillary microsampling (CMS) is one technique used to obtain blood from juvenile rats during a study (Chapman et al. 2014; Jonsson et al. 2012). For PND 4 and PND 10 pups, the submandibular vein was reported to be the best location, and for PND 17 pups, the tail vein was recommended (Niu et  al. 2016). CMS allows the investigator to collect about 30 μL of blood using EDTA-treated hematocrit tubes. By using capillary microsampling, it is feasible

840

Catherine A. Picut and Amera K. Remick

to collect multiple daily blood samples from PND 4, 10, and 17 rats without disrupting key endpoints. CMS may, however, cause an acute and temporary drop in red cells, hemoglobin, or hematocrit with increased reticulocytes when done three times a day, but these changes will correct within 7 days (Niu et al. 2016). References Acciani M, Kopp C, Palmer JL, Davis T, Picut C (2016) Effects of immersion fixation on post-­ mortem rat brain. 55th Annual meeting of Society of Toxicology, New Orleans Acevedo N, Davis B, Schaeberle CM, Sonnenschein C, Soto AM (2013) Perinatally administered bisphenol a as a potential mammary gland carcinogen in rats. Environ Health Perspect 121(9):1040–1046. https://doi. org/10.1289/ehp.1306734 Adikwu E, Bokolo B (2017) Possible hepatotoxic consequence of nevirapine use in juvenile albino rats. J Pharm Pharmacog Res 5(4):217–226 Agrawal AK, Shapiro BH (2005) Neonatal phenobarbital imprints overexpression of cytochromes P450 with associated increase in tumorigenesis and reduced life span. FASEB J  19(3):470–472. https://doi.org/10.1096/ fj.04-2550fje Akcali C, Inaloz S, Karakok M, Demirtas OC, Kirtak N, Inaloz S (2007) The effects of anastrozole on neonatal rat skin. Eur J  Gynaecol Oncol 28(6):534–536 Allais L, Condevaux F, Fant P, Barrow PC (2009) Juvenile toxicity of cyclosporin in the rat. Reprod Toxicol 28(2):230–238. https://doi. org/10.1016/j.reprotox.2009.04.012 Anand KJ, Soriano SG (2004) Anesthetic agents and the immature brain: are these toxic or therapeutic? Anesthesiology 101(2):527–530 Anway MD, Cupp AS, Uzumcu M, Skinner MK (2005) Epigenetic transgenerational actions of endocrine disruptors and male fertility. Science 308(5727):1466–1469. https://doi. org/10.1126/science.1108190 Ashton N, Ward B, Serpell G (1953) Role of oxygen in the genesis of retrolental fibroplasia; a preliminary report. Br J Ophthalmol 37(9):513–520 Bailey GP, Marien D (2011) The value of juvenile animal studies “What have we learned from preclinical juvenile toxicity studies? II”. Birth Defects Res B Dev Reprod Toxicol 92(4):273– 291. https://doi.org/10.1002/bdrb.20328 Baldrick P (2010) Juvenile animal testing in drug development  – is it useful? Regul Toxicol Pharmacol 57(2–3):291–299. https://doi. org/10.1016/j.yrtph.2010.03.009 Baldrick P (2013) The evolution of juvenile animal testing for small and large molecules. Regul

Toxicol Pharmacol 67(2):125–135. https:// doi.org/10.1016/j.yrtph.2013.07.009 Banu SK, Govindarajulu P, Aruldhas MM (2002) Developmental profiles of TSH, sex steroids, and their receptors in the thyroid and their relevance to thyroid growth in immature rats. Steroids 67(2):137–144 Barone S Jr, Stanton ME, Mundy WR (1995) Neurotoxic effects of neonatal triethyltin (TET) exposure are exacerbated with aging. Neurobiol Aging 16(5):723–735 Barrow PC (2007) Toxicology testing for products intended for pediatric populations. In: Slikker W, Chang LW (eds) Handbook of developmental neurotoxicology. Academic, San Diego, pp 403–426 Beck MJ, Padgett EL, Parker GA, Maginnis GM, Toot JD, Varsho BJ, Varsho JS (2012) Nonclinical juvenile toxicity testing. In: Hood RD (ed) Developmental and reproductive toxicology: a practical approach, 3rd edn. Informa Healthcare, New York, pp 302–345 Bode G, Clausing P, Gervais F, Loegsted J, Luft J, Nogues V, Sims J, Steering Group of the RP (2010) The utility of the minipig as an animal model in regulatory toxicology. J  Pharmacol Toxicol Methods 62(3):196–220. https://doi. org/10.1016/j.vascn.2010.05.009 Bolon B, Garman R, Jensen K, Krinke G, Stuart B (2006) A ‘best practices’ approach to neuropathologic assessment in developmental neurotoxicity testing  – for today. Toxicol Pathol 34(3):296–313 Bolton SJ, Perry VH (1998) Differential blood-­ brain barrier breakdown and leucocyte recruitment following excitotoxic lesions in juvenile and adult rats. Exp Neurol 154(1):231–240. https://doi.org/10.1006/exnr.1998.6927 Bondy SC, Campbell A (2005) Developmental neurotoxicology. J  Neurosci Res 81(5):605– 612. https://doi.org/10.1002/jnr.20589 Boucherat O, Morissette MC, Provencher S, Bonnet S, Maltais F (2016) Bridging lung development with chronic obstructive pulmonary disease. Relevance of developmental pathways in chronic obstructive pulmonary disease pathogenesis. Am J Respir Crit Care Med 193(4):362–375. https://doi.org/10.1164/ rccm.201508-1518PP

Pathology of Juvenile Animals Boyce RW, Varela A, Chouinard L, Bussiere JL, Chellman GJ, Ominsky MS, Pyrah IT (2014) Infant cynomolgus monkeys exposed to denosumab in utero exhibit an osteoclastpoor osteopetrotic-­like skeletal phenotype at birth and in the early postnatal period. Bone 64:314–325. https://doi.org/10.1016/j. bone.2014.04.002 Branes D, Altman J  (1973) Effects of different schedules of early undernutrition on the preweaning growth of the rat cerebellum. Exp Neurol 38(3):406–419 Brisken C, Kaur S, Chavarria TE, Binart N, Sutherland RL, Weinberg RA, Kelly PA, Ormandy CJ (1999) Prolactin controls mammary gland development via direct and indirect mechanisms. Dev Biol 210(1):96–106. https:// doi.org/10.1006/dbio.1999.9271 Brown DL, Walling BE, Mattix ME (2016) Urinary system. In: Parker GA, Picut CA (eds) Atlas of histology of the juvenile rat. Elseveir, San Diego, pp 395–398 Bulger WH, Kupfer D (1983) Estrogenic action of DDT analogs. Am J Ind Med 4(1–2):163–173 Burri PH (1997) Structural aspects of prenatal and postnatal development and growth of the lung. In: McDonald JA (ed) Lung growth and development. M. Decker, New York, pp 1–35 Bussiere JL, Pyrah I, Boyce R, Branstetter D, Loomis M, Andrews-Cleavenger D, Farman C, Elliott G, Chellman G (2013) Reproductive toxicity of denosumab in cynomolgus monkeys. Reprod Toxicol 42:27–40. https://doi. org/10.1016/j.reprotox.2013.07.018 Capen CC (1997) Mechanistic data and risk assessment of selected toxic end points of the thyroid gland. Toxicol Pathol 25(1):39–48 Carney EW, Zablotny CL, Marty MS, Crissman JW, Anderson P, Woolhiser M, Holsapple M (2004) The effects of feed restriction during in utero and postnatal development in rats. Toxicol Sci 82(1):237–249 Chakraborty PP, Biswas SN, Patra S, Santra G (2017) “Zebra stripe” sign and “bone in bone” sign in cyclical bisphosphonate therapy. J  Clin Diagn Res 11(2):RJ01–RJ02. https://doi. org/10.7860/JCDR/2017/24349.9177 Chamanza R, Marxfeld HA, Blanco AI, Naylor SW, Bradley AE (2010) Incidences and range of spontaneous findings in control cynomolgus monkeys (Macaca fascicularis) used in toxicity studies. Toxicol Pathol 38(4):642–657 Chapman K, Chivers S, Gliddon D, Mitchell D, Robinson S, Sangster T, Sparrow S, Spooner N, Wilson A (2014) Overcoming the barriers to the uptake of nonclinical microsampling in regulatory safety studies. Drug Discov Today 19(5):528–532. https://doi.org/10.1016/j. drudis.2014.01.002 Charil A, Laplante DP, Vaillancourt C, King S (2010) Prenatal stress and brain development.

841

Brain Res Rev 65(1):56–79. https://doi. org/10.1016/j.brainresrev.2010.06.002 Chen H, Sun J, Buckley S, Chen C, Warburton D, Wang XF, Shi W (2005) Abnormal mouse lung alveolarization caused by Smad3 deficiency is a developmental antecedent of centrilobular emphysema. Am J Physiol Lung Cell Mol Physiol 288(4):L683–L691. https://doi. org/10.1152/ajplung.00298.2004 Choi K, Chang J, Lee MJ, Wang S, In K, Galano-­ Tan WC, Jun S, Cho K, Hwang YH, Kim SJ, Park W (2016) Reference values of hematology, biochemistry, and blood type in cynomolgus monkeys from cambodia origin. Lab Anim Res 32(1):46–55. https://doi.org/10.5625/ lar.2016.32.1.46 Coder P, Groeber EA (2018) Quantitative methods for monitoring thyroid hormones in late-­ fetal and early-neonatal rat specimens. 37th Annual meeting of the Society of Toxicologic Pathology, Indianapolis, 16–21 June 2018 Coe S, Vidal J, Nelson K (2017) Incidence and age association of increased stromal collagen of testes in cynomolgus monkeys. 37th Annual meeting of Society of Toxicologic Pathologists, Montreal Cohen JA, Stommel EW (2015) Demyelinating diseases of the peripheral nerves. In: Tubbs RS, Rizk E, Shoja M, Loukas M, Spinner RJ, Barabaro N (eds) Nerves and nerve injuries, vol 2. Elsevier, San Diego, pp 895–934 Comereski CR, Bregman CL, Buroker RA (1987) Testicular toxicity of N-methyltetrazolethiol cephalosporin analogs in the juvenile rat. Fundam Appl Toxicol 8(2):280–289 Cook CS (2013) Ocular embryology and congenital malformations. In: Gelatt KN, Gilger BC, Kern TJ (eds) Veterinary ophthalmology. Wiley, Ames Crabbs TA, Frame SR, Laast VA, Patrick DJ, Thomas J, Zimmerman B, Hardisty JF (2013) Occurrence of spontaneous amphophilic-­ vacuolar renal tubule tumors in sprague-­ dawley rats from subchronic toxicity studies. Toxicol Pathol 41(6):866–871. https://doi. org/10.1177/0192623312467523 Creasy D (2012) Reproduction of the rat, mouse, dog, non-human primate, and minipig. In: McInnes EF (ed) Background lesions in laboratory animals. A color atlas. Elseveir, New York, pp 109–111 CVemireddi V, Creasy D (2017) Exacerbation of seminiferous tubule dilatation by fibrous hypoplasia in cynomolgus monkey testes. 37th Annual meeting of Society of Toxicologic Pathologists, Montreal Davis B, Fenton S (2013) Mammary gland. In: Haschek WM, Rousseaux CG, Wallig MA (eds) Haschek and Rousseaux’s handbook of toxicologic pathology. Elsevier Inc./Academic, New York

842

Catherine A. Picut and Amera K. Remick

de Groot DM, Hartgring S, van de Horst L, Moerkens M, Otto M, Bos-Kuijpers MH, Kaufmann WS, Lammers JH, O’Callaghan JP, Waalkens-Berendsen ID, Pakkenberg B, Gundersen HG (2005) 2D and 3D assessment of neuropathology in rat brain after prenatal exposure to methylazoxymethanol, a model for developmental neurotoxicity. Reprod Toxicol 20(3):417–432 De Schaepdrijver L, Rouan MC, Raoof A, Bailey GP, De Zwart L, Monbaliu J, Coogan TP, Lammens L, Coussement W (2008) Real life juvenile toxicity case studies: the good, the bad and the ugly. Reprod Toxicol 26(1):54–55. https:// doi.org/10.1016/j.reprotox.2008.04.002 DeLahunta A, Glass E (2009) Visual system. In: DeLahunta A, Glass E (eds) Veterinary neuroanatomy and clinical neurology, 3rd edn. Saunders Elsevier, St. Louis, pp 389–432 Deveci E, Inaloz HS, Inaloz SS, Unal B (2000) Effects of clomiphene citrate on neonatal rat skin. Clin Exp Obstet Gynecol 27(3–4):238–240 Dietert RR (2006) Developmental immunotoxicity testing and protection of children’s health. PLoS Med 3(8):e296 Dietert RR, Etzel RA, Chen D, Halonen M, Holladay SD, Jarabek AM, Landreth K, Peden DB, Pinkerton K, Smialowicz RJ, Zoetis T (2000) Workshop to identify critical windows of exposure for children’s health: immune and respiratory systems work group summary. Environ Health Perspect 108(Suppl 3):483–490 Downes N (2012) Juvenile toxicity: are we asking the right questions? Toxicol Pathol 40(5):830–837 Downes N, Mullins P (2014) The development of myelin in the brain of the juvenile rat. Toxicol Pathol 42(5):913–922 EDSP test guidelines OPPTS 890.1450 Pubertal development and thyroid function in intact juvenile/peripubertal female rats (2009) U.  S. Environmental Protection Agency. http://www. regulations.gov/#!documentDetail;D=EPAHQ-OPPT-2009-0576-0009. EMA (2008) Guideline on the need for non-­clinical testing in juvenile animals of pharmaceuticals for paediatric indications. European Medicines Agency, London, UK EPA (1998) Health Effects Test guidelines: OPPTS 870.6300. Developmental Neurotoxicity Study Washington, D.C. EPA (2005) Guidance for thyroid assays in pregnant animals, fetuses and postnatal animals, and adult animals. Office of Pesticide Programs, Heath Effects Division Washington, D.C. EPA (2011) Toxicological review of trichloroethylene (CASRN 79-01-6) in support of summary information on the integrated risk information system. Washington, D.C.

Fabian RJ, Bond JM, Drobeck HP (1967) Induced corneal opacities in the rat. Br J  Ophthalmol 51(2):124–129 Fan C, Georgiou KR, King TJ, Xian CJ (2011) Methotrexate toxicity in growing long bones of young rats: a model for studying cancer chemotherapy-induced bone growth defects in children. J  Biomed Biotechnol 2011:903097. https://doi.org/10.1155/2011/903097 Fata JE, Kong YY, Li J, Sasaki T, Irie-Sasaki J, Moorehead RA, Elliott R, Scully S, Voura EB, Lacey DL, Boyle WJ, Khokha R, Penninger JM (2000) The osteoclast differentiation factor osteoprotegerin-ligand is essential for mammary gland development. Cell 103(1):41–50 Fattah S, Augustijns P, Annaert P (2015) Age-­ dependent activity of the uptake transporters Ntcp and Oatp1b2  in male rat hepatocytes: from birth till adulthood. Drug Metab Dispos 43(1):1–8. https://doi.org/10.1124/ dmd.114.059212 FDA (2006) Guidance for industry: nonclinical safety evaluation of pediatric drug products. U.S. Food and Drug Administration, Rockville Fenton SE (2006) Endocrine-disrupting compounds and mammary gland development: early exposure and later life consequences. Endocrinology 147(6 Suppl):S18–S24. https://doi.org/10.1210/en.2005-1131 Fenton SE (2009) The mammary gland: a tissue sensitive to environmental exposures. Rev Environ Health 24(4):319–325 Fernandez E, Perez R, Hernandez A, Tejada P, Arteta M, Ramos JT (2011) Factors and mechanisms for pharmacokinetic differences between pediatric population and adults. Pharmaceutics 3(1):53–72. https://doi.org/10.3390/ pharmaceutics3010053 Filgo AJ, Foley JF, Puvanesarajah S, Borde AR, Midkiff BR, Reed CE, Chappell VA, Alexander LB, Borde PR, Troester MA, Bouknight SA, Fenton SE (2016) Mammary gland evaluation in juvenile toxicity studies: temporal developmental patterns in the male and female Harlan Sprague-Dawley rat. Toxicol Pathol 44(7):1034–1058. https://doi. org/10.1177/0192623316663864 Follett PL, Rosenberg PA, Volpe JJ, Jensen FE (2000) NBQX attenuates excitotoxic injury in developing white matter. J  Neurosci 20(24):9235–9241 Forster C, Kociok K, Shakibaei M, Merker HJ, Vormann J, Gunther T, Stahlmann R (1996) Integrins on joint cartilage chondrocytes and alterations by ofloxacin or magnesium deficiency in immature rats. Arch Toxicol 70(5):261–270 Fossey S, Vahle J, Long P, Schelling S, Ernst H, Boyce RW, Jolette J, Bolon B, Bendele A, Rinke M, Healy L, High W, Roth DR, Boyle

Pathology of Juvenile Animals M, Leininger J  (2016) Nonproliferative and proliferative lesions of the rat and mouse skeletal tissues (bones, joints, and teeth). J Toxicol Pathol 29(3 Suppl):49S–103S. https://doi. org/10.1293/tox.29.3S-2 Fouser L, Avner ED (1993) Normal and abnormal nephrogenesis. Am J Kidney Dis 21(1):64–70 Frazier KS (2017) Species differences in renal development and associated developmental nephrotoxicity. Birth Defects Res 109(16):1243–1256. https://doi.org/10.1002/bdr2.1088 Frazier KS, Seely JC, Hard GC, Betton G, Burnett R, Nakatsuji S, Nishikawa A, Durchfeld-Meyer B, Bube A (2012) Proliferative and nonproliferative lesions of the rat and mouse urinary system. Toxicol Pathol 40(4 Suppl):14S–86S Gao B, St Pierre MV, Stieger B, Meier PJ (2004) Differential expression of bile salt and organic anion transporters in developing rat liver. J  Hepatol 41(2):201–208. https://doi. org/10.1016/j.jhep.2004.04.029 Garman RH, Li AA, Kaufmann W, Auer RN, Bolon B (2016) Recommended methods for brain processing and quantitative analysis in rodent developmental neurotoxicity studies. Toxicol Pathol 44(1):14–42. https://doi. org/10.1177/0192623315596858 Gaytan F, Lucena MC, Munoz E, Paniagua R (1986) Morphometric aspects of rat testis development. J Anat 145:155–159 Goedken MJ, Kerlin RL, Morton D (2008) Spontaneous and age-related testicular findings in beagle dogs. Toxicol Pathol 36(3):465–471 Gold E, Gordis L, Tonascia J, Szklo M (1978) Increased risk of brain tumors in children exposed to barbiturates. J  Natl Cancer Inst 61(4):1031–1034 Gomez RA, Sequeira Lopez ML, Fernandez L, Chernavvsky DR, Norwood VF (1999) The maturing kidney: development and susceptibility. Ren Fail 21(3–4):283–291 Gough A, Barsoum NJ, Mitchell L, McGuire EJ, de la Iglesia FA (1979) Juvenile canine drug-­ induced arthropathy: clinicopathological studies on articular lesions caused by oxolinic and pipemidic acids. Toxicol Appl Pharmacol 51(1):177–187 Gough A, Johnson R, Campbell E, Hall L, Tylor J, Carpenter A, Black W, Basrur PK, Baragi VM, Sigler R, Metz A (1996) Quinolone arthropathy in immature rabbits treated with the fluoroquinolone, PD 117596. Exp Toxicol Pathol 48(4):225–232. https://doi.org/10.1016/ S0940-2993(96)80003-0 Gray LE Jr, Furr J, Tatum-Gibbs KR, Lambright C, Sampson H, Hannas BR, Wilson VS, Hotchkiss A, Foster PM (2016) Establishing the “biological relevance” of dipentyl phthalate reductions in fetal rat testosterone production and plasma and testis testosterone levels. Toxicol Sci 149(1):178–191. https://doi.org/10.1093/ toxsci/kfv224

843

Greeley GH Jr, Kizer JS (1980) The effects of chronic methylphenidate treatment on growth and endocrine function in the developing rat. J Pharmacol Exp Ther 215(3):545–551 Grier RL, Wise GE (1998) Inhibition of tooth eruption in the rat by a bisphosphonate. J Dent Res 77(1):8–15. https://doi.org/10.1177/00 220345980770011201 Groeber EA, Bell SR, Lucarell J, Quang C, Moran L, Badamy M, Coder P (2018) Development of a sensitive LC/MS assay for measuring thyroid hormones T3 and T4  in late-fetal and neonatal rat samples. 37th Annual meeting of Society of Toxicologic Pathology, Indianapolis, 16–21 June 2018 Groseclose MR, Laffan SB, Frazier KS, Hughes-­ Earle A, Castellino S (2015) Imaging MS in toxicology: an investigation of juvenile rat nephrotoxicity associated with dabrafenib administration. J  Am Soc Mass Spectrom 26(6):887–898. https://doi.org/10.1007/ s13361-015-1103-4 Guillet R, Wyatt J, Baggs RB, Kellogg CK (1988) Anesthetic-induced corneal lesions in developmentally sensitive rats. Invest Ophthalmol Vis Sci 29(6):949–954 Hahnel H, Modis L, Levai G (1978) Histological and histochemical investigations of the epiphyseal cartilage in rats after administration of heparin, coumarin as well as coumarin and diphosphonate (EHDP). Exp Pathol (Jena) 15(4):196–207 Heinrichs C, Colli M, Yanovski JA, Laue L, Gerstl NA, Kramer AD, Uyeda JA, Baron J  (1997) Effects of fasting on the growth plate: systemic and local mechanisms. Endocrinology 138(12):5359–5365. https://doi. org/10.1210/endo.138.12.5603 Henning SJ (1981) Postnatal development: coordination of feeding, digestion, and metabolism. Am J Physiol 241(3):G199–G214 Holladay SD, Smialowicz RJ (2000) Development of the murine and human immune system: differential effects of immunotoxicants depend on time of exposure. Environ Health Perspect 108(Suppl 3):463–473 Hoover DM, Buening MK, Tamura RN, Steinberger E (1989) Effects of cefamandole on spermatogenic development of young CD rats. Fundam Appl Toxicol 13(4):737–746 Hurtt ME, Daston G, Davis-Bruno K, Feuston M, Silva Lima B, Makris S, McNerney ME, Sandler JD, Whitby K, Wier P, Cappon GD (2004) Juvenile animal studies: testing strategies and design. Birth Defects Res B Dev Reprod Toxicol 71(4):281–288. https://doi.org/10.1002/ bdrb.20017 Ikonomidou C, Bosch F, Miksa M, Bittigau P, Vockler J, Dikranian K, Tenkova TI, Stefovska V, Turski L, Olney JW (1999) Blockade of NMDA receptors and apoptotic neurodegeneration in the developing brain. Science 283(5398):70–74

844

Catherine A. Picut and Amera K. Remick

Inaloz HS, Deveci E, Inaloz SS, Unal B, Eralp A, Can I (2002) The effects of tamoxifen on rat skin. Eur J Gynaecol Oncol 23(1):50–52 Ingulli EG, Mak RH (2014) Growth in children with chronic kidney disease: role of nutrition, growth hormone, dialysis, and steroids. Curr Opin Pediatr 26(2):187–192. https://doi. org/10.1097/MOP.0000000000000070 James RW, Crook D, Heywood R (1979) Canine pituitary-testicular function in relation to toxicity testing. Toxicology 13(3):237–247 Johri A, Dhawan A, Singh RL, Parmar D (2008) Persistence in alterations in the ontogeny of cerebral and hepatic cytochrome P450s following prenatal exposure to low doses of lindane. Toxicol Sci 101(2):331–340. https://doi. org/10.1093/toxsci/kfm269 Jonsson O, Palma Villar R, Nilsson LB, Norsten-­ Hoog C, Brogren J, Eriksson M, Konigsson K, Samuelsson A (2012) Capillary microsampling of 25 microl blood for the determination of toxicokinetic parameters in regulatory studies in animals. Bioanalysis 4(6):661–674. https:// doi.org/10.4155/bio.12.25 Karen T, Schlager GW, Bendix I, Sifringer M, Herrmann R, Pantazis C, Enot D, Keller M, Kerner T, Felderhoff-Mueser U (2013) Effect of propofol in the immature rat brain on short- and long-term neurodevelopmental outcome. PLoS One 8(5):e64480. https://doi.org/10.1371/ journal.pone.0064480 Kato M, Onodera T (1988) Morphological investigation of osteochondrosis induced by ofloxacin in rats. Fundam Appl Toxicol 11(1):120–131 Kato M, Takada S, Kashida Y, Nomura M (1995) Histological examination on Achilles tendon lesions induced by quinolone antibacterial agents in juvenile rats. Toxicol Pathol 23(3):385–392 Kaufmann W, Bolon B, Bradley A, Butt M, Czasch S, Garman RH, George C, Groters S, Krinke G, Little P, McKay J, Narama I, Rao D, Shibutani M, Sills R (2012) Proliferative and nonproliferative lesions of the rat and mouse central and peripheral nervous systems. Toxicol Pathol 40(4 Suppl):87S–157S Keane KA, Parker GA, Regan KS, Picut C, Dixon D, Creasy D, Giri D, Hukkanen RR (2015) Scientific and Regulatory Policy Committee (SRPC) Points to Consider: histopathology evaluation of the pubertal development and thyroid function assay (OPPTS 890.1450, OPPTS 890.1500) in rats to screen for endocrine disruptors. Toxicol Pathol 43(8):1047–1063. https:// doi.org/10.1177/0192623315579943 Kerlin R, Bolon B, Burkhardt J, Francke S, Greaves P, Meador V, Popp J  (2016) Scientific and Regulatory Policy Committee: recommended (“best”) practices for determining, communicating, and using adverse effect data from nonclinical

studies. Toxicol Pathol 44(2):147–162. https:// doi.org/10.1177/0192623315623265 Kim IS, Ariyaratne HB, Chamindrani Mendis-­ Handagama SM (2001) Effects of continuous and intermittent exposure of lactating mothers to aroclor 1242 on testicular steroidogenic function in the adult male offspring. Tissue Cell 33(2):169–177 Kortenkamp A, Evans R, Martin O, McKinlay R, Orton F, Frosivatz E (2012) State of the art assessment of endocrine disrupters. Final report. Project contract number 070307/2009/550687/SER/D3 Kumar A, Beazley S (2017) Common spontaneous histopathologic findings in juvenile domestic pigs used in nonclinical research studies. 26th Annual meeting of the Society of Toxicologic Pathologist, Montreal Landreth KS (2002) Critical windows in development of the rodent immune system. Hum Exp Toxicol 21(9–10):493–498 Landrigan PJ, Kimmel CA, Correa A, Eskenazi B (2004) Children’s health and the environment: public health issues and challenges for risk assessment. Environ Health Perspect 112(2):257–265 Liu Y, Silverstein FS, Skoff R, Barks JD (2002) Hypoxic-ischemic oligodendroglial injury in neonatal rat brain. Pediatr Res 51(1):25–33. https://doi. org/10.1203/00006450-200,201,000-00007 Lucas JN, Rudmann DG, Credille KM, Irizarry AR, Peter A, Snyder PW (2007) The rat mammary gland: morphologic changes as an indicator of systemic hormonal perturbations induced by xenobiotics. Toxicol Pathol 35(2):199–207 Macon MB, Fenton SE (2013) Endocrine disruptors and the breast: early life effects and later life disease. J  Mammary Gland Biol Neoplasia 18(1):43–61. https://doi.org/10.1007/ s10911-013-9275-7 Mandrup KR, Hass U, Christiansen S, Boberg J  (2012) Perinatal ethinyl oestradiol alters mammary gland development in male and female Wistar rats. Int J  Androl 35(3):385–396. https://doi. org/10.1111/j.1365-2605.2012.01258.x Markey CM, Luque EH, Munoz De Toro M, Sonnenschein C, Soto AM (2001) In utero exposure to bisphenol A alters the development and tissue organization of the mouse mammary gland. Biol Reprod 65(4):1215–1223 Masso-Welch PA, Darcy KM, Stangle-Castor NC, Ip MM (2000) A developmental atlas of rat mammary gland histology. J  Mammary Gland Biol Neoplasia 5(2):165–185 McCausland JE, Ryan GB, Alcorn D (1996) Angiotensin converting enzyme inhibition in the postnatal rat results in decreased cell pro-

Pathology of Juvenile Animals liferation in the renal outer medulla. Clin Exp Pharmacol Physiol 23(6–7):552–554 McHugh NA, Vercesi HM, Egan RW, Hey JA (2003) In vivo rat assay: bone remodeling and steroid effects on juvenile bone by pQCT quantification in 7  days. Am J  Physiol Endocrinol Metab 284(1):E70–E75. https:// doi.org/10.1152/ajpendo.00102.2002 McLeod F, Marzo A, Podpolny M, Galli S, Salinas P (2017) Evaluation of synapse density in hippocampal rodent brain slices. J  Vis Exp 128. https://doi.org/10.3791/56153 Mendis-Handagama SM, Ariyaratne HB (2004) Effects of thyroid hormones on Leydig cells in the postnatal testis. Histol Histopathol 19(3):985–997 Miyawaki T, Moriya N, Nagaoki T, Taniguchi N (1981) Maturation of B-cell differentiation ability and T-cell regulatory function in infancy and childhood. Immunol Rev 57:61–87 Muller-Rover S, Handjiski B, van der Veen C, Eichmuller S, Foitzik K, McKay IA, Stenn KS, Paus R (2001) A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages. J  Invest Dermatol 117(1):3–15. https://doi. org/10.1046/j.0022-202x.2001.01377.x Murphy RL, Sommadossi JP, Lamson M, Hall DB, Myers M, Dusek A (1999) Antiviral effect and pharmacokinetic interaction between nevirapine and indinavir in persons infected with human immunodeficiency virus type 1. J  Infect Dis 179(5):1116–1123. https://doi. org/10.1086/314703 Niu X, Beekhuijzen M, Schoonen W, Emmen H, Wenker M (2016) Effects of capillary microsampling on toxicological endpoints in juvenile rats. Toxicol Sci 154(1):69–77. https://doi. org/10.1093/toxsci/kfw146 NTP (2011) Specifications for the conduct of studies to evaluate the reproductive and developmental toxicity of chemical, biological and physical agents in laboratory animals for the National Toxicology Program (NTP). National Institute of Environmental Health Sciences, RTP, N.C. Nurmio M, Toppari J, Zaman F, Andersson AM, Paranko J, Soder O, Jahnukainen K (2007) Inhibition of tyrosine kinases PDGFR and C-Kit by imatinib mesylate interferes with postnatal testicular development in the rat. Int J  Androl 30(4):366–376.; ; discussion 376. https://doi. org/10.1111/j.1365-2605.2007.00755.x OECD (2007) Test No. 426: Developmental Neurotoxicity Study. OECD Publishing, Paris OECD (2011) Test No. 443: Extended One-­ Generation Reproductive Toxicity Study. OECD Publishing, Paris OECD (2013) Guidance Document supporting Test Guideline 443 on the Extended-One-­

845

Generation Reproductive Toxicity Test, vol GD 151. OECD Publishing, Paris OECD (2016) Test No. 421: Reproduction/ Developmental Toxicity Screening Test. OECD Publishers, Paris OECD (2018) Test No. 443: Extended One-­ Generation Reproductive Toxicity Study. OECD Publishing, Paris Okayama Y, Wakui S, Wempe MF, Sugiyama M, Motohashi M, Mutou T, Takahashi H, Kume E, Ikegami H (2017) In utero exposure to di(n-butyl)phthalate induces morphological and biochemical changes in rats postpuberty. Toxicol Pathol 45(4):526–535. https://doi. org/10.1177/0192623317709091 Olney JW, Farber NB, Wozniak DF, JevtovicTodorovic V, Ikonomidou C (2000) Environmental agents that have the potential to trigger massive apoptotic neurodegeneration in the developing brain. Environ Health Perspect 108(Suppl 3):383–388 Osborne G, Rudel R, Schwarzman M (2015) Evaluating chemical effects on mammary gland development: a critical need in disease prevention. Reprod Toxicol 54:148–155. https://doi.org/10.1016/j. reprotox.2014.07.077 Oxenhandler RW, Adelstein EH, Haigh JP, Hook RR Jr, Clark WH Jr (1979) Malignant melanoma in the Sinclair miniature swine: an autopsy study of 60 cases. Am J  Pathol 96(3):707–720 Parker GA (2016a) Introduction. In: Parker GAaP CA (ed) Atlas of histology of the juvenile rat. Academic, San Diego, pp xi–xiii Parker GA (2016b) Development of immune system organs. In: Parker GA (ed) Immunopathology in toxicology and drug development. Springer, New York, NY Parker GA, Gibson WB (1995) Liver lesions in rats associated with wrapping of the torso. Toxicol Pathol 23(4):507–512 Parker GA, Papenfuss T (2016) Immune system. In: Parker GA, Picut C (eds) Atlas of histology of the juvenile rat. Elsevier, San Diego, California Pathak S, Multani AS, McConkey DJ, Imam AS, Amoss MS Jr (2000) Spontaneous regression of cutaneous melanoma in sinclair swine is associated with defective telomerase activity and extensive telomere erosion. Int J  Oncol 17(6):1219–1224 Patyna S, Arrigoni C, Terron A, Kim TW, Heward JK, Vonderfecht SL, Denlinger R, Turnquist SE, Evering W (2008) Nonclinical safety evaluation of sunitinib: a potent inhibitor of VEGF, PDGF, KIT, FLT3, and RET receptors. Toxicol Pathol 36(7):905–916 Paxinos G, Watson C (1986) The Rat Brain in stereotaxic coordinates, 2nd edn. Academic, Orlando

846

Catherine A. Picut and Amera K. Remick

Penna A, Buchanan N (1991) Paracetamol poisoning in children and hepatotoxicity. Br J  Clin Pharmacol 32(2):143–149 Petersen M, Thorikay M, Deckers M, van Dinther M, Grygielko ET, Gellibert F, de Gouville AC, Huet S, ten Dijke P, Laping NJ (2008) Oral administration of GW788388, an inhibitor of TGF-beta type I and II receptor kinases, decreases renal fibrosis. Kidney Int 73(6):705–715. https://doi.org/10.1038/ sj.ki.5002717 Pfister K, Mazur D, Vormann J, Stahlmann R (2007) Diminished ciprofloxacin-induced chondrotoxicity by supplementation with magnesium and vitamin E in immature rats. Antimicrob Agents Chemother 51(3):1022–1027. https:// doi.org/10.1128/AAC.01175-06 Picut CA, Coleman GD (2016) Gastrointestinal tract. In: Parker GA, Picut CA (eds) Atlas of histology of the juvenile rat. Elseveir, San Diego, pp 127–131 Picut CA, Remick AK (2017) Impact of age on the male reproductive system from the pathologist’s perspective. Toxicol Pathol 45(1):195–205. https://doi. org/10.1177/0192623316672744 Picut CA, Remick AK, de Rijk EP, Simons ML, Stump DG, Parker GA (2015a) Postnatal development of the testis in the rat: morphologic study and correlation of morphology to neuroendocrine parameters. Toxicol Pathol 43(3):326–342. https://doi. org/10.1177/0192623314547279 Picut CA, Dixon D, Simons ML, Stump DG, Parker GA, Remick AK (2015b) Postnatal ovary development in the rat: morphologic study and correlation of morphology to neuroendocrine parameters. Toxicol Pathol 43(3):343–353. https://doi. org/10.1177/0192623314544380 Picut CA, Brown DL, Remick AK (2016a) Nervous system. In: Parker GA, Picut CA (eds) Atlas of histology of the juvenile rat. Elsevier, San Diego, pp 45–48 Picut CA, Parker EF, Swanson C (2016b) Prenatal and early postnatal development of the thyroid gland in the rat: histologic and morphometric endpoints. 37th Annual meeting of the American College of Toxicology, Baltimore Picut CA, Ziejewski MK, Stanislaus D (2017a) Comparative aspects of pre- and postnatal development of the male reproductive system. Birth Defects Res. https://doi.org/10.1002/ bdr2.1133 Picut CA, Brown D, CSwanson C, Greeley M, Kirpkpatrick D, Palmer JL, Nussbaum J (2017b) Animal model of bronchopulmonary dysplasia in juvenile rats. Annual meeting of Society of Toxicologic Pathology, Montreal

Picut CA, Parker EF, White-Hunt S, Szabo K, Keen J, Coder P, McElroy P (2018) Renal lesions associated with soy-deficient diet in rats. Paper presented at the 37th annual meeting of Society of Toxicologic Pathologists, Indianapolis, June 16–21, 2018 Porter RM (2003) Mouse models for human hair loss disorders. J Anat 202(1):125–131 Poulton AS, Bui Q, Melzer E, Evans R (2016) Stimulant medication effects on growth and bone age in children with attention-deficit/hyperactivity disorder: a prospective cohort study. Int Clin Psychopharmacol 31(2):93–99. https:// doi.org/10.1097/YIC.0000000000000109 Price PA, Williamson MK, Haba T, Dell RB, Jee WS (1982) Excessive mineralization with growth plate closure in rats on chronic warfarin treatment. Proc Natl Acad Sci U S A 79(24):7734–7738 Radde IC (1985) Mechanisms of drug absorption and their development. In: Macleod SMaR IC (ed) Textbook of pediatric clinical pharmacology. PSG Publishing Co., Littleton, pp 17–43 Raghavan S, Bauer C, Mundschau G, Li Q, Fuchs E (2000) Conditional ablation of beta1 integrin in skin. Severe defects in epidermal proliferation, basement membrane formation, and hair follicle invagination. J Cell Biol 150(5):1149–1160 Rasmussen AD, Richmond E, Wegener KM, Downes N, Mullins P (2015) Vigabatrin-induced CNS changes in juvenile rats: Induction, progression and recovery of myelin-related changes. Neurotoxicology 46:137–144. https://doi. org/10.1016/j.neuro.2014.12.008 Reeves PG, Rossow KL, Lindlauf J  (1993) Development and testing of the AIN-93 purified diets for rodents: results on growth, kidney calcification and bone mineralization in rats and mice. J Nutr 123(11):1923–1931. https://doi. org/10.1093/jn/123.11.1923 Rehm S (2000) Spontaneous testicular lesions in purpose-bred beagle dogs. Toxicol Pathol 28(6):782–787 Rehm S, White TE, Zahalka EA, Stanislaus DJ, Boyce RW, Wier PJ (2008) Effects of food restriction on testis and accessory sex glands in maturing rats. Toxicol Pathol 36(5):687–694 Reich B, Hoeber D, Bendix I, Felderhoff-Mueser U (2016) Hyperoxia and the immature brain. Dev Neurosci 38(5):311–330. https://doi. org/10.1159/000454917 Report on Neurontin (Gabapentin) (2009). https://www.accessdata.fda.gov/drugsatfda_ docs/label/2009/020235s041,020882s028,0 21129s027lbl.pdf. Accessed 18 June 2018 Rice D, Barone S Jr (2000) Critical periods of vulnerability for the developing nervous system: evidence from humans and animal models. Environ Health Perspect 108(Suppl 3):511–533

Pathology of Juvenile Animals Richter-Reichhelm HB, Althoff J, Schulte A, Ewe S, Gundert-Remy U (2002) Workshop report. Children as a special subpopulation: focus on immunotoxicity. Federal Institute for Health Protection of Consumers and Veterinary Medicine (BgVV), 15–16 November 2001, Berlin, Germany. Arch Toxicol 76(7):377–382 Robinson PB, Harvey W, Belal MS (1988) Inhibition of cartilage growth by the anticonvulsant drugs diphenylhydantoin and sodium valproate. Br J Exp Pathol 69(1):17–22 Rodier PM (1995) Developing brain as a target of toxicity. Environ Health Perspect 103(Suppl 6):73–76 Roerig DL, Hasegawa AT, Harris GJ, Lynch KL, Wang RI (1980) Occurrence of corneal opacities in rats after acute administration of l-alpha-­ acetylmethadol. Toxicol Appl Pharmacol 56(2):155–163 Rudel RA, Fenton SE, Ackerman JM, Euling SY, Makris SL (2011) Environmental exposures and mammary gland development: state of the science, public health implications, and research recommendations. Environ Health Perspect 119(8):1053–1061. https://doi. org/10.1289/ehp.1002864 Russo IH, Russo J (1996) Mammary gland neoplasia in long-term rodent studies. Environ Health Perspect 104(9):938–967 Sampson HW, Spears H (1999) Osteopenia due to chronic alcohol consumption by young actively growing rats is not completely reversible. Alcohol Clin Exp Res 23(2):324–327 Sampson HW, Gallager S, Lange J, Chondra W, Hogan HA (1999) Binge drinking and bone metabolism in a young actively growing rat model. Alcohol Clin Exp Res 23(7):1228–1231 Semple BD, Blomgren K, Gimlin K, Ferriero DM, Noble-Haeusslein LJ (2013) Brain development in rodents and humans: identifying benchmarks of maturation and vulnerability to injury across species. Prog Neurobiol 106-107:1–16. https://doi.org/10.1016/j. pneurobio.2013.04.001 Sengupta A, Lichti UF, Carlson BA, Ryscavage AO, Gladyshev VN, Yuspa SH, Hatfield DL (2010) Selenoproteins are essential for proper keratinocyte function and skin development. PLoS One 5(8):e12249. https://doi.org/10.1371/journal.pone.0012249 Sharma AK, Morrison JP, Rao DB, Pardo ID, Garman RH, Bolon B (2016) Toxicologic pathology analysis for translational neuroscience: improving human risk assessment using optimized animal data. Int J  Toxicol 35(4):410–419. https://doi. org/10.1177/1091581816636372

847

Sharpe RM, Rivas A, Walker M, McKinnell C, Fisher JS (2003) Effect of neonatal treatment of rats with potent or weak (environmental) oestrogens, or with a GnRH antagonist, on Leydig cell development and function through puberty into adulthood. Int J Androl 26(1):26–36 Shigami N, Ishimouchi K, Hashimoto A, Katagi J  (2016) Focal chondrocyte dysplasia in the femoral metaphysis in young sprague-dawley rats.. 36th Annual meeting of the Society of Toxicologic Pathologists, San Diego Shimizu T, Fujita S, Izumi K, Koja T, Ohba N, Fukuda T (1984) Corneal lesions induced by the systemic administration of capsaicin in neonatal mice and rats. Naunyn Schmiedebergs Arch Pharmacol 326(4):347–351 Sidhu RS, Del Bigio MR, Tuor UI, Seshia SS (1997) Low-dose vigabatrin (gamma-vinyl GABA)-induced damage in the immature rat brain. Exp Neurol 144(2):400–405. https:// doi.org/10.1006/exnr.1997.6412 Sizonenko SV, Sirimanne E, Mayall Y, Gluckman PD, Inder T, Williams C (2003) Selective cortical alteration after hypoxic-ischemic injury in the very immature rat brain. Pediatr Res 54(2):263–269. https://doi.org/10.1203/01. PDR.0000072517.01207.87 Skoff RP, Bessert DA, Barks JD, Song D, Cerghet M, Silverstein FS (2001) Hypoxic-ischemic injury results in acute disruption of myelin gene expression and death of oligodendroglial precursors in neonatal mice. Int J  Dev Neurosci 19(2):197–208 Smith EJ, Little DG, Briody JN, McEvoy A, Smith NC, Eisman JA, Gardiner EM (2005) Transient disturbance in physeal morphology is associated with long-term effects of nitrogen-containing bisphosphonates in growing rabbits. J  Bone Miner Res 20(10):1731–1741. https://doi. org/10.1359/JBMR.050604 Snodgrass WR (1992) Physiological and biochemical differences between children and adults as determinant of toxic response to environmental pollutants. In: Guzelian PS, Henry CJ, Olin SS (eds) Similarities and differences between children and adults: implications for risk assessment. ILSI Press, Washington, D.C., pp 35–42 Stahlmann R, Chahoud I, Thiel R, Klug S, Forster C (1997) The developmental toxicity of three antimicrobial agents observed only in nonroutine animal studies. Reprod Toxicol 11(1):1–7 Stanko JP, Kissling GE, Chappell VA, Fenton SE (2016) Differences in the rate of in situ mammary gland development and other developmental endpoints in three strains of female rat commonly used in mammary carcinogenesis studies: implications for timing of carcinogen exposure.

848

Catherine A. Picut and Amera K. Remick

Toxicol Pathol 44(7):1021–1033. https://doi. org/10.1177/0192623316655222 Staska LMaP JT (2016) Skin and mammary gland. In: Parker GAaP CA (ed) Atlas of histology of the juvenile rat. Elsevier, San Diego, pp 1–5 Stump DG, Nemec MD, Parker GA, Coder PS, Sloter ED, Varsho BJ (2012) Significance, reliability, and interpretation of developmental and reproductive toxicity study findings. In: Hood RD (ed) Developmental and reproductive toxicology: a practical approach, 3rd edn. Informa Healthcare, New York, pp 229–301 Sundberg JP, Peters EM, Paus R (2005) Analysis of hair follicles in mutant laboratory mice. J Investig Dermatol Symp Proc 10(3):264–270. https:// doi.org/10.1111/j.1087-0024.2005.10126.x Sundberg JP, Silva KA, King LE Jr, Pratt CH (2016) Skin diseases in laboratory mice: approaches to drug target identification and efficacy screening. Methods Mol Biol 1438:199–224. https://doi. org/10.1007/978-1-4939-3661-8_12 Svensson O, Hjerpe A, Reinholt FP, Engfeldt B (1985) The effect of manganese ingestion, phosphate depletion, and starvation on the morphology of the epiphyseal growth plate. A stereologic study. Clin Orthop Relat Res 197:286–294 Swierkot J, Gruszecka K, Matuszewska A, Wiland P (2015) Assessment of the effect of methotrexate therapy on bone metabolism in patients with rheumatoid arthritis. Arch Immunol Ther Exp (Warsz) 63(5):397–404. https://doi. org/10.1007/s00005-015-0338-x Teitelbaum SL, Ross FP (2003) Genetic regulation of osteoclast development and function. Nat Rev Genet 4(8):638–649. https://doi. org/10.1038/nrg1122 Teitelbaum SL, Belpoggi F, Reinlib L (2015) Advancing research on endocrine disrupting chemicals in breast cancer: expert panel recommendations. Reprod Toxicol 54:141–147. https://doi.org/10.1016/j. reprotox.2014.12.015 Tendron-Franzin A, Gouyon JB, Guignard JP, Decramer S, Justrabo E, Gilbert T, Semama DS (2004) Long-term effects of in utero exposure to cyclosporin A on renal function in the rabbit. J  Am Soc Nephrol 15(10):2687– 2693. https://doi.org/10.1097/01. ASN.0000139069.59466.D8 Thompson DD, Simmons HA, Pirie CM, Ke HZ (1995) FDA Guidelines and animal models for osteoporosis. Bone 17(4 Suppl):125S–133S Thuilliez C, Tortereau A, Perron-Lepage MF, Howroyd P, Gauthier B (2014) Spontaneous testicular tubular hypoplasia/atrophy in the Gottingen minipig: a retrospective study. Toxicol Pathol 42(6):1024–1031. https://doi. org/10.1177/0192623313512430

Towfighi J, Mauger D, Vannucci RC, Vannucci SJ (1997) Influence of age on the cerebral lesions in an immature rat model of cerebral hypoxia-­ ischemia: a light microscopic study. Brain Res Dev Brain Res 100(2):149–160 Traebert M, Lotscher M, Aschwanden R, Ritthaler T, Biber J, Murer H, Kaissling B (1999) Distribution of the sodium/phosphate transporter during postnatal ontogeny of the rat kidney. J Am Soc Nephrol 10(7):1407–1415 Trinh VA, Davis JE, Anderson JE, Kim KB (2014) Dabrafenib therapy for advanced melanoma. Ann Pharmacother 48(4):519–529. https:// doi.org/10.1177/1060028013513009 Tripier MF, Berard M, Toga M, Martin-Bouyer G, Le Breton R, Garat J (1981) Hexachlorophene and the central nervous system. Toxic effects in mice and baboons. Acta Neuropathol 53(1):65–74 Tucker DK, Foley JF, Hayes-Bouknight SA, Fenton SE (2016) Preparation of high-­ quality hematoxylin and eosin-stained sections from rodent mammary gland whole mounts for histopathologic review. Toxicol Pathol 44(7):1059–1064. https://doi. org/10.1177/0192623316660769 van der Brugge-Gamelkoorn GJ, Sminia T (1985) T-cells and T-cells subsets in rat bronchus associated lymphoid tissue (BALT) in situ and in suspension. Adv Exp Med Biol 186:323–329 van Leeuwen BL, Hartel RM, Jansen HW, Kamps WA, Hoekstra HJ (2003) The effect of chemotherapy on the morphology of the growth plate and metaphysis of the growing skeleton. Eur J Surg Oncol 29(1):49–58 Vettorazzi A, Wait R, Nagy J, Monreal JI, Mantle P (2013) Changes in male rat urinary protein profile during puberty: a pilot study. BMC Res Notes 6:232. https://doi. org/10.1186/1756-0500-6-232 Vonvoigtlander PF, Kolaja GJ, Block EM (1982) Corneal lesions induced by antidepressants: a selective effect upon young Fischer 344 rats. J Pharmacol Exp Ther 222(1):282–286 Walling BE, Picut CA, Remick AK (2016) The endocrine system. In: Parker GA, Picut CA (eds) Atlas of histology of the juvenile rat. Elsevier, San Diego, pp 257–263 Walthall K, Cappon GD, Hurtt ME, Zoetis T (2005) Postnatal development of the gastrointestinal system: a species comparison. Birth Defects Res B Dev Reprod Toxicol 74(2):132–156. https://doi.org/10.1002/ bdrb.20040 Walzer M, Bekersky I, Wanaski S, Collins S, Jortner B, Patterson R, Garman R, Sagar S, Tolbert D (2011) Oral toxicity of vigabatrin in immature rats: characterization of intramyelinic edema.

Pathology of Juvenile Animals Neurotoxicology 32(6):963–974. https://doi. org/10.1016/j.neuro.2011.03.014 Weinstock D, Lewis DB, Parker GA, Beyer J, Collinge M, Brown TP, Dybdal N (2010) Toxicopathology of the developing immune system: investigative and development strategies. Toxicol Pathol 38(7):1111–1117 Whitney KM (2012) Testicular histopathology in juvenile rat toxicity studies. Syst Biol Reprod Med 58(1):51–56 Yamasaki K (1995) Histologic study of the femoral growth plate in beagle dogs. Toxicol Pathol 23(5):612–616 You L, Sar M, Bartolucci EJ, McIntyre BS, Sriperumbudur R (2002) Modulation of mam-

849

mary gland development in prepubertal male rats exposed to genistein and methoxychlor. Toxicol Sci 66(2):216–225 Ytrehus B, Carlson CS, Ekman S (2007) Etiology and pathogenesis of osteochondrosis. Vet Pathol 44(4):429–448 Zoetis T, Hurtt ME (2003) Species comparison of anatomical and functional renal development. Birth Defects Res B Dev Reprod Toxicol 68(2):111–120. https://doi.org/10.1002/ bdrb.10013 Zoetis TaW I (2003) Principles and practices for direct dosing of pre-weaning mammals in toxicity testing and research. ILSI Press, Washington, D.C.

Chapter 20 Non-mammalian Laboratory Species: Fish, Frogs, and Beyond Shannon M. Wallace and Jeffrey C. Wolf Abstract For many decades, bioassays that incorporate mammalian test subjects such as rodents, rabbits, and nonhuman primates have been mainstays of preclinical toxicology research. Benefits of using these traditional laboratory models include relative taxonomic and anatomic proximity to humans, well-established husbandry requirements, and a wealth of associated biological, physiological, and toxicological data. Despite these advantages, however, studies based on fish, amphibian, or avian models are becoming increasingly more mainstream. Although many of these experiments have an ecotoxicological focus, with an emphasis on the detection or characterization of effects caused by environmental contamination, nonmammalian subjects such as zebrafish (Danio rerio) and Xenopus laevis frogs are also being used to screen for potential pharmacological candidate compounds, to investigate the effects of test substances in genetically altered animals with gain- or loss-of-function mutations, or to study therapeutic models of tissue regeneration, for example. Additionally, there is a need to evaluate the safety of pharmaceutical agents in food production animals such as poultry and aquacultured fishes. This chapter will discuss the benefits, limitations, considerations, and applications that pertain to the use of nonmammalian species such as frogs, birds, and fish in toxicological studies. Key words Fish, Amphibians, Reptiles, Birds, Nontraditional, Nonmammalian, Wildlife, Toxicology, Pathology, Bioassay, Transgenic, Environmental

1  Introduction For scientists accustomed to traditional laboratory models such as laboratory rodents and other domestic mammals, research involving nontraditional species may seem marginally interesting and somewhat quaint. The phylogenetic distance between these creatures and humans is often cited as a reason for dismissing the value of fish, amphibians, birds, and other nontraditional species as research subjects, despite the fact that a large percentage of anatomic features and physiological processes are highly conserved. For example, there is approximately 70% homology between the zebrafish (Danio rerio) and human genomes, and it is estimated that 84% of genes responsible for human disease have a zebrafish Thomas J. Steinbach et al. (eds.), Toxicologic Pathology for Non-Pathologists, https://doi.org/10.1007/978-1-4939-9777-0_20, © Springer Science+Business Media, LLC, part of Springer Nature 2019

851

852

Shannon M. Wallace and Jeffrey C. Wolf

counterpart (Howe et al. 2013). Many researchers might also be surprised to learn that most endocrine system feedback loops, toxicologically relevant biotransformation reactions, and mechanisms of carcinogenesis are quite comparable between fish and humans. Additionally, it is often the differences between so-called “lower vertebrates” and mammals that provide significant research ­opportunities and advantages. For example, species in which certain metabolizing enzymes or pathways are naturally omitted relative to humans can serve as de facto “knockout” models without having to resort to genetic manipulation. In other instances, lower animals can fill in gaps for which traditional laboratory species fall short. For example, although there is no naturally occurring rodent model for human melanoma, comparable melanotic tumors are known to occur in certain platy fish x swordtail hybrids (Xiphophorus spp.), in which the genetic basis for tumorigenesis has been elucidated (Meierjohann and Schartl 2006). In addition to serving as human disease surrogates, there are other reasons for employing nonmammalian species in toxicological or carcinogenicity studies. Aquatic animals are particularly suitable for studies of potential health effects associated with environmental contaminants, because many of those substances eventually end up in surface waters, and aquatic animals such as fish and amphibians may be exposed to pollutants simultaneously through several routes, e.g., respiratory, dietary, and/or dermal absorption. Consequently, such species may serve as sentinels for possible human hazard, or they may be studied to determine if they themselves, or related wildlife species, are being negatively impacted by environmental pollution. Wildlife surveys can also be used to determine the effectiveness of efforts to remediate environmental contamination, by periodically monitoring the health of nonmigratory indicator species within well-defined geographic areas. Other nontraditional animals that are cultivated for food or sport may be used in toxicological studies to assess the safety and/or efficacy of pharmaceutical products intended for therapeutic use in those particular species. Finally, the decision to use nontraditional species as experimental subjects may be based on cost and animal-use considerations. For example, small fish and amphibian embryos have demonstrated utility for the high-throughput screening of pharmaceutical compounds and toxicologic substances (Zhu et  al. 2014). Additionally, because of their relatively small size, juvenile or even adult stages of small aquarium species can be used in numbers sufficient to provide ample statistical power for toxicologic bioassays and still be cost-effective. The wide array of potential test subjects that currently exists includes countless commercially available strains of genetically modified zebrafish (Aleström et al. 2006). Furthermore, the research use of zebrafish embryos is not currently regulated; consequently, this model has been proposed as a refinement alternative to traditional animal experimentation

Non-mammalian Laboratory Species: Fish, Frogs, and Beyond

853

(Scholz et al. 2008). And as efforts to refine, replace, and reduce animal testing continue, it is likely that the role of nontraditional vertebrate species as research subjects will increase. The ensuing chapter will provide a brief overview and further description of the many advantages and fewer disadvantages ­associated with the use of nontraditional animals in toxicological research, with an emphasis on the histopathology endpoint.

2  Advantages of Using Nonmammalian Species for Toxicologic Studies On a daily basis, we make choices based on what we identify as advantageous or disadvantageous. For example, being short-­ statured may be perceived as a disadvantage in sports, but in reality this determination is dependent upon the context: a height-­ challenged center is not likely to excel in basketball, while a gymnast, wrestler, or jockey with that same build will more likely experience positive outcomes. Similarly, species selection for toxicology studies should be geared to the exploitation of those differences that allow for the best possible outcome. “Comparative medicine is a discipline in which the similarities and differences in biology among animal species are studied to enhance the understanding of mechanisms of human and animal disease.” (AVMA 2018). Mice and rats have a long history as animal models for human diseases. One reason is the degree to which elements of anatomy and physiology are conserved; for example, approximately 95% of 30,000 rodent genes are shared with humans (Bryda 2013). Consequently, it is often the case that only minor tweaking of the rodent genome is required to mimic the phenotype of a variety of human afflictions such as obesity, diabetes, and cardiovascular disease. Furthermore, rodents bring to the table decades of historical testing data, well-established husbandry procedures and nutritional requirements, options for ancillary testing (e.g., behavioral), unique strains and transgenic lines, and regulatory agency acceptance. Rodents can, however, pose occasional disadvantages in toxicity studies. Male rats produce a low molecular protein, alpha 2u-globulin (α2UG) in their liver. In toxicity studies, certain chemicals can bind to this protein, forming a protein chemical complex that causes eosinophilic globules to form in kidney tubules. These complexes can ultimately result in nephropathy and in some cases renal tumors. For example, dietary administration of diisononyl phthalate (DINP), a plasticizer, resulted in kidney tumors in high dose-treated male rats, while similarly treated female rats, and male and female mice, did not develop renal tumors (Caldwell et  al. 1999). A retrospective study was performed that investigated the relationship between cell proliferation induced by α2UG accumulation/nephropathy in male rats and tumor formation. The study confirmed the existence of this relationship in male rats, and because

854

Shannon M. Wallace and Jeffrey C. Wolf

humans do not produce α2UG, these renal tumors were not considered a relevant human risk. Additionally, the Environmental Protection Agency has accepted the recommendations of its science advisory board and technical panel to disregard male rat kidney tumors associated with α2UG nephropathy in weight of evidence determinations for human risk assessment (Caldwell et al. 1999) Many conventional animal models are larger than their nonmammalian counterparts, which is advantageous for surgical procedures. Rabbits, for example, are excellent subjects for atherosclerosis, bone implantation, ocular, and intervertebral disk disease research. Additionally, they are docile and have short gestation, lactation, and puberty cycles. However, they require special accommodations in terms of housing and handling, and they may become easily dehydrated. There are various examples of inherited diseases for which dogs are suitable models [e.g., bone cancer, aging and dementia, surgical valve replacement (NABR 2017)]. Dogs’ genomic architecture, detailed health information, shared environment with humans, and active private owner participation in clinical research studies make them excellent comparative models for treatment of human disorders (Hytönen 2016). There are a number of human conditions and disorders for which nonhuman primates are the most appropriate model due to their high degree of physiologic similarity to Homo sapiens. Noteworthy examples include AIDS, lung disorders, atherosclerosis, and drug metabolism (Phillips et al. 2014). However, the use of nonhuman primates in research studies can be limited by ethical concerns. Although subclinical disease can complicate research outcomes in studies involving traditional laboratory mammals, this may be mitigated by obtaining specific pathogen-free animals and by maintaining concurrent sentinel animals for common types of infections. Examples of subclinical diseases occasionally observed in traditional laboratories include pulmonary infections with Mycoplasma pulmonis in rats, liver disease due to Eimeria stiedae in rabbits, and immune suppression due to simian immunodeficiency virus in nonhuman primates. Several advantages exist when considering the use of nonmammalian species, such as the ability to dose large numbers of animals simultaneously (e.g., via water bath exposure) with the possible added benefit of reduced costs. Costs may also be relatively reduced for studies that rely on injection or gavage administration, due to the need for lesser amounts of the test article. Given the small body size of some adult fish and amphibians, and juveniles of larger species, there is an opportunity to evaluate numerous organ systems at once on a single histologic slide, using either excised or whole-­ body sections. Small body size can also be a disadvantage, for example in studies that require repeated intravenous sampling, although that need can usually be fulfilled through the use of larger

Non-mammalian Laboratory Species: Fish, Frogs, and Beyond

855

species, such as rainbow trout. However, the comparatively long time to maturity and larger space requirements for trout can be problematic for some laboratories and institutions. The following is a discussion of the relative advantages of various taxa as models for toxicological studies that have pathology endpoints. 2.1  Birds

Compared to mammals, advantages for the use of birds in toxicological studies include embryo development without maternal influences, easy embryo manipulation, and two patterns of embryo development, altricial and precocial. Altricial species (songbirds) are less developed, and their young hatch earlier compared to those of precocial (ducks and geese) birds. Interspecies differences in the rate of physiologic development, including maturation of the endocrine system, may be interesting aspects of exploitation and comparison (Scanes and McNabb 2003). Altricial birds, for example, demonstrate little histologic or functional thyroid development during embryonic or peri-hatch stages, rendering them less likely to be impacted by thyroid-active endocrine disrupting compounds (EDCs) during embryonic development and making them poorer candidates than their precocial counterparts for such studies (Jaspers 2015). Birds offer several advantages for use in specific types of research, in particular, inhalation and reproductive studies. The avian trachea is on average 2.7 times longer than that of comparably sized mammals, with complete cartilaginous rings, and birds have a constant-volume flow-through lung. Compared to mammals, the avian pulmonary gas-exchange barrier is only half as thick, yet the ventilation of gas-exchange tissues is similarly efficient, and thus, birds can extract approximately twice the amount of a given gas from the ventilatory stream (Brown et al. 1997). The effective (parabronchial) ventilation in nonpasserine birds at resting conditions is 30–160% greater than alveolar ventilation in comparably sized mammals (Brown et  al. 1997). Bird pulmonary airflow is unidirectional and gas exchange occurs as a cross-current system, which allows for efficient exchange of carbon dioxide and oxygen; however, this enhanced efficiency also holds true for other airborne gases, which, if toxic, can lead to respiratory decompensation (Shuster et  al. 2012). Experimental or incidental inhaled exposure to Teflon (polytetrafluorethylene, PTFE) fumes provides an example of how anatomical features in birds may result in increased sensitivity and toxicosis relative to mammals; while birds exposed to PTFE fumes develop severe, often fatal, pulmonary hemorrhage, pulmonary edema, and hepatic necrosis, humans exposed to comparable airborne concentrations are likely to develop relatively milder flu-like symptoms and lesser degrees of pulmonary edema. In addition to anatomical considerations, it should be recognized that enzymatic differences between birds

856

Shannon M. Wallace and Jeffrey C. Wolf

and mammals may impact responses to inhaled toxicants (Brown et al. 1997). Avian male and female reproductive physiology offers unique opportunities relevant to toxicological research (Scanes and McNabb 2003; Touart 2004). The novelty of external egg laying and the ability to manipulate breeding cycles facilitate the study of multigenerational effects caused by reproductive toxicants or teratogens and effects that may occur during embryogenesis. A variety of avian species are employed for reproductive studies, including the chicken Gallus gallus domesticus, mallard duck Anas platyrhynchos, domestic turkey Meleagris gallopavo, quail (Colinus and Coturnix spp.), and various species of psittacines (Kalmar et al. 2010), and each has beneficial attributes that can be utilized. For example, Coturnix spp. quail have an androgen-dependent cloacal gland, the relative size of which in males and females can be used as a potential indicator of estrogenic or androgenic substance exposure, respectively (Massa et  al. 1980). Transgenic Japanese quail, Coturnix japonica, provide advantages as compared to traditionally used transgenic mice (superior model for embryonic developmental biology) or transgenic chickens (smaller size and greater reproductive capacity) (Poynter et al. 2009). Laying hens develop spontaneous ovarian tumors similar to ovarian cancers in women and thus may serve as a valuable preclinical model (Bradaric et al. 2011). Reproductive endocrine disruption research is aimed at determining the effects of chemicals that mimic or interfere with the function of endogenous hormones, and as a consequence, may be capable of adversely altering the hypothalamic-pituitary-gonadal axis during critical windows of sexual differentiation and reproductive capacity in adult animals. At least experimentally, embryonic and adult birds have demonstrated clear histopathologically evident responses to administered xenoestrogens (Berg et al. 1999; Furuya et al. 2003). Differences in sex determination in birds as compared to mammals should be considered when performing this type of research, as the female is the heterogametic gender in birds, as opposed to the male in mammals (male birds have matching ZZ sex chromosomes and are, therefore, homogametic, while female birds have a ZW chromosomes and are the heterogametic sex). One might assume that this determinative difference would cause effects in birds to be opposite those of mammals; however, studies have demonstrated that this is not necessarily the case (Scanes and McNabb 2003). Although bird models offer a variety of advantages relative to mammals, they are more prone to stress; therefore, diligent experimental design is imperative to help reduce stressors that have the potential to confound research outcomes. 2.2  Reptiles

With the notable exception of the African clawed frog, Xenopus laevis, various reptile and amphibian species have historically played a lesser role in toxicologic research as compared to mammals, birds,

Non-mammalian Laboratory Species: Fish, Frogs, and Beyond

857

or fish (Johnson et al. 2017). This may be due to various factors, including, but not limited to, inexperience in the use of these s­ pecies and the uncertainty of translating study outcomes to humans and other animals. Compared to amphibians, reptiles offer few advantages as research subjects. In addition to impervious skin, they tend to have demanding dietary and husbandry needs, and their biology and physiology are often less well-understood (Wolf 2013). In crocodilians, some turtles, and lizards, embryonic sex is determined chiefly by the temperature of egg incubation (temperature-­dependent sex determination, TDSD). Incubation temperature regulates expression of aromatase enzymes, which in turn affects estrogen production and sexual differentiation of the gonads (Bishop et  al. 2010). For example, aromatase inhibition induces male development at female-producing temperatures, whereas exogenous exposure to testosterone, which is convertible to estrogen via aromatization, produces females at male-producing temperatures (Bishop et al. 2010). The renal portal system of reptiles, amphibians, and fish should be considered when dosing with any parenteral test article. Venous blood from the hindlimb and/ or tail circulates directly to the kidney, and substances injected in the caudal portion of the animals are potentially eliminated via first pass through the urinary system prior to entering the systemic circulation. Consequently, if the injected chemical is a therapeutic agent, it may not be efficacious, or if it has nephrotoxic potential, it may cause substantial renal damage. 2.3  Amphibians

Amphibians probably offer some of the most novel options for toxicology research, given unique aspects of their physiology and metamorphic development. They often have short generational times, and the aquatic phases of certain anurans (frogs and toads) can be exposed to test agents via water bath as well as simultaneously thru multiple absorption routes. They lay permeable eggs in water which is conducive for environmental contaminant exposure studies, their skin is permeable to fluids/gases, a water and land life cycle allows for multiple exposure routes, an algal diet of the young and carnivorous diet of adults enhance the opportunity to ingest contaminated food, and there is the opportunity to capitalize on animals that hibernate by exposing them to pollutant-rich sediment (Wolf 2013). Various time point-based sediment toxicity assessments exist, such as the USEPA Office of Prevention, Pesticides and Toxic Substances 850.1800 and ASTM guide E2591-07, and when coupled with other types of testing, such as measurements of sediment contaminant concentrations, a more complete picture of potential impacts to the aquatic community may be realized (Johnson et al. 2017). Studies have demonstrated that dermal exposures during terrestrial life stages can contribute significantly to total body burden in amphibians, as their ventral dermal surfaces are adept at water absorption, allowing for considerable exposure to water-solu-

858

Shannon M. Wallace and Jeffrey C. Wolf

ble compounds (Johnson et al. 2017). Differences in the timing of life cycle stage development among amphibian species can be leveraged, and exposures can be timed to take advantage of certain developmental windows. This is more readily accomplished for common laboratory subjects such as Xenopus spp., because information on the physiology and life history of many native amphibian species tends to be less available. Despite their lesser role in research, physiologic attributes of amphibians offer significant advantages, many of which have been successfully exploited. Amphibian metamorphosis is a well-defined thyroid-dependent process, and as a result, amphibians are often used to assess substances that may affect the hypothalamic-­pituitary-­ thyroid axis. The Amphibian Metamorphosis Assay, developed jointly by the US Environmental Protection Agency (USEPA) in conjunction with the Organization for Economic Cooperation and Development (OECD), exists to identify chemicals that may disrupt the thyroid axis, consequently resulting in developmental and histopathologic effects (Grim et al. 2009). Reptiles and amphibians are ectotherms; therefore, temperature and humidity can be used in various species to control embryonic development, growth, metamorphosis, and/or immune responses (Linder et al. 2010). Because amphibians can tolerate a range of ambient temperatures and oxygen saturations, they often serve as subjects for experiments involving heat shock proteins or hypoxia-inducing environments. Amphibians have long been a model for experimental embryology due to the ease of embryonic observation and manipulation. Xenopus spp., in particular, have been the focus of genetic sequencing efforts and studies involving in situ expression of organogenesis. Additional research benefits include ease of breeding, large and rapidly developing larvae, and amenability to genetic transformation. These benefits outweigh their reduced ability to tolerate surgical procedures as compared to other species. The well-known ability of amphibians to regenerate tissues such as myocytes makes them ideal candidates for studies that concern recovery from myocardial infarction. Defining the molecular basis of cardiac regeneration in amphibians may provide insight into myocyte regeneration, an ability that appears to have been lost through evolution in mammals (Parmacek and Epstein 2005). Species selection is important, as the regenerative capacity of anurans generally exceeds that of urodeles (salamanders and newts) (Burggren and Warburton 2007). The seasonal spawning habits and low laboratory survival of some wildcaught amphibian species pose significant challenges for the researcher, which must be taken into account when planning husbandry procedures and creating experimental designs. 2.4  Fish

Fish are among the most intensively studied nonmammalian research subjects, and carcinogenesis studies represent a fundamental area of early fish research. Few molecular differences exist

Non-mammalian Laboratory Species: Fish, Frogs, and Beyond

859

between fish and humans; for example, the human HRAS oncogene, a frequent cancer-causing offender when mutated, shares greater than 95% identity with the corresponding gene in a fish known as the Japanese medaka (Schartl 2014). Fish have generally demonstrated greater sensitivity to carcinogenic chemical exposure as compared to amphibians (Johnson et al. 2017). The rainbow trout (Oncorhynchus mykiss) is one of the older surrogate models for human cancer research and has demonstrated remarkable sensitivity to environmental carcinogens (Schartl 2014). Compared to humans, trout express comparable changes in gene expression in response to liver damage and demonstrate similar (KRAS) oncogene mutations in liver cancer (Schartl 2014). Small aquarium fish, in particular, offer a variety of advantages as research models. They are hardy, prolific, have short life cycles, and large numbers can be kept in relatively small spaces. They can be dosed via immersion, feed, gavage, intramuscular, or intraperitoneal routes. If chemicals are administered by water bath, exposure can be continuous, which is a feat that is difficult to achieve in mammals. For histopathologic examination, tissues may be excised, or if small fish species are used, the animals may be processed and sectioned whole in various planes (e.g., sagittal, frontal, or transverse). If fish are sectioned sagittally, it may be possible to evaluate greater than 30 tissue types in as few as two or three histologic slides. As in birds, external fertilization and embryo development allow for the observation and manipulation of developing embryos (Spitsbergen and Kent 2003). Teleost (bony) fish have the ability to functionally regenerate severed axons in the central nervous system; by studying the molecular mechanisms that control axon regeneration in fish, researchers may one day develop treatment options for triggering nerve repair in mammals (Diekmann et al. 2015). Female fish produce vitellogenin (Vtg), a precursor egg yolk protein required for oocyte maturation. Male fish typically have little Vtg, and thus, elevated levels of this phospholipoglycoprotein in males can be used to monitor for estrogenic substance exposure (Canesi and Fabbri 2015). Anatomic differences between mammals and fish may provide both advantages and disadvantages. For example, although the fish liver possesses similar biotransformation enzymes, it lacks the classic hepatic lobular architecture of mammals, its metabolic enzymes tend to be evenly distributed (versus zonally), it has a lower capacity to metabolize substances by these enzymes, and it has less sinusoidal perfusion. All of these factors suggest that fish may require greater concentrations of certain toxicants to elicit effects in challenge studies (Wolf and Wolfe 2005). Fish also lack certain tissues present in mammals, such as lungs, lymph nodes, hair follicles, adrenal glands, a true urinary bladder, uterus, thyroid gland c-cells, diaphragm, bone marrow, mammary glands, and salivary glands, to name a few. Some species, for example, such as medaka and

860

Shannon M. Wallace and Jeffrey C. Wolf

zebrafish, lack a glandular stomach and therefore are not ideal for gastric studies (Bunton 1996). However, for some research aims, certain anatomic differences may be beneficial. For example, the lack of glomeruli in the kidneys of seahorses and toadfish can facilitate the study of renal tubular function or toxicity (Reimschuessel 2001).

3  Disadvantages, Limitations, and Considerations Associated with the Use of Nonmammalian Species The use of nonmammalian species for toxicological research can involve some disadvantages and limitations. Laboratory experiments involving aquatic animals can be highly labor-intensive, and modern complex studies may require large initial investments in housing and dosing equipment. Long-term studies can be especially challenging, and the potential for errant environmental parameters to cause studies to be delayed or canceled is higher than that for most types of mammalian assays. Certain populations of nonmammalian laboratory animals continue to be plagued by infectious diseases that are difficult, if not impossible, to eradicate. Although the severity of these infections is minimal in most cases, their effects as potentially confounding study factors can be difficult to estimate. Well-established examples include microsporidian infections in the nervous tissue of zebrafish and the ovaries of fathead minnows, helminth parasitic infections in zebrafish (some of which have been associated with enteric tumor formation), and acid-fast bacterial infections, which can be problematic for both aquatic and terrestrial research species maintained in captivity. Opportunistic mycobacterial infection can be a significant problem in long-term aging or tumor studies in fish, and since mycobacterial antigens are immunostimulants, they can confound disease resistance or immune response research (Spitsbergen and Kent 2003). Nonmammalian species tend to have less historical data to draw from, which can affect the interpretation of histopathology results and determinations of causality in some instances. As compared to pathologists who evaluate mammalian (e.g., rodent) studies, there are far fewer nonmammalian pathologists, and comprehensive references concerning macroscopic and microscopic anatomy, physiology, and pathology are not as widely available, especially given the broad variety of species used as test subjects. Less is also known about the husbandry of some nonmammalian species, and this is especially true for many wildlife species that have not yet been widely cultivated. Although not necessarily a disadvantage or limitation, specialized nonstandard methods may be advisable for the histologic preparation of nonmammalian tissues. One example is the p ­ reparation of whole fish specimens, for which processing, embedding, and

Non-mammalian Laboratory Species: Fish, Frogs, and Beyond

861

sectioning can be challenging if procedures have not been fully optimized. For example, minor differences in the size/age of the test subjects can greatly influence the technical approach to be used for a given study. If extra specimens are available, it may be helpful to process a small number of non-study cohort animals ahead of the main project in order to fully validate the histologic methodology. A major consideration in the use of nonmammalian test subjects is the ability, real or perceived, to extrapolate the study results to other taxa, particularly humans. Despite a fairly high degree of genetic conservation (as discussed in the Introduction), there are study types for which nonmammalian species are clearly unsuitable due to translation issues. In some cases, this is attributable to the lack of comparable organ systems or tissues (as previously mentioned), but it can also be due to basic differences in physiology, natural route(s) of toxicant exposure, the types or numbers of receptor ligands, and mechanisms of xenobiotic metabolism, to name just a few variables. Interestingly, however, a high degree of predictivity has been demonstrated for some zebrafish models. For example, Vliegenthart et al. (2014) demonstrated that the metabolism of eight of nine studied liver toxicants was similar between zebrafish and humans, and McGrath and Li (2008) found that zebrafish correctly predicted the teratogenicity of 10 of 12 tested compounds. However, the applicability of data derived from fish bioassays for human chemical hazard and risk assessments has not yet been firmly established (Embry et al. 2010).

4  Applications of Nonmammalian Species in Toxicological Studies 4.1  Carcinogenicity Studies

Given the long-standing intense level of interest in preclinical and clinical cancer research, the relatively short lifespan of many nonmammalian species, and their propensity to develop a wide array of spontaneous and chemically induced tumor types, many of which strongly resemble comparable human tumors, animals such as fish and frogs have long been attractive subjects for carcinogenicity experiments. For example, studies in the 1920s first identified a spontaneous melanoma model in hybrid poecilid aquarium fishes (Xiphophorus sp.) (Patton et al. 2010). Further research based on such findings has resulted in the creation of transgenic zebrafish that can accelerate the detection of novel pharmaceutical candidate compounds. More recently, a number of studies have documented links between environmental contaminant exposure and the development of neoplasia in aquatic animals. Examples include hepatocellular adenomas, carcinomas, and cholangiocarcinomas in winter flounder (Pleuronectes americanus) collected from sewage outfall sites that had sediments high in mixtures of PAHs, polychlorinated biphenyls (PCBs), and heavy metals (Bunton 1996), and degenerative megalocytic hepatosis with foci of alteration and benign

862

Shannon M. Wallace and Jeffrey C. Wolf

and malignant hepatocellular and biliary neoplasms in adult English sole (Parophrys vetulus) from areas of Puget Sound, Washington, which were contaminated with PCBs, heavy metals, and other potential carcinogens (Myers 1991). Interestingly, responses of different fish species may vary even when they occupy an identical habitat niche in the same environment. For example, the prevalence of hepatic neoplasms is substantially greater in English sole (high prevalence) than it is in starry flounder (Platichthys stellatus) from the same contaminated locations (Myers 1991). In some cases, fish may be relatively more susceptible to tumor induction than mammals. Rainbow trout produce liver tumors in response to probable human carcinogens such as aflatoxins, nitrosamines, and PAHs, which makes them convenient carcinogenesis models; however, trout are relatively deficient in their ability to repair DNA adducts which may enhance their sensitivity (Williams et al. 2003). Fish often used in carcinogenicity studies conducted in laboratory settings include rivulus (Rivulus marmoratus), sheepshead minnow (Cyprinodon variegatus), and Japanese medaka (Oryyzias latipes). Each of these species has a low background tumor incidence and yet each is highly sensitive to carcinogen exposure. A major disadvantage of fish models is the lack of tissues that are of major human importance such as mammary gland, prostate, and lung (Bunton 1996). However, fish tend to have a shorter latency period than mammals for tumor development and often a lower spontaneous tumor incidence (Baumann and Okihiro 2000). Chickens serve as an experimental model for the spontaneous onset and progression of ovarian cancer (OVC). Prevalences range from 10% to 35% dependent on age, strain, reproductive history, and diet, with most tumors occurring in birds at or greater than 2 years of age (Hawkridge 2014). Morphologically, the subtypes of OVC are quite similar in humans and chickens, and both species have an elevated risk of OVC development with increasing ovulations (Hawkridge 2014). Compared to humans, chickens have a compressed ovulation cycle, which allows for a greater degree of OVC occurrence during a narrower time window (Hawkridge 2014). In contrast, the microscopic appearance and progression of OVC in rodents and humans are relatively dissimilar (Barua et al. 2009). 4.2  Metal Exposures

Although metals occur naturally in the environment, hazardous levels of metal contamination often result from anthropogenic activities such as mining and smelting operations, industrial use and production, and the agricultural use of metals and metal-­ containing compounds. The high degree of toxicity associated with these substances warrants the monitoring of environmental metal concentrations and metal-induced disease, as exposure can pose a significant risk to public health (Tchounwou et al. 2012). Heavy metals such as arsenic (As), cadmium (Cd), lead (Pb), and

Non-mammalian Laboratory Species: Fish, Frogs, and Beyond

863

mercury (Hg) are highly toxic to multiple organs, and some are classified as human carcinogens by the US Environmental Protection Agency (Tchounwou et al. 2012). In the case of mercury, all forms are toxic. In particular, methylmercury, which can be obtained from eating contaminated fish, is readily absorbed through the gastrointestinal tract and is able to cross both the placental and blood-brain barriers. Methylmercury accumulates in the kidneys, neural tissue, and liver, and significant exposure can result in gastrointestinal toxicity, neurotoxicity, and nephrotoxicity (Tchounwou et al. 2012). Wildlife species that reside within potentially contaminated environments serve as important first-line indicators of toxic metal effects. Birds have been used as both sentinel species for environmental metal contamination and as test subjects for investigating toxic effects. Laboratory exposure of chickens to metals such as lead and zinc and accidental poisonings in captive birds have led to changes in the exocrine pancreas such as necrosis, fibrosis, and atrophy (Sileo et al. 2004; Puschner and Poppenga 2009). In Sileo et al. (2004), waterfowl were collected at a site considered to be contaminated with lead, cadmium, and zinc. Although zinc intoxication had not previously been diagnosed in free-ranging wild birds, findings of atrophy and fibrosis of the exocrine pancreas were similar to lesions reported for experimental and environmental exposures. These morphologic indicators, when combined with the presence of significantly elevated zinc levels in the pancreas and liver, pointed to a diagnosis of zinc toxicosis. However, caution should be exercised when attributing causality to lesions observed in wild-caught specimens. For example, Spanish storks living within an environmental disaster site were exposed to potentially toxic levels of lead, zinc, arsenic, copper, and cadmium, and bill and leg deformities were observed in nestling storks collected following a spill that occurred in 1998 (Smits et  al. 2016). However, when additional storks were harvested from within metal-contaminated and comparatively clean reference sites, the flesh of the reference site birds often contained higher levels of toxic metals than those residing near the spill (Smits et al. 2016). Metal exposure can have varied effects on amphibians. Amphibian embryos are sensitive to aquatic environments contaminated by metals in addition to conditions of low pH. For example, effects of aluminum toxicity are highly dependent on pH, and exposed embryos exhibit one of two scenarios depending on the degree of acidity: at very low pH, development stops following initial exposure, whereas at higher (but still lethal) pH levels, embryos develop normally, but the vitelline membrane ceases to expand (Freda 1991). Constricting membranes either cause the death of the embryo or a curling effect created by progressive growth in a confined space; consequently, surviving embryos exhibit severe scoliosis (Freda 1991). Other metals such as copper have been shown

864

Shannon M. Wallace and Jeffrey C. Wolf

to significantly affect survival and development of southern leopard frog embryos, with significant effects at 100 ug/L or greater (Lance et al. 2012). Amphibian responses to metal exposures can also be different in the field when compared to the laboratory. Under natural conditions, amphibians are rarely exposed to just one metal; therefore, additive, synergistic, or antagonistic effects should be considered (Hopkins and Rowe 2010). In fish, the embryo and larval stages are generally considered to be the most sensitive to toxic substances; however, metal exposures of adults can also lead to contamination of eggs and sperm, resulting in reduced fertility and delayed embryonic development (Sfakianakis et al. 2015). Metals can be responsible for a variety of toxic effects in fish. In most experimental exposures conducted across a wide range of species, cadmium, copper, and zinc tended to affect hatching and larval survival, and each of those three metals and lead was found to cause spinal deformities. Similar deformities have been observed in wild fish collected from polluted regions (Sfakianakis et  al. 2015). Although various metals may produce similar toxic effects, they often do so via different mechanisms. Spinal deformities, for example, may be caused by disruption of calcium balance and subsequent bone fragility, which may be attributable to the genotoxic activities of metals such as copper and cadmium. In heavily polluted surface waters, metal exposure can be an indirect cause of nutritional deficiency, as a result of decreased or altered food consumption (Sfakianakis et al. 2015). 4.3  Endocrine Disruption

Endocrine-active substances (EASs) include natural and synthetic chemicals that, under certain conditions, have the potential to interact with one or more of the various endocrine systems of animals or humans. Alternatively, endocrine disrupting chemicals (EDCs) are exogenous compounds that cause adverse hormone-­ like effects when subjects are exposed to environmentally relevant concentrations. Exposure to EDCs may target growth and development, or ultimately interfere with normal homeostatic functions of the reproductive, immune, or neurologic systems, among others. Although the number of established EDCs is still relatively small, there are thousands of known or suspected EAS. Examples of anthropomorphogenic EAS include alkylphenols, bisphenol-A (BPA), dioxins, natural and synthetic estrogens, some pesticides, some metals, organochlorines, organophosphate flame retardants, some plastics and nanomaterials, some petroleum components, some pharmaceuticals (e.g., diethylstilbesterol), polyaromatic hydrocarbons, and polybrominated diphenyl ethers (Diamanti-­ Kandarakis et  al. 2009; Wolf and Wheeler 2018). Additionally, natural EASs include plant-based phytoestrogens that are frequently present in food products. Reproductive abnormalities observed in wild birds were among the major harbingers of ecological impairment which inspired pub-

Non-mammalian Laboratory Species: Fish, Frogs, and Beyond

865

lic and scientific scrutiny of EASs. The most publicized culprits, the organochlorine DDT (1,1,1-trichloro-2,2-bis[4-­chlorophenyl] ethane) and its metabolite DDE (1,1-dichloro-2,2-di[4-­ chlorophenyl]ethylene), were demonstrated to cause perturbations such as eggshell thinning and oviduct malformations, which are thought to have had adverse consequences on bird populations (Holm et al. 2006). The health of wild birds has been monitored for greater than 30 years in the Great Lakes region due to historically high levels of organochlorine contamination. Herring gulls (Larus argentatus) and terns (Sterna hirundo) in this region were observed to have developmental abnormalities and thyroid lesions, suggestive of hypothalamus-pituitary-thyroid (HPT) axis activation (Scanes and McNabb 2003). However, while some laboratory studies of PCB exposure in various species have been associated with thyroid effects, other results have been negative or even contradictory (Scanes and McNabb 2003). A standardized multigenerational assay, the Avian Two-generation Toxicity Test in the Japanese Quail (US EPA 2015a), can be used to investigate potential endocrine effects in birds such as Japanese quail or Northern bobwhite quail. Tissues collected for histopathologic analysis in chicks and adults include hormonally sensitive organs such as the thyroid glands, adrenal glands, cloacal glands, and reproductive tract, plus tissues that may exhibit evidence of systemic toxicity such as the liver, kidney, spleen, thymus, bursa of Fabricius, and gastrointestinal tract. The cloacal glands of Japanese quail are sexually dimorphic; consequently, the glands of males can be feminized by exposure to exogenous estrogenic compounds, and those of females may be masculinized by xenoandrogen exposure. Non-­ morphologic endpoints of this assay include adult and embryonic mortality, egg production, egg-shell thickness, fertility, hatchability, embryonic mortality, and viability. Reproductive data must be collected from birds prior to treatment start to ensure that egg production meets acceptable criteria to serve as a baseline for evaluating treatment effects on fecundity. Because amphibian metamorphosis is considered to be a thyroid hormone-dependent process, wild anuran frogs and laboratory species such as Xenopus laevis have become premier models for evaluating the potential of various EAS to disrupt the thyroid axis. In studies with X. laevis, thyroid-active EDCs have been shown to produce clear and reliable effects in the thyroids of tadpoles sacrificed near the peak of metamorphosis, including follicular cell hypertrophy, follicular cell hyperplasia, thyroid hypertrophy, thyroid atrophy, and colloid changes (Grim et al. 2009). Currently, two standardized tests that are available for evaluating thyroid function in X. laevis are the Amphibian Metamorphosis Assay (AMA; US EPA 2011a) and the Larval Amphibian Growth and Development Assay (LAGDA; US EPA 2015b). Endpoints other than histopathology that are incorporated in these tests include

866

Shannon M. Wallace and Jeffrey C. Wolf

measurements of hind limb length, developmental stage, and ­time-­to-­metamorphosis. The LAGDA assay additionally includes collection and histopathologic analysis of the liver, kidney, and reproductive tract in post-metamorphic juveniles. A variety of fish species are used to investigate potential effects of endocrine-active chemicals, with particular emphasis on perturbations of the reproductive system. Examples include the zebrafish, fathead minnow, Japanese medaka, and stickleback (Gasterosteidae). Wild fishes such as the European common roach (Rutilus rutilus) and domestic centrarchids such as the smallmouth bass (Micropterus dolomieu) have also been studied due to the predilection of their males to develop oocytes within their testes, either in response to environmental contamination, or possibly spontaneously in some cases. This phenomenon should not be confused with the sequential hermaphroditism that occurs naturally in a variety of marine fishes, such as clownfish, wrasses, and groupers. Most of the EAS tested to date in fish have been estrogenic substances of low to moderate potency, but other investigated modes of action have included androgenic, antiestrogenic, antiandrogenic, progestogenic, aromatase inhibition, and steroidogenesis inhibition (Scholz and Klüver 2009). In addition to testicular oocytes, EDCs may induce a variety of gonadal and extra-gonadal changes in fish, depending on the substance assayed, species and sex of the fish, and the timing and duration of exposure (Scholz and Klüver 2009). Examples of gonadal changes in testes include spermatogenic cell degeneration, changes in spermatogenic cell proportions, alterations in Leydig cell numbers, and disturbances in sex ratio (e.g., due to complete gonadal feminization). Exposure-­related findings in ovaries can include degenerative or proportional changes in oogenic cell populations, decreased incorporation of yolk into oocytes, hyperplasia or hypertrophy of perifollicular cells (e.g., granulosa cells), and gonad masculinization. Two standardized tests developed for the US EPA’s Endocrine Disruptor Screening Program are the Fish Short-Term Reproduction Assay (FSTRA; US EPA 2011b) and the Medaka Extended One Generation Reproduction Test (MEOGRT; US EPA 2015c). Further endpoints incorporated in these guidelines include survival, growth, fecundity and fertility, time to hatch, external sex ratio, assessment of secondary sex characteristics, and measurement of vitellogenin (Vtg; a phospholipoglycoprotein required for oocyte maturation). The MEORGT assay includes microscopic examination of the liver and kidney in addition to the gonads. One example of a substance that has been studied in fish is bisphenol A (BPA), which is a product of flame retardants and epoxy resins and has had a variety of commercial applications. At relatively high concentrations, BPA is known to have estrogenic effects, which in fish may translate to elevated Vtg levels, especially in males, and altered sex ratios (Canesi and Fabbri

Non-mammalian Laboratory Species: Fish, Frogs, and Beyond

867

2015). Compared to mammals, the potency of BPA may be enhanced in fish, because exposure via the gills bypasses metabolic degradation by the liver (Canesi and Fabbri 2015). 4.4  Developmental Toxicity

Despite continuous advances in computational and informational biology, the field of developmental toxicity testing remains highly dependent on studies that are conducted using pregnant animals (DeSesso 2017). Multigenerational studies can be designed to detect effects that are evident at birth (e.g., potential teratogens) plus latent effects that may not manifest until later in life. Exposures can occur prior to conception (F0 parental generation), prenatally, or at any point prior to sexual maturation. One example of a substance studied in alternative species is the degreaser/solvent trichloroethylene. Trichloroethylene (TCE) is a common contaminant of groundwater and soil, and it is present in air emissions. Although suspected to be a human cardiac teratogen, results of rodent studies and epidemiological investigations in humans have been mixed. However, TCE has been demonstrated to have cardiac teratogenic activity in birds (Rufer et al. 2010). Exposures performed during organogenesis in chick embryos at test concentrations near the EPA’s maximum contamination level (MCL) caused high mortality and functional dysmorphologies in surviving chicks that included a significant number of ventricular septal defects. Although no longer produced, PCBs continue to persist in the environment and in animals, warranting continued attention. For example, diet studies have shown absorption and subsequent transfer of PCBs from hen to developing embryos (Scanes and McNabb 2003), and teratogenic effects have been demonstrated following maternal or in ovo exposure (Golub et al. 1991). Amphibians are utilized as models in developmental toxicity for teratogenic effects in wildlife and in humans. For example, the Frog Embryo Teratogenesis Assay (FETAX) uses the African clawed frog (Xenopus laevis) as a test subject to rapidly identify developmental toxicants (ASTM 2017). A FETAX study (Brennan et  al. 2005) determined that exposure to low concentrations of bromodichloromethane (BDCM) and chloroform (by-products of water disinfection) resulted in a variety of adverse developmental effects that included inhibited growth, developmental defects of the notochord and face, and cardiac edema. In that same study, sodium chlorate exposure was associated with growth inhibition and inhibition of gut coiling, while dibromoacetic acid (DBAA) produced defects or growth inhibition only at lethal test concentrations. Although epidemiologic studies associate chronic exposure to DWDB with increased stillbirths and spontaneous ­ abortions in pregnant women, a clear causal relationship in humans has not yet been established (Brennan et al. 2005).

868

Shannon M. Wallace and Jeffrey C. Wolf

The use of small fish models such as zebrafish embryos for the assessment of potential teratogenic and developmental toxicity is gaining traction, based on favorable characteristics such as high fecundity, rapid embryogenesis, and transparency of the embryos (Rajini and Revathy 2015). A zebrafish embryo model was used to investigate TCDD (2,3,7,8-tetrachlorodibenzo-pdioxin), a halogenated aromatic hydrocarbon (HAH). TCDD is a prototype compound known to affect the survival, growth, and reproduction of wildlife species in contaminated areas. Fish are among the most sensitive vertebrates to TCDD effects, and reported consequences of exposure include wasting syndrome, delayed mortality, epithelial and lymphomyeloid lesions, cardiovascular dysfunction, edema, hemorrhage, craniofacial malformations, and impaired reproduction (King-Heiden et al. 2012). King-Heiden et  al. (2012) successfully utilized zebrafish as a model to predict the risk of exposure to dioxin-like compounds for wild fish populations, and it may be relevant to note that an increased risk of valvular stenosis and other cardiac malformations has also been established for humans living near inland waters contaminated with HAHs (King-Heiden et al. 2012). Direct testing of zebrafish embryos is also unique in that the mother is not required to administer a toxicant, whereas in mammalian models that require toxicant dosing of the mother, determining exact exposure amounts to embryos can be difficult. Exact exposure amounts can be helpful when determining risk of sensitive populations such as children (DeMicco et al. 2010). Pyrethroids, although considered a safer pesticide due to lower toxicity to nontarget species, can persist in household dust and air for months after application (Leng et al. 2005), making children a population of concern. In DeMicco et al. (2010), only high doses of pyrethroids in zebrafish were observed to show similar mild effects observed in mammalian models, with Type I and Type II pyrethroids similar to rodents. In contrast, minor alterations of cartilaginous structures were observed at high doses with specific permethrins, such as deltamethrin, while fetotoxicity or teratogenicity had not been found in rats or mouse dams similarly exposed to deltamethrin. The differences in findings between species are most likely due to the difference in dosing regimens. Similar to toxicity testing in amphibians, direct embryo exposure in fish can result in greater exposures to embryos compared to mammals where the exposed dam can reduce the amount of exposure to her embryo. With similar effects demonstrated between mammalian and zebrafish exposure to pyrethroids, this study demonstrated the suitability of the zebrafish as a toxicity model; in addition, it also demonstrated utility of direct embryo testing that mammalian models cannot provide.

Non-mammalian Laboratory Species: Fish, Frogs, and Beyond

869

4.5  Therapeutics and Feed Additives for Nonmammalian Species (Target Animal Safety Studies)

Studies are conducted to investigate the safety of chemicals or other treatments used to enhance the health, welfare, and/or growth of animals. For nonmammalian species, such efforts are generally geared toward production animals, and thus, domestic fowl and aquacultured fish are overrepresented. Target animal safety studies (TAS) are required for registration of veterinary products (VICH 2008), and guidance for the conduct of TAS has been established by the International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products. This guidance exists to establish standards between participating regions’ regulatory agencies and minimize duplication of research efforts, thus reducing overall cost and animal use (VICH 2008). Further guidance is available from the US FDA at https://www.fda.gov/downloads/ A n i m a l Ve t e r i n a r y / G u i d a n c e C o m p l i a n c e E n f o r c e m e n t / GuidanceforIndustry/ucm052464.pdf. Target animal safety studies are designed to mimic administration of the test substance under “real world” conditions. Common test articles include antibiotic or antifungal agents, parasiticides, and hormonal substances used to promote growth or regulate reproduction. Genetically modified animals may also be assessed to determine if the specific genetic change has adverse health effects. Typically, the lowest tested concentration (1X) simulates the dose proposed for use of the candidate product, and two multiples of the low dose (e.g., 2X and 3X, or 3X and 5X) are incorporated in the experimental protocol to establish the margin of safety.

4.6  Impacts of Environmental Toxicants on At-Risk Species

As a consequence of habitat degradation, dispersal of pathogens, introduction of nonnative predators, hunting by humans, climate change, and toxic substance exposure, a number of wildlife species have either disappeared or faced serious population declines. Included are nonmammalian taxa such as birds and fish; however, of the vertebrates, amphibians and reptiles are considered to be at greatest risk (Ortiz-Santaliestra et al. 2018). Although the relative degree to which toxicants impact wildlife populations as compared to other factors is often unknown (Köhler and Triebskorn 2013), they are still the subject of intense study, in part because the use of anthropomorphogenic chemicals can be potentially controlled by regulation. Because it is not feasible to perform testing on each of the myriad at-risk species, and duplicative efforts are expensive in terms of monetary cost and animal life, scientists often rely on data obtained from assays that use domesticated laboratory models or suitable wildlife surrogates. The selection of an appropriate surrogate for a given combination of species, test article, and exposure route can be challenging, and the relative sensitivity of various taxa to different classes of chemicals can vary. For example, there is evidence that fish may be the proxy of choice for investigating effects of waterborne toxicants in aquatic amphibian and reptiles (or during their aquatic life cycle stages) (Ortiz-Santaliestra et al. 2018),

870

Shannon M. Wallace and Jeffrey C. Wolf

whereas information gleaned from mammalian and avian studies may be more applicable for modeling oral exposures in terrestrial amphibians (Crane et al. 2016).

5  Future Considerations and Challenges Currently, the universe of well-trained comparative pathologists who focus on toxicology studies that involve nonmammalian test subjects is relatively small. Consequently, many morphologic investigations in nontraditional species are performed by scientists for whom histopathology is not necessarily their primary area of expertise. As a result, the quality and credibility of published histopathology data for such studies can be highly variable and not always dependable (Wolf and Maack 2017; Wolf and Wheeler 2018). There are a number of ideas and initiatives that can be instated to combat this knowledge gap (Wolf 2018). Not surprisingly, supplementary training of anatomic and clinical pathologists so that they may knowledgeably and confidently evaluate species beyond the typical domesticated and laboratory test subjects is key. For both classically trained pathologists and non-pathologist scientists, this type of advanced training may be accomplished through specialized coursework, but given the frenetic pace of contemporary research when compared to the relatively limited extent of universally available educational opportunities, many professionals will need to rely on training materials in the form of well-constructed texts and scientifically accurate articles. The generation of such materials will require the collaboration of pathologists with colleagues from multiple disciplines, including biologists, anatomists, toxicologists, and physiologists. Standardization of diagnostic terminology and criteria can help to ensure that pathologists with diverse training experience can communicate study results accurately and effectively. One current initiative dedicated to this purpose is the International Harmonization of Nomenclature and Diagnostic Criteria (INHAND 2019) which is sponsored internationally by several professional societies of toxicologic pathology. Presently, standardized nomenclature and criteria are being developed individually for avian and fish studies. Review of initial study materials (e.g., glass slides, images, and narrative report) by a second pathologist who has expertise in the evaluation of the nonmammalian target species (pathology peer review) can help to improve the quality of the experimental data and diagnostic interpretations. Finally, the ultimate responsibility for ensuring the ­scientific integrity of published pathology results defaults to journal reviewers and editors. The growth in literature and numbers of conferences dedicated to this topic suggests that interest in the health impacts of toxic and therapeutic substances in nonmammalian species appears to be increasing. Although there is commendable effort toward

Non-mammalian Laboratory Species: Fish, Frogs, and Beyond

871

replacing in vivo testing with in vitro methods, such methods will require validation against morphologic responses before they can be considered as viable substitutes. Additionally, there may be exposure scenarios that remain difficult or impossible to replicate using tissue cultures or in silico models exclusively. Thus, for the foreseeable future, there will likely continue to be a need, albeit reduced, for the “animal as laboratory.” References Aleström et  al (2006) Zebrafish in functional genomics and aquatic biomedicine. Trends Biotechnol 24:15–21 ASTM (American Society for Testing and Materials) E1439-98 (2004) Standard guide for conducting the Frog Embryonic Teratogenesis Assay-­ Xenopus (FETAX). www.astm.org. Accessed 10 Oct 2017 AVMA (2018) (American Veterinary Medical Association) Comparative medicine and translational research, https://www.avma.org/KB/ Policies/Pages/Comparative-Medicine-andTranslational-Research.aspx. Accessed 11 Jan 2018 Barua A et  al (2009) Histopathology of ovarian tumors in laying hens, a preclinical model of human ovarian cancer. Int J  Gynecol Cancer 19(4):531–539 Baumann PC, Okihiro MS (2000) Cancer, experimental models. In: Laboratory fish. Academic Press, London, pp 591–616 Berg C, Halldin K, Fridolfsson AK, Brandt I, Brunström B (1999) The avian egg as a test system for endocrine disrupters: effects of diethylstilbestrol and ethynylestradiol on sex organ development. Sci Total Environ 233(1–3):57–66 Bishop CA, McDaniel TV, de Solla SR (2010) Atrazine in the environment and its implications for amphibians and reptiles. In: Ecotoxicology of amphibians and reptiles, 2nd edn. CRC Press and SETAC, Pensacola, pp 227–259 Bradaric MJ, Barua A, Penumatsa K, Yi Y, Edassery SL, Sharma S, Abramowicz JS, Bahr JM, Luborsky JL (2011) Sphingosine-1 phosphate receptor (S1p1), a critical receptor controlling human lymphocyte trafficking, is expressed in hen and human ovaries and ovarian tumors. J Ovarian Res 4:1–12 Brennan LM et  al (2005) Developmental toxicity of drinking water disinfection by-­products to embryos of the African Clawed Frog (Xenopus laevis). Bull Environ Contam Toxicol 75:361–367 Brown RE, Brain JD, Wang N (1997) The avian respiratory system: a unique model for studies of respiratory toxicosis and for monitoring air quality. Environ Health Perspect 105(2):188–200

Bryda EC (2013) The Mighty Mouse: the impacts of rodents on advances in biomedical research. Mo Med 110(3):207–211 Bunton TE (1996) Experimental chemical carcinogenesis in fish. Toxicol Pathol 24(5):603–618 Burggren WW, Warburton S (2007) Amphibians as animal models for laboratory research in physiology. ILAR J 48(3):260–269 Caldwell DJ, Eldridge SR, Lington AW, McKee RH (1999) Retrospective evaluation of alpha 2u-globulin accumulation in male rat kidneys following high doses of diisononyl phthalate. Toxicol Sci 51:153–160 Canesi L, Fabbri E (2015) Environmental effects of BPA: focus on aquatic species. Dose Response 3:1–14 Crane M, Finnegan M, Weltje L, Kosmala-­ Grzechnik S, Gross M, Wheeler JR (2016) Acute oral toxicity of chemicals in terrestrial life stages of amphibians: comparisons to birds and mammals. Regul Toxicol Pharmacol 80:335–341 DeMicco A, Cooper KR, Richardson JR, White LA (2010) Developmental neurotoxicity of pyrehtroid insecticides in Zebrafish embryos. Toxicol Sci 113(1):177–186 DeSesso JM (2017) Future of developmental toxicity testing. Curr Opin Toxicol 3:1–5 Diamanti-Kandarakis E, Bourguignon J-P, Giudice LC, Hauser R, Prins GS, Soto AM, Zoeller RT, Gore AC (2009) Endocrine-disrupting chemicals: an endocrine society scientific statement. Endocr Rev 30(4):293–342 Diekmann H et al (2015) Characterization of optic nerve regeneration using transgenic Zebrafish. Front Cell Neurosci 9(118):1–11. https://doi. org/10.3389/fncel.2015.00118 Embry MR, Belanger SE, Braunbeck TA, Galay-­ Burgos M, Halder M, Hinton DE, Léonard MA, Lillicrap A, Norberg-King T, Whale G (2010) The fish embryo toxicity test as an animal alternative method in hazard and risk assessment and scientific research. Aquat Toxicol 97(2):79–87 Freda J (1991) The effects of aluminum and other metals on amphibians. Environ Pollut 71(2–4):305–328 Furuya M, Sasaki F, Hassanin AM, Kuwahara S, Tsukamoto Y (2003) Effects of bisphenol-A on

872

Shannon M. Wallace and Jeffrey C. Wolf

the growth of comb and testes of male chicken. Can J Vet Res 67(1):68–71 Golub MS, Donald JM, Reyes JA (1991) Reproductive toxicity of commercial PCB mixtures: LOAELs and NOAELs from animal studies. Environ Health Perspect 94:245–253 Grim KC et  al (2009) Thyroid histopathology assessments for the amphibian metamorphosis assay to detect thyroid-active substances. Toxicol Pathol 37:415–424 Hawkridge AM (2014) The Chicken Model of spontaneous ovarian cancer. Proteomics Clin Appl 8(9–10):689–699 Holm L, Blomqvist A, Brandt I, Brunström B, Ridderstråle Y, Berg C (2006) Embryonic exposure to o,p′-DDT causes eggshell thinning and altered shell gland carbonic anhydrase expression in the domestic hen. Environ Toxicol Chem 25(10):2787 Hopkins WA, Rowe CL (2010) Interdisciplinary and hierarchical approaches for studying the effects of metals and metalloids on amphibians. In: Ecotoxicology of amphibians and reptiles, 2nd edn. CRC Press and SETAC, Pensacola, pp 325–336 Howe K et  al (2013) The zebrafish reference genome sequence and its relationship to the human genome. Nature 496(7446):498–503. https://doi.org/10.1038/nature12111 Hytonen MK, Lohi H (2016) Canine models of human rare disorders. Rare Dis 4(1):e1241362-e1241362-6. https://doi.org/ 10.1080/21675511.2016.1241362 INHAND (2019). https://www.goreni.org/. Accessed 25 March 2019 Jaspers VLB (2015) Selecting the right bird model in experimental studies on endocrine disrupting chemicals. Front Environ Sci 3(35):1–7 Johnson MS, Aubee C, Salice CJ, Leigh KB, Liu E, Pott U, Pillard D (2017) A review of ecological risk assessment methods for amphibians: comparative assessment of testing methodologies and available data. Integr Environ Assess Manag 13(4):601–613 Kalmar ID, Janssens GP, Moons CP (2010) Guidelines and ethical considerations for housing and management of psittacine birds used in research. ILAR J 51(4):409–423 King-Heiden TC et  al (2012) Reproductive and developmental toxicity of dioxin in fish. Mol Cell Endocrinol 354(1–2):121–138 Köhler HR, Triebskorn R (2013) Wildlife ecotoxicology of pesticides: can we track effects to the population level and beyond? Science 341(6147):759–765 Lance SL, Erickson MR, Flynn RW, Mills GL, Tuberville TD, Scott DE (2012) Effects of chronic copper exposure on development and survival in the southern leopard frog (Lithobates [Rana] sphenocephalus). Environ Toxicol Chem 31(7):1587–1594

Leng G, Berger-Preiss E, Levsen K, Ranft U, Sugiri D, Hadnagy W, Idel H (2005) Pyrethroids used indoor  - ambient monitoring of pyrethroids following a pest control operation. Int J  Hyg Environ Health 208:193–199 Linder GE et  al (2010) Physiological ecology of amphibians and reptiles, natural history and life history attributes framing chemical exposure in the field. In: Ecotoxicology of amphibian and reptiles, 2nd edn. CRC Press and SETAC, Pensacola, pp 107–149 Massa R, Davies DT, Bottoni L (1980) Cloacal gland of the Japanese quail: androgen dependence and metabolism of testosterone. J Endocrinol 84(2):223–230 McGrath P, Li CQ (2008) Zebrafish: a predictive model for assessing drug-induced toxicity. Drug Discov Today 13(9–10):394–401 Meierjohann S, Schartl M (2006) From Mendelian to molecular genetics: the Xiphophorus melanoma model. Trends Genet 22(12):654–661 Myers MS (1991) Relationships between hepatic neoplasms and related lesions and exposure to toxic chemicals in marine fish from the U.S. West Coast. Environ Health Perspect 90:7–15 NABR (National Association for Biomedical Research 2010–2017). http://www.nabr.org/ biomedical-research/laboratory-animals/species-in-research/dogs. Accessed 24 May 2018 Ortiz-Santaliestra ME, Maia JP, Egea-Serrano A, Lopes I (2018) Validity of fish, birds and mammals as surrogates for amphibians and reptiles in pesticide toxicity assessment. Ecotoxicology 27(7):819–833 Parmacek MS, Epstein JA (2005) Pursuing cardiac progenitors: regeneration Redux. Cell 120:295–298 Patton EE, Mitchell DL, Nairn RS (2010) Genetic and environmental melanoma models in fish. Pigment Cell Melanoma Res 23:314–337 Phillips KA et al (2014) Why primate models matter. Am J Primatol 76(9):801–827. https://doi. org/10.1002/ajp.22281 Poynter G, Huss D, Lansford R (2009) Japanese quail: an efficient animal model for the production of transgenic avians. Cold Spring Harb Protoc. https://doi.org/10.1101/pdb. emo112 Puschner B, Poppenga RH (2009) Lead and zinc intoxication in companion birds. Compend Contin Educ Vet 31(1):E1–12 Rajini A, Revathy K (2015) Teratogenic and developmental toxicity of combination pesticide to Danio rerio embryo-larval stages. Res J Chem Environ 19(8):1–7 Reimschuessel R (2001) A fish model of renal regeneration and development. ILAR J 42(4):285–291 Rufer ES et  al (2010) Altered cardiac function and ventricular septal defect in avian embryos

Non-mammalian Laboratory Species: Fish, Frogs, and Beyond exposed to low-dose trichloroethylene. Toxicol Sci 113(2):444–452 Scanes CG, McNabb FMA (2003) Models for research in toxicology and endocrine disruption. Avian Poult Biol Rev 14(1):21–52 Schartl M (2014) Beyond the zebrafish: diverse fish species for modeling human disease. Dis Model Mech 7:181–192 Scholz S, Fischer S, Gündel U, Küster E, Luckenbach T, Voelker D (2008) The zebrafish embryo model in environmental risk assessment-applications beyond acute toxicity testing. Environ Sci Pollut Res Int 15(5):394–404 Scholz S, Klüver N (2009) Effects of endocrine disrupters on sexual, gonadal development in fish. Sex Dev 3(2–3):136–151 Sfakianakis DG, Renieri E, Kentouri M, Tsatsakis AM (2015) Effect of heavy metals on fish larvae deformities. Environ Res 137:246–255 Shuster KA, Brock KL, Dysko RC, DiRita VJ, Bergin IL (2012) Polytetrafluoroethylene toxicosis in recently hatched Chickens (Gallus domesticus). Comp Med 62(1):49–52 Sileo L, Wn B, Mateo R (2004) Pancreatitis in wild zinc-poisoned waterfowl. Avian Pathol 32(6):655–660 Smits JEG, Bortolotti GR, Baos R, Blas J, Hiraldo  F, Xie Q (2016) Skeletal pathology in white storks ( ) associated with heavy metal contamination in southwestern Spain. Toxicol Pathol 33(4):441–448 Spitsbergen JM, Kent ML (2003) The state of the art of the Zebrafish Model for toxicology and toxicologic pathology research-advantages and current limitations. Toxicol Pathol 31S:62–87 Tchounwou PB, Yejou CG, Patlolla AK, Sutton (2012) Heavy metal toxicity and the environment. Exp Suppl 101:133–364 Touart LW (2004) Factors considered in using birds for evaluating endocrine-disrupting chemicals. ILAR J 45:462–468 U.  S. EPA (U.S.  Environmental Protection Agency)) (2011a) Amphibian metamorphosis assay. Endocrine disruptor screening program: Office of Chemical Safety and Pollution Prevention (OCSPP) OCSPP Guideline 890.1100, https://www.epa.gov/endocrinedisruption/amphibian-metamorphosis-assaysep-and-dest.html). Accessed 11 Jan 2018 U.  S. EPA (U.S.  Environmental Protection Agency)) (2011b) Fish short-term reproduction assay. Endocrine disruptor screening program: Office of Chemical Safety and Pollution Prevention (OCSPP) OCSPP Guideline 890.1350., https://www.epa.gov/ sites/production/files/2015-07/documents/ final_890.1350_fish_short_term_reproduction_ assay_sep_10.6.11.pdf). Accessed 24 May 2018 U.  S. EPA (U.S.  Environmental Protection Agency) (2015a) Avian two-generation toxicity

873

test in the Japanese Quail. Endocrine Disruptor Screening Program: Office of Chemical Safety and Pollution Prevention (OCSPP) OCSPP Guideline 890.2100, https://www.epa.gov/ sites/production/files/2015-07/documents/ final_890.1350_fish_short_term_reproduction_assay_sep_10.6.11.pdf). Accessed 24 May 2018 U.  S. EPA (U.S.  Environmental Protection Agency)) (2015b) Larval amphibian growth and development assay. Endocrine disruptor screening program: Office of Chemical Safety and Pollution Prevention (OCSPP) OCSPP guideline 890.2300., https://www. regulations.gov/document?D=EPA-HQOPPT-2009-0576-0018. Accessed 11 Jan 2018 U. S. EPA (U.S. Environmental Protection Agency) (2015c) Endocrine disruptor screening program test guidelines. OCSPP 890.2200: Medaka extended one-generation reproduction test (MEOGRT), https://www.regulations.gov/ document?D=EPA-HQ-OPPT-2009-0576-0019. Accessed 24 May 2018 VICH (Veterinary International Center Harmonization Steering Committee) (2008) Target Animal Safety (TAS) for Veterinary Pharmaceutical Products. VICH GL 43 (Target Animal Safety) – Pharmaceuticals July 2008. 1–19 Vliegenthart AD, Tucker CS, Del Pozo J, Dear JW (2014) Zebrafish as model organisms for studying drug-induced liver injury. Br J  Clin Pharmacol 78(6):1217–1227 Williams DE, Bailey GS, Reddy A, Hendricks JD, Oganesian A, Oner GA, Pereira CB, Swenberg JA (2003) The Rainbow Trout (Oncorhynchus mykiss) Tumor Model: recent applications in low-dose exposures to tumor initiators and promoters. Toxicol Pathol 31S:58–61 Wolf JC (2013) Alternative animal models. In: Haschek and Rousseaux’s handbook of toxicologic pathology, 3rd edn. Academic Press, Waltham, pp 477–518 Wolf JC (2018) Fish toxicologic pathology: the growing credibility gap and how to bridge it. Bull Eur Ass Fish Pathol 38(2):51–64 Wolf JC, Maack G (2017) Evaluating the credibility of histopathology data in environmental endocrine toxicity studies. Environ Toxicol Chem 36(3):601–611 Wolf JC, Wheeler JR (2018) A critical review of histopathological findings associated with endocrine and non-endocrine hepatic toxicity in fish models. Aquat Toxicol 197:60–78 Wolf JC, Wolfe MJ (2005) A brief overview of nonneoplastic hepatic toxicity in fish. Toxicol Pathol 33:75–85 Zhu F et  al (2014) Fishing on chips: up-and-­ coming technological advances in analysis of Zebrafish and Xenopus embryos. Cytometry A 85A:921–932

 ppendix: Fundamental Pathology Terminology A Based on the Standard for the Exchange of Nonclinical Data (SEND) Any seasoned world traveler will tell you that one of the biggest obstacles to fully appreciating a new culture is understanding the language. To toxicologists, the “culture” of pathology can seem very foreign due to the unique language used by pathologists with terms and syntax that are unfamiliar to those who have not trained in the discipline. A focus of this book is to bridge this cultural gap. Thankfully, in the past decade, pathologists have made significant strides in removing the variances in terminology. The Society of Toxicologic Pathology (STP) along with British, Japanese, and European STPs has undertaken a significant effort to standardize diagnostic terminology through the International Harmonization of Nomenclature and Diagnostic Criteria (INHAND). This global effort has made tremendous progress in standardizing the language of toxicologic pathology and ultimately produced the anatomic pathology lexicon for Standard for the Exchange of Nonclinical Data (SEND). The SEND project is led by the Clinical Data Interchange Standards Consortium (CDISC). CDISC partners with the National Cancer Institute and makes all of its terminology freely downloadable at the following website: https:// www.cancer.gov/research/resources/terminology/cdisc#4 Below we have provided a portion of the current list of pathology terminology for SEND data, including nonneoplastic diagnoses and their modifiers; but please note that this list is not intended to be the definitive list of all current or future terms. Our intent is to provide the reader with a handy source of some of the terms and definitions pathologists use in creating pathology data tables and in discussing the results within the pathology report. We hope that this list can serve as a convenient and readily accessible reference and help to make the language of pathology data provided in pathology tables and reports less foreign.

Thomas J. Steinbach et al. (eds.), Toxicologic Pathology for Non-Pathologists, https://doi.org/10.1007/978-1-4939-9777-0, © Springer Science+Business Media, LLC, part of Springer Nature 2019

875

876

Appendix: Fundamental Pathology Terminology Based on the Standard…

Modifier/ diagnosis Type

Term

Modifier

Chronicity

Acute

Morphologic changes that have a rapid onset

Modifier

Chronicity

Chronic

Morphologic changes that are persistent or long standing

Modifier

Chronicity

Chronic active

Morphologic changes that are persistent or long standing, superimposed with areas of acute change

Modifier

Chronicity

Peracute

Morphologic changes of very short or immediate onset. This onset is more rapid than that which is seen in an acute chronicity

Modifier

Chronicity

Subacute

Morphologic changes containing characteristics of both acute and chronic but predominantly acute

Modifier

Chronicity

Subchronic

Morphologic changes containing characteristics of both acute and chronic but predominantly chronic

Modifier

Distribution

Diffuse

Modifier

Distribution

Focal

Limited to a specific area

Modifier

Distribution

Focal/multifocal

A finding that generally has features of focal and multifocal distribution

Modifier

Distribution

Locally extensive

Modifier

Distribution

Multifocal

Arising from, pertaining to, or having many locations

Modifier

Distribution

Multiple

More than one

Modifier

Distribution

Single

One

Modifier

Neoplastic classification

Benign

For neoplasms, a noninfiltrating and nonmetastasizing neoplastic process that is characterized by the absence of morphologic features associated with malignancy (e.g., severe atypia, nuclear pleomorphism, tumor cell necrosis, and abnormal mitoses). For other conditions, a process that is mild in nature and not dangerous to health

Synonym(s)

Widespread

Focally extensive; regionally extensive

Definition

Widely spread; not localized or confined

Being widespread throughout a specific area

(continued)

Appendix: Fundamental Pathology Terminology Based on the Standard…

877

Modifier/ diagnosis Type

Term

Modifier

Neoplastic classification

Malignant

Refers to abnormal cell activity manifested by decreased control over growth and function, causing tumor growth or spread into surrounding tissue and adverse effects to the host

Modifier

Neoplastic classification

Metastatic

A term referring to the pathologic observation of a tumor extension or migration from its original site of growth to another nonadjacent site

Diagnosis Neoplasm type

Adenoma

A benign neoplasm arising from epithelium

Diagnosis Neoplasm type

Carcinoma

Diagnosis Neoplasm type

Fibroma

A benign neoplasm arising from fibrous tissue

Diagnosis Neoplasm type

Fibrosarcoma

A malignant mesenchymal neoplasm of the soft tissue and bone

Diagnosis Neoplasm type

Lymphoma

Lymphoma (Hodgkin and non-Hodgkin); lymphoma (Hodgkin’s and non-Hodgkin’s); malignant lymphoma

A malignant neoplasm composed of lymphocytes of B- or T-/NK-cell phenotype

Diagnosis Neoplasm type

Osteosarcoma

Osteogenic sarcoma

A malignant neoplasm usually arising from the bone

Diagnosis Neoplasm type

Papilloma

Diagnosis Neoplasm type

Sarcoma

Synonym(s)

Definition

Epithelial carcinoma; A malignant epithelial neoplasm epithelioma malignant; malignant epithelial neoplasm; malignant epithelial tumor; malignant epithelioma

A benign epithelial neoplasm that projects above the surrounding epithelial surface Mesenchymal tumor, A malignant mesenchymal malignant; neoplasm. A general term for sarcoma; sarcoma which the transformed cell type of soft tissue and has not been specified bone; sarcoma of the soft tissue and bone (continued)

878

Appendix: Fundamental Pathology Terminology Based on the Standard…

Modifier/ diagnosis Type

Term

Synonym(s)

Definition

Diagnosis Neoplastic status Benign

For neoplasms, a noninfiltrating and nonmetastasizing neoplastic process that is characterized by the absence of morphologic features associated with malignancy (e.g., severe atypia, nuclear pleomorphism, tumor cell necrosis, and abnormal mitoses). For other conditions, a process that is mild in nature and not dangerous to health

Diagnosis Nonneoplastic finding type

Abscess

An inflammatory response represented by a focal collection of leukocytes (predominantly neutrophils) that can be encapsulated

Diagnosis Nonneoplastic finding type

Accessory tissue

A supernumerary tissue in addition to normal tissues

Diagnosis Nonneoplastic finding type

Accumulation

An increase of substance (e.g., proteinaceous fluid and glycogen) in either the intracellular space, extracellular space, or within a hollow organ or structure

Diagnosis Nonneoplastic finding type

Adenomyosis

The growth of endometrial tissue inside the muscular wall of the uterus

Diagnosis Nonneoplastic finding type

Adenosis

The presence of small collections of epithelial cells with or without microlumens in the stroma adjacent to ducts or acini in glandular tissues

Diagnosis Nonneoplastic finding type

Adhesion

A fibrinous or fibrous connection between two surfaces or tissues, connecting tissues or organs that are not normally attached

Diagnosis Nonneoplastic finding type

Adnexal dysplasia

Abnormal development of the adnexal appendages of the skin

Diagnosis Nonneoplastic finding type

Aggregate

Diagnosis Nonneoplastic finding type

Alpha 2u-globulin nephropathy

Aggregation

A collection of cells or particles forming a cohesive mass or cluster Increase in eosinophilic cytoplasmic droplets of alpha 2u-globulin in the S2 segment of the proximal tubules in the cortex with exfoliation of cells, an increase in mitotic figures in affected portions of the proximal tubules, tubular basophilia in some cases, and formation of granular casts at the junction of the inner and outer stripes of the medulla (continued)

Appendix: Fundamental Pathology Terminology Based on the Standard… Modifier/ diagnosis Type

879

Term

Synonym(s)

Definition

Amyloid

Amyloidosis

An accumulation of amyloid protein

Diagnosis Nonneoplastic Angiectasis Finding Type

Hemangiectasis

Dilatation of the blood vessels or endothelial lined sinusoids

Diagnosis Nonneoplastic finding type

Aplasia

Agenesis

A congenital abnormality resulting in the absence of an anatomical structure

Diagnosis Nonneoplastic finding type

Apoptosis

A form of programmed cell death triggered by internal or external signals that results in a series of characteristic morphological changes

Diagnosis Nonneoplastic finding type

Apoptosis/necrosis

A finding that generally has features of apoptosis and necrosis

Diagnosis Nonneoplastic finding type

Artifact

A structure or appearance that is not naturally present but has been introduced through manipulation

Diagnosis Nonneoplastic finding type

Astrocytosis

Diagnosis Nonneoplastic finding type

Atelectasis

The partial or total collapse of alveoli and/or airways

Diagnosis Nonneoplastic finding type

Atrophy

A decrease in size of organ, tissue, or cell

Diagnosis Nonneoplastic finding type

Atypical residual bodies

Abnormally large, misshapen, and/ or clumped vacuoles containing cell debris in the testis, or present in stages of spermatogenesis when not normally seen

Diagnosis Nonneoplastic finding type

Autophagic vacuoles

Vacuoles containing segregated cytoplasmic organelles or contents, characterized by intracytoplasmic globules surrounded by a thin, clear halo

Diagnosis Nonneoplastic finding type

Basophilia

A blue-purple tinctorial change associated with staining with basic dyes

Diagnosis Nonneoplastic finding type

Basophilic focus

A localized group of cells that exhibit some type of cytologic alteration resulting in basophilia

Diagnosis Nonneoplastic finding type

Astrogliosis; gemistocytosis

Reactive astrocytic proliferation often associated with degenerative, inflammatory, or neoplastic changes in the central nervous system

(continued)

880

Appendix: Fundamental Pathology Terminology Based on the Standard…

Modifier/ diagnosis Type

Term

Synonym(s)

Definition

Diagnosis Nonneoplastic finding type

Basophilic granules

Intracytoplasmic phagolysosomes that are strongly basophilic. These structures are usually seen within the tubular epithelium and glomeruli of the kidney in response to antisense oligonucleotides

Diagnosis Nonneoplastic finding type

Basophilic hypertrophic focus

Discrete unencapsulated noncompressing focus/foci involving one or more acini with enlarged basophilic cells and occasionally enlarged nuclei

Diagnosis Nonneoplastic finding type

Bronchiectasis

Segmental, irreversible dilation of the bronchial tree

Diagnosis Nonneoplastic finding type

Calculus

A concretion of material in the body, usually composed of mineral salts. Representative examples include gallbladder stones, kidney stones, and salivary gland stones

Diagnosis Nonneoplastic finding type

Cast

A mold of a hollow structure (e.g., renal tubule, bronchiole). The casts may be composed of various materials (e.g., protein, granular substance, cellular debris)

Diagnosis Nonneoplastic finding type

Cell debris

Diagnosis Nonneoplastic finding type

Cholangiofibrosis

Diagnosis Nonneoplastic finding type

Cholesterol cleft

Diagnosis Nonneoplastic finding type

Chromatolysis

Cellular debris

An accumulation of cell fragments A hepatotoxin-induced finding in the liver consisting of dilated/cystic bile ducts filled with mucus and cellular debris and surrounded by inflammatory cell infiltrates and often sclerotic connective tissue. Epithelium is pleomorphic and, in cystic glands, may be partially lost resulting in crescent-­shaped structures

Acicular cleft

Presence of flat, thin, rhomboid spaces in tissue created by the dissolution of cholesterol crystals during processing The disintegration of the chromophil substance (Nissl bodies) in a nerve cell body which may occur after injury to the cell (continued)

Appendix: Fundamental Pathology Terminology Based on the Standard…

881

Modifier/ diagnosis Type

Term

Diagnosis Nonneoplastic finding type

Chronic progressive nephropathy

A spontaneous, age-related renal disease of rats and mice, characterized by morphological changes such as degeneration of the epithelium lining of the tubules, cast formation, thickening of glomerulus, Bowman and proximal tubular basement membranes, and lesions in the glomeruli, leading to mesangial overload and glomerulosclerosis

Diagnosis Nonneoplastic finding type

Congestion

Increased number of erythrocytes in the capillary bed or larger vessels of an organ

Diagnosis Nonneoplastic finding type

Corpora amylacea

Small hyaline masses of degenerated cells that develop into compact concretions due to pressure dehydration and/or compaction of dead cells. May appear mineralized

Diagnosis Nonneoplastic finding type

Cribriform change

Pseudoglandular formation

Formation of epithelial pseudoglandular structures with lumens

Diagnosis Nonneoplastic finding type

Crust

Scab

A covering or layer of solid matter formed by dried bodily exudate or secretion

Diagnosis Nonneoplastic finding type

Crystals

A clear or pale solid having a highly regular structure, which may present as a crystal profile

Diagnosis Nonneoplastic finding type

Cyst

A sac-like closed pocket of tissue that may be empty or may be filled with fluid, gas, semisolid, or amorphous material. It typically has an outer epithelial-lined capsule

Diagnosis Nonneoplastic finding type

Cystic degeneration

A finding consisting of multilocular cysts lined by fine septa containing fine flocculent eosinophilic material or, in some tissues, blood. The cysts are not lined by endothelial cells and do not compress the surrounding parenchyma. This does not include congenital polycystic change

Diagnosis Nonneoplastic finding type

Cytoplasmic alteration

A cytoplasmic change that may be characterized by, but is not limited to, increased cytoplasmic granularity, eosinophilia, and/or cell swelling

Synonym(s)

Definition

(continued)

882

Appendix: Fundamental Pathology Terminology Based on the Standard…

Modifier/ diagnosis Type

Term

Synonym(s)

Definition

Diagnosis Nonneoplastic finding type

Decidual reaction

A primarily uterine reaction with generally indistinct borders and two recognizable regions. These regions are an antimesometrial region containing closely packed mesenchymal cells and a mesometrial region containing mesometrial cells with long cytoplasmic processes and abundant glycogen

Diagnosis Nonneoplastic finding type

Decidualization

A focal lesion within the uterus consisting of markedly hypertrophied stromal cells with cytoplasmic glycogen and prominent nuclei

Diagnosis Nonneoplastic finding type

Degeneration

Disturbance of cell integrity and deterioration of normal tissue, cells, or organs

Diagnosis Nonneoplastic finding type

Degenerative joint disease

A disease process characterized by degeneration of the articular cartilage, hypertrophy of the bone at the margins, and changes in the synovial membrane

Diagnosis Nonneoplastic finding type

Demyelination

Loss of myelin with relative preservation of the ensheathed axon, characterized by the presence of myelin ovoids and reduced myelin staining

Diagnosis Nonneoplastic finding type

Dental dysplasia

Aberrant development of odontogenic tissues without accompanying fracture

Diagnosis Nonneoplastic finding type

Denticle

Toothlike structure formed from displaced odontogenic tissue, which may include dental papilla

Diagnosis Nonneoplastic finding type

Dentin niches

Focal or multifocal recesses within the dentin

Diagnosis Nonneoplastic finding type

Dilatation

Diagnosis Nonneoplastic finding type

Diverticulum

A saclike protrusion in the wall of a hollow organ or tissue

Diagnosis Nonneoplastic finding type

Ectasia

Expansion of substructures (such as ducts, glands, sinuses, alveoli) within the tissue

Diagnosis Nonneoplastic finding type

Ectopic tissue

An otherwise normal tissue or portion of tissue that forms in a location of the body at or in which it is not normally present

Dilation

Expansion of the cavity, ducts, or lumen of a hollow organ or vessel

(continued)

Appendix: Fundamental Pathology Terminology Based on the Standard… Modifier/ diagnosis Type

Term

Synonym(s)

883

Definition

Diagnosis Nonneoplastic finding type

Edema

Excessive amount of watery fluid in tissues or cavities, generally characterized microscopically as clear spaces separating tissue components

Diagnosis Nonneoplastic finding type

Elastosis

Degeneration of elastin with accumulation of irregular, thickened elastic fibers

Diagnosis Nonneoplastic finding type

Embolus

Diagnosis Nonneoplastic finding type

Emphysema

Abnormal enlargement of the air space distal to the terminal bronchiole accompanied by destructive changes in the alveolar septa

Diagnosis Nonneoplastic finding type

Eosinophilic globules

Intracytoplasmic droplets that are strongly eosinophilic

Diagnosis Nonneoplastic finding type

Epithelial alteration

Diagnosis Nonneoplastic finding type

Erosion

A shallow or superficial destruction of a surface, without destruction of the basement membrane

Diagnosis Nonneoplastic finding type

Erosion/ulcer

A finding that generally has features of erosion and ulceration

Diagnosis Nonneoplastic finding type

Erythrophagocytosis

Macrophages containing phagocytized intact or fragmented erythrocytes, with or without nuclei, and/or erythrocyte ghosts

Diagnosis Nonneoplastic finding type

Exfoliation

Shedding or sloughing of cells from an epithelial surface, including the skin, mucosa, and testis

Diagnosis Nonneoplastic finding type

Extramedullary hematopoiesis

Formation of blood cells that occurs outside of the bone marrow

Diagnosis Nonneoplastic finding type

Fatty change

Increased lipid within the cytoplasm of cells

Emboli

An intravascular mass, such as clotted blood or other elements, that was carried in the blood and occludes distal vessels

Respiratory tract A change or slight modification in epithelial alteration respiratory and/or cuboidal/ transitional epithelial cells in the respiratory system, characterized mainly by loss of cilia (respiratory epithelium), flattening and horizontal orientation of epithelial cells, and a slight increase in cell layers

(continued)

884

Appendix: Fundamental Pathology Terminology Based on the Standard…

Modifier/ diagnosis Type

Term

Synonym(s)

Definition

Diagnosis Nonneoplastic finding type

Fibro-osseous lesion

Accumulation of a mixed cell population of nonneoplastic mesenchymal cells along endosteal surfaces which may be associated with focal osteoclastic bone resorption and marrow fibroplasia

Diagnosis Nonneoplastic finding type

Fibroplasia

The formation of fibrous tissue characterized by an increased number of active, plump fibroblasts, and variable amounts of collagen

Diagnosis Nonneoplastic finding type

Fibrosis

Increase in collagen and low numbers of fibrocytes

Diagnosis Nonneoplastic finding type

Fibrous osteodystrophy

The replacement of cortical bone by fibrous connective tissue and stromal cells

Diagnosis Nonneoplastic finding type

Focus of cellular alteration

Foci of cellular alteration; focus/ foci of cellular alteration

A localized proliferation of hepatocytes phenotypically different from surrounding hepatocyte parenchyma with no or minimal compression of surrounding tissue

Diagnosis Nonneoplastic finding type

Foreign material

Foreign body

An occurrence where any object originating inside or outside the body is not in its physiological or intended location

Diagnosis Nonneoplastic finding type

Fracture

Diagnosis Nonneoplastic finding type

Fungus

Diagnosis Nonneoplastic finding type

Germ cell degeneration

Disturbance of cell integrity and deterioration of germ cells

Diagnosis Nonneoplastic finding type

Germ cell depletion

Partial or complete absence of germ cell layer(s)

Diagnosis Nonneoplastic finding type

Gliosis

Nonspecific reactive response of nervous system glial cells, chiefly astrocytes, and microglia rather than oligodendroglia

Diagnosis Nonneoplastic finding type

Glomerulonephritis

Localized disruption of bone or tooth structure resulting in partial or complete discontinuity Fungi

Glomerular nephritis

The presence of fungi

Inflammatory changes in the renal glomeruli characterized by thickening of the glomerular basement membrane, mesangial cell proliferation, and/or mononuclear inflammatory cell infiltration. In some forms, the glomerular epithelial cells may also proliferate and form adhesions (continued)

Appendix: Fundamental Pathology Terminology Based on the Standard… Modifier/ diagnosis Type

Term

Synonym(s)

885

Definition

Diagnosis Nonneoplastic finding type

Glomerulopathy

Chronic degenerative changes in the glomeruli characterized by loss of cellularity of glomerular capillary tufts and acellular deposition of immunoglobulins

Diagnosis Nonneoplastic finding type

Glomerulosclerosis

Diagnosis Nonneoplastic finding type

Granulation tissue

A finding associated with tissue repair, characterized by the presence of ingrowth of fibroblasts, and new blood vessels

Diagnosis Nonneoplastic finding type

Granuloma

An organized chronic inflammatory reaction characterized by the presence of epithelioid macrophages. Giant cells and/or necrosis can be observed

Diagnosis Nonneoplastic finding type

Growth plate closed

Physis closed

Cartilage of the physis is replaced by bone

Diagnosis Nonneoplastic finding type

Growth plate open

Physis open

A physis consisting of hyaline cartilage, without complete osseous fusion

Diagnosis Nonneoplastic finding type

Hamartoma

An excessive but focal overgrowth of cells and tissues native to the organ in which it occurs

Diagnosis Nonneoplastic finding type

Helicobacter

The presence of any species of Helicobacter

Diagnosis Nonneoplastic finding type

Hemorrhage

The presence of extravascular erythrocytes

Diagnosis Nonneoplastic finding type

Hepatodiaphragmatic nodule

A congenital abnormality of the liver, characterized by grossly visible nodule(s) usually located on the median lobe

Diagnosis Nonneoplastic finding type

Hydrocephalus

An enlargement of the ventricles relative to brain tissue

Diagnosis Nonneoplastic finding type

Hydromyelia

Dilation of the central canal of the spinal cord

Diagnosis Nonneoplastic finding type

Hyperkeratosis

Diagnosis Nonneoplastic finding type

Hyperplasia

Glomerular sclerosis

Increased keratinization

Hyaline deposits or scarring within the renal glomeruli

Thickening of the outermost layer of stratified squamous epithelium Increase in the number of resident cells, generally with an increase in mitotic figures present, per unit area in an organ or tissue (continued)

886

Appendix: Fundamental Pathology Terminology Based on the Standard…

Modifier/ diagnosis Type

Term

Synonym(s)

Definition

Diagnosis Nonneoplastic finding type

Hypertrophy

Cell size enlargement due to the increase in the amount of cytoplasm and its constituent organelles. The cells are larger, but otherwise the appearance is unchanged

Diagnosis Nonneoplastic finding type

Hypoplasia

Incomplete or underdevelopment of a tissue or organ

Diagnosis Nonneoplastic finding type

Immaturity

In an early period of life or development or growth; not fully developed

Diagnosis Nonneoplastic finding type

Imperforate vagina

Embryologic remnant consisting of a persistent connective tissue membrane within the vaginal vault

Diagnosis Nonneoplastic finding type

Inclusion

Inclusion bodies; inclusion body

A general term used to describe abnormal structures present within the cytoplasm or nucleus of a cell

Diagnosis Nonneoplastic finding type

Infarct

Infarction

Localized necrosis of tissue resulting from obstruction of the blood supply usually by a thrombus, an embolus, or vascular torsion

Diagnosis Nonneoplastic finding type

Infiltrate

Cellular infiltration; infiltration

An influx of cells, generally leukocytes, in locations or numbers not normally found, without other features of inflammation

Diagnosis Nonneoplastic finding type

Inflammation

A response to an injury or abnormal stimuli characterized by inflammatory cell infiltration and varying degrees of vascular and tissue reactions (hyperemia, edema, fibrin, and/or fibrosis)

Diagnosis Nonneoplastic finding type

Interstitial nephritis

Generalized inflammation of the renal interstitium characterized by a diffuse or patchy distribution of lymphocytes, plasma cells, and/or macrophages and variable degrees of edema

Diagnosis Nonneoplastic finding type

Intimal thickening

An increase in matrix, without an increase in cell numbers, between the endothelium and the internal elastic lamina

Diagnosis Nonneoplastic finding type

Intrahepatocelluar erythrocytes

The presence of red blood cells within hepatocytes (continued)

Appendix: Fundamental Pathology Terminology Based on the Standard… Modifier/ diagnosis Type

Term

Synonym(s)

887

Definition

Diagnosis Nonneoplastic finding type

Intramural plaque

A plaque located in the tunica intima of vessels characterized by the presence of granular material, collagenous fibers with interspersed spindle cells, and focal protrusion of a variably mineralized matrix into the vascular lumen

Diagnosis Nonneoplastic finding type

Intussusception

Telescoping or invagination of a portion of a tubuluar organ into an adjacent segment

Diagnosis Nonneoplastic finding type

Karyocytomegaly

An increase in nuclear size and amount of cytoplasm of a cell. The cells or nucleus may be slightly irregular and/or may be polyploid

Diagnosis Nonneoplastic finding type

Karyomegaly

Diagnosis Nonneoplastic finding type

Lipoproteinosis

The abnormal, excessive accumulation of acellular, periodic acid-Schiff positive, pale eosinophilic material (lipoprotein-­ type). This is typically found in the pulmonary alveoli

Diagnosis Nonneoplastic finding type

Luteinized follicle

A corpus luteum-like structure with a retained oocyte and variably luteinized granulosa cells

Diagnosis Nonneoplastic finding type

Lymphangiectasis

Dilatation of the lymphatic vessels

Diagnosis Nonneoplastic finding type

Malformation

A permanent structural change that is likely to adversely affect the form, survival or health of the species under study. (Gupta, R. C. ed. (2011) Reproductive and Developmental Toxicology. London, UK: Elsevier, Inc.)

Diagnosis Nonneoplastic finding type

Mesangiolysis

A finding in the glomerulus of the kidney, characterized by the degeneration of mesangial cells and the dissolution of the mesangial matrix

Diagnosis Nonneoplastic finding type

Mesonephric duct remnant

The persistence of the mesonephric duct beyond embryogenesis

Diagnosis Nonneoplastic finding type

Metaplasia

Conversion of a mature, normal cell or groups of mature cells to other forms of mature cells

Nuclear enlargement

An increase in the size of a cellular nucleus

(continued)

888

Appendix: Fundamental Pathology Terminology Based on the Standard…

Modifier/ diagnosis Type

Term

Synonym(s)

Definition

Diagnosis Nonneoplastic finding type

Microabscess

A very small, circumscribed collection of white blood cells, predominantly neutrophils

Diagnosis Nonneoplastic finding type

Microgliosis

An accumulation of microglial cells in nervous system tissue

Diagnosis Nonneoplastic finding type

Mineralization

Diagnosis Nonneoplastic finding type

Mucification, increased

Increase in the number of mucusproducing epithelial cells, which may form a distinct mucified layer

Diagnosis Nonneoplastic finding type

Multinucleated giant cell

An abnormally large cell with more than one nucleus, generally seen in the testis

Diagnosis Nonneoplastic finding type

Multinucleated hepatocytes

Hepatocytes that have multiple nuclei present

Diagnosis Nonneoplastic finding type

Murine obstructive uropathy

Diagnosis Nonneoplastic finding type

Necrosis

Death of a group of cells in an organ or tissue

Diagnosis Nonneoplastic finding type

Needle tract lesion

Focal lesion in the tissue due to insertion and/or withdrawal of the needle

Diagnosis Nonneoplastic finding type

Neovascularization

The formation of new blood vessels

Diagnosis Nonneoplastic finding type

Nephroblastematosis

Small, focal, or locally extensive basophilic cell mass of blast cells with ill-defined cytoplasm and nuclei, which may be present in one or both kidneys. May arise from remnant of developing metanephric blastema

Diagnosis Nonneoplastic finding type

Neuronophagia

The phagocytosis of degenerating neurons

Diagnosis Nonneoplastic finding type

Obstruction

Complete or partial blockage of the lumen of a tubular structure

Calcification; mineral Basophilic, granular deposits of inorganic material in tissue

Mouse urological syndrome (mus)

A constellation of findings in male mice characterized by ulceration and/or inflammation of the penis and prepuce, proteinaceous material with inflammatory cells, spermatozoa, or desquamated urothelial cells forming a plug in the urethra and generally dilatation of the bladder, hydroureter, and hydronephrosis

(continued)

Appendix: Fundamental Pathology Terminology Based on the Standard…

889

Modifier/ diagnosis Type

Term

Diagnosis Nonneoplastic finding type

Obstructive nephropathy

Renal damage secondary to crystal deposition in the tubular lumen or blockage of urinary outflow in the bladder or urethra. Lesion is characterized by interstitial granulomatous inflammation often with epithelioid cells and multinucleated giant cells, crystal deposition or evidence of blockage of the ureters (e.g., proteinaceous plug in male mice)

Diagnosis Nonneoplastic finding type

Osteoblastic surface, increased

Increase in the remodeling or modeling-based bone formation

Diagnosis Nonneoplastic finding type

Osteoid, increased

Increase in the amount of unmineralized bone matrix

Diagnosis Nonneoplastic finding type

Osteophyte

Periarticular nonneoplastic osseous protuberance with or without a cartilage cap located along the epiphyseal margins

Diagnosis Nonneoplastic finding type

Ovotestis

A rare condition characterized by the unequivocal presence of both testicular and ovarian tissues in a gonad

Diagnosis Nonneoplastic finding type

Parasite

The presence of parasites and/or parasitic ova

Diagnosis Nonneoplastic finding type

Perforation

A hole or opening through a membrane or other tissue that is not normally present

Diagnosis Nonneoplastic finding type

Periodontal pocket

An abnormal dilation and/or expansion of the periodontium resulting in destruction of the supporting periodontal tissue

Diagnosis Nonneoplastic finding type

Phospholipidosis

Disorder caused by defects in the function of the lysosomes resulting in the presence of small clear vacuoles containing phospholipids within the cytoplasm of various cells

Diagnosis Nonneoplastic finding type

Pigment

Diagnosis Nonneoplastic finding type

Polyovular follicle

An ovarian follicle that contains more than one oocyte

Diagnosis Nonneoplastic finding type

Prolapse

A condition in which an organ drops or bulges out of place

Diagnosis Nonneoplastic finding type

Proliferation, intima

Thickening of the tunica intima of a vessel by smooth muscle cells or, less commonly, fibroblasts

Synonym(s)

Pigmentation

Definition

Accumulation of exogenous or endogenous colored material within an organ, tissue, or cell

(continued)

890

Appendix: Fundamental Pathology Terminology Based on the Standard…

Modifier/ diagnosis Type

Term

Diagnosis Nonneoplastic finding type

Proliferation, stroma, valve

A noninflammatory increase in valvular stromal cells accompanied by increased matrix

Diagnosis Nonneoplastic finding type

Prostatic rudiment

An embryological structure composed of epithelial cells surrounded by mesenchyme that gives rise, in the male, to the prostate gland

Diagnosis Nonneoplastic finding type

Proteinaceous plug

Diagnosis Nonneoplastic finding type

Pulp concretion

Concentric layers of mineralized tissue surrounding dead/injured cells or collagen fibers in the dental pulp

Diagnosis Nonneoplastic finding type

Pustule

A circumscribed skin or mucosal epithelial lesion filled with purulent material

Diagnosis Nonneoplastic finding type

Pyelonephritis

A tubulointerstitial inflammatory disease involving a spectrum of lesions affecting the tubules, interstitium and/or the pelvis of the kidney. Pyelonephritis can result from infections, both ascending and descending and following papillary necrosis and urothelial ulceration. Certain strains of rodents are particularly susceptible to developing spontaneous pyelonephritis and are used as animal models to assess antibiotic therapy

Diagnosis Nonneoplastic finding type

Pyometra

The accumulation of inflammatory cells, predominantly neutrophils, within the uterus and lumen

Diagnosis Nonneoplastic finding type

Rarefaction

Intracytoplasmic accumulation of material such as glycogen or xenobiotics, characterized by clear, not well-defined spaces in the cytoplasm around a centrally located nucleus

Diagnosis Nonneoplastic finding type

Regeneration

A reparative process to replace lost or damaged cells, commonly characterized by cellular basophilia, increased nuclear cytoplasmic ratio and/or irregular architecture

Diagnosis Nonneoplastic finding type

Resorption

A process in which tissue is absorbed by the body

Synonym(s)

Seminal plug

Definition

Eosinophilic proteinaceous material in male urinary bladder or urethra

(continued)

Appendix: Fundamental Pathology Terminology Based on the Standard…

891

Modifier/ diagnosis Type

Term

Diagnosis Nonneoplastic finding type

Retrograde nephropathy

Constellation of tubule changes extending from papilla to cortex. In the cortex, the lesions consist of linear patches of tubular basophilia coupled with tubular dilation and tracts of basophilic, hyperplastic collecting ducts, often with mitotic figures. Inflammation is usually not a prominent component. Differentiated from obstructive nephropathy by absence of granulomatous inflammation and crystals

Diagnosis Nonneoplastic finding type

Salpingitis isthmica nodosa

Nodules and diverticuli in the isthmus of the fallopian tube

Diagnosis Nonneoplastic finding type

Satellitosis

A finding characterized by the presence of rings or clusters of primarily oligodendroglia near a degenerating neuron cell body

Diagnosis Nonneoplastic finding type

Septal deviation

An alteration of the septum from the midline. This is typically seen in the nasal cavity

Diagnosis Nonneoplastic finding type

Sperm stasis

Luminal aggregation of released sperm generally within an atrophic tubule

Diagnosis Nonneoplastic finding type

Spermatid retention

Persistence of mature elongating spermatids in the seminiferous tubule after the normal stage of physiologic release

Diagnosis Nonneoplastic finding type

Spermatocele

A benign cystic dilatation in the epididymis or testis that contains fluid and spermatozoa

Diagnosis Nonneoplastic finding type

Squamous cyst

A saclike structure lined by stratified squamous epithelium

Diagnosis Nonneoplastic finding type

Squamous plaque/cyst

A focus of squamous epithelium in or near the surface of the heart, generally believed to be an embryonic rest

Diagnosis Nonneoplastic finding type

Syringomyelia

Cavitation of the spinal cord parenchyma

Diagnosis Nonneoplastic finding type

Tension lipidosis

A focus of hepatocytes containing well delineated circular clear spaces in the liver, often near mesenteric attachments such as the falciform ligament

Synonym(s)

Definition

(continued)

892

Appendix: Fundamental Pathology Terminology Based on the Standard…

Modifier/ diagnosis Type

Term

Synonym(s)

Definition

Diagnosis Nonneoplastic finding type

Thrombus

Thrombi; thrombosis An intravascular aggregation of blood components, primarily platelets, and fibrin with entrapment of cellular elements, which is attached to the vessel wall

Diagnosis Nonneoplastic finding type

Type II astrocytes

Diagnosis Nonneoplastic finding type

Ulcer

Ulceration

Destruction of an epithelial surface extending into or beyond the basement membrane

Diagnosis Nonneoplastic finding type

Vacuolation

Vacuoles

The presence of vacuoles within the cytoplasm of cells

Diagnosis Nonneoplastic finding type

Vesicle

Cytotoxic response of astrocytes characterized by swollen nuclei with central clearing, marginated heterochromatin, prominent/ swollen nucleoli and indistinct cytoplasm

An abnormal fluid-filled cleft (e.g., as in the epidermis) or membrane-­bound space

Index A Aberrant crypt foci (ACF)�������������������������������� 439–440, 168 ABP, see Androgen-binding protein ACE, see Angiotensin-converting enzyme Acetazolamide������������������������������������������������������������������240 Acetylation������������������������������������������������������������������� 98, 99 Acetylcholine (ACh)������������������������ 158–159, 198, 206, 176, 337, 562, 593, 601 ACF, see Aberrant crypt foci ACh, see Acetylcholine Acid burns������������������������������������������������������������������������638 Acidophils���������������������������������������������������������������� 544, 586 Acinar cells, pancreatic�����������������������������176–183, 189, 190, 194, 185, 187 apoptosis of������������������������������������������������� 179, 183, 187 necrosis of��������������������������������������������������� 150, 178, 183 Acinus, of liver���������������������������������������������������������� 114, 115 Acquired immunodeficiency syndrome (AIDS)���������������854 Acrolein������������������������������������������������������������ 126, 335, 336 Acrylonitrile���������������������������������������������������������������������562 ACTH, see Adrenocorticotropic hormone Action potential, cardiomyocyte������������������������������� 281, 288 Activin���������������������������������������������������������������������� 288, 421 Acute beryllium disease����������������������������������������������������345 Acute pancreatitis��������������������������������������������� 183–185, 189 ACVP, see American College of Veterinary Pathology Adaptation, cell injury and�������������������������������������������������64 atrophy and�������������������������������������������������������������������64 hypertrophy and������������������������������������������������������������64 Adducts, DNA formation of��������������������������������������������862 Adenocarcinomas colon���������������������������������������������������������������������������169 mammary glands��������������������������������������������������������446 Adenohypophysis�������������������������������������������������������������545 proliferative lesions of�����������������������������������������588–589 Adenomas adrenal cortex����������������������������������������������������� 559, 561 colon������������������������������������������������������������������� 168, 169 gallbladder�������������������������������������������������� 129, 133, 134 hepatocellular���������������������������������������������� 129–133, 757 islet cells���������������������������������������������������������������������565 kidney�������������������������������������������������������������������������230 Leydig cell������������������������������������������������������������������762 mammary glands��������������������������������������������������������546

pancreas endocrine pancreas�������������������������176, 543, 563–565 exocrine pancreas������������������������������������������175–188 parathyroid������������������������������������������������� 551, 552, 555 pars intermedia��������������������������������������������������� 545, 546 pituitary spontaneous��������������������������� 544–546, 559, 561, 762 stomach, mouse����������������������������������������������������������159 thyroid������������������������������������������������������������������������551 Adenomyosis������������������������������������������������������������ 448, 878 Adenosine triphosphate (ATP)������������������������������� 219, 222, 593–595, 640 Adenosis, cervix����������������������������������������������������������������878 Adipose tissue atrophy���������������������������������������������������������������� 186, 387 inflammation��������������������������������������������������������������186 Adnexa atrophy of�������������������������������������������������������������������499 inflammation of����������������������������������������������������������499 neoplasms of���������������������������������������������������������������522 structure and function of���������������������������� 484, 488–489 Adrenal cortex adenomas of����������������������������������������������������������������561 carcinomas of��������������������������������������������������������������561 hyperplasia of���������������������������������������������� 387, 559, 561 hypertrophy, ACTH stimulation and��������� 387, 560, 561 Adrenal cortical cells������������������������������������������������ 559, 561 Adrenal gland brown degeneration����������������������������������������������������559 cortex accessory adrenal cortical tissue����������������������������559 adenoma���������������������������������������������������������������559 atrophy reticularis���������������������������������������������� 557, 559, 561 zona fasciculata��������������������������������������������� 556, 561 zona glomerulosa��������������������������������������������������556 hemorrhage��������������������������������������������������������� 559, 562 hyperplasia diffuse����������������������������������������������������������� 559, 561 focal�������������������������������������������������������������� 559, 561 subcapsular������������������������������������������������������������559 hypertrophy�������������������������������������������������������� 560, 561 medulla histology���������������������������������������������������������������556 necrosis��������������������������������������������������������������� 561, 562

Thomas J. Steinbach et al. (eds.), Toxicologic Pathology for Non-Pathologists, https://doi.org/10.1007/978-1-4939-9777-0, © Springer Science+Business Media, LLC, part of Springer Nature 2019

893

oxicologic Pathology for Non-Pathologists 894  ITndex



  

Adrenal gland (cont.) neoplasia pheochromocytoma����������������������������������������������552 steroid synthesis����������������������������������������������������������557 Adrenal medulla ganglioneuromas of�����������������������������������������������������563 hyperplasia of��������������������������������������������������������������552 pheochromocytomas of������������������������������� 552, 563, 768 Adrenal steroidogenesis�������������������������������������������� 558, 560 Adrenergic agonists����������������������������������������������������������149 Adrenocorticotropic hormone (ACTH)������������������ 485, 539, 541, 544, 546, 557, 559–561, 566 adrenal hypertrophy and���������������������������������������������560 Adriamycin������������������������������������������������������� 225, 227, 235 Adult respiratory distress syndrome (ARDS)����������� 342, 343 Adverse events, risk assessment and���������������������������������242 Aerosols, lung absorption of������������������������������������� 330, 349 Aflatoxin B1 (AFB1)��������������������������������������������������������130 Age-related macular degeneration (AMD)�������������� 636, 655 Aging lymphoid organ changes with�������������������������������������384 AIDS, see Acquired immunodeficiency syndrome Alanine aminotransferase (ALT)��������������119, 599, 713–716, 725, 789, 839 as hepatocellular injury indicator������������������������714–716 Albuminuria���������������������������������������������������������������������208 Aldehyde dehydrogenases�������������������������������������������������815 Aldose reductase (AR)������������������������������������������������������639 Aldosterone��������������������������������������� 556–558, 560, 561, 566 Alkylating agents���������������������������������������������� 418, 642, 760 Allergy, immune system and���������������������������������������������376 Alloxan��������������������������������������������������������������������� 555, 564 Alopecia��������������������������������������491, 497, 515, 517, 518, 527 Alpha-naphthylthiourea (ANTU)������������������������������������342 Alpha 2u-Globulin nephropathy���������������������� 212, 214, 879 Alveolar ducts�����������������������������313, 317, 319, 320, 322, 345 Alveolar macrophages (AMs)����������������������� 27, 31, 312, 321, 322, 324, 330, 331, 333, 334, 343 AMD, see Age-related macular degeneration American College of Veterinary Pathology (ACVP)�������������������������������������������������������� 3, 71 Amikacin��������������������������������������������������������������������������670 Aminoglutethimide����������������������������������������������������������560 Aminoglycoside antibiotics������������������������������� 670, 672, 717 Aminophenoxyalkanes������������������������������������������������������642 Amiodarone��������������������������������������� 167, 289, 549, 607, 642 Amphetamine�������������������������������������������������������������������603 Amylase�������������������������������������������������������������������� 147, 175 Amyloidosis�������������������������������������� 122, 158, 209–211, 224, 559, 565, 879 islet cell�����������������������������������������������������������������������565 Anaphylaxis�������������������������������������������������������������� 329, 375 Anastrozole����������������������������������������������������������������������826 Androgen-binding protein (ABP)������������������������������������401

Anemia autoimmune hemolytic�����������������������������������������������375 hemolytic��������������������������������������������������������������������375 Angiography, fluorescein������������������������������������������ 654, 655 Angiotensin-converting enzyme (ACEi) inhibitors���������221 Animal in vivo imaging��������������������������������������������103–110 Apoptosis cardiomyocytes���������������������������������������������������284–286 exocrine pancreas���������������������������������������� 179, 183, 187 hepatocyte�������������������������������������������������������������������124 Arteritis, see Vasculitis Arthritis����������������������������������������������������������������������������592 Aspirin�������������������������������������������������������������� 161, 286, 717 Atrial lesions, see Heart Atrophy adipose tissue���������������������������������������������� 186, 387, 605 adrenal cortex reticularis��������������������������������������������������������������561 zona fasciculata�����������������������������������������������������561 zona glomerulosa��������������������������������������������������556 bone marrow���������������������������������������������������������������512 cornea�������������������������������������������������������������������������646 epidermal������������������������������������������������������������ 499, 826 esophagus�������������������������������������������������������������������152 gastric mucosa�������������������������������������������������������������158 heart���������������������������������������������������������������������������512 intestinal villi������������������������������������������������������ 166, 173 lymph node��������������������������������������������������������� 385, 512 mammary gland�������������������������������������������������� 460, 463 muscle��������������������������������������������������599, 600, 604, 605 ovary dog���������������������������������������������������������������� 518, 817 monkey�����������������������������������������������������������������817 rodents������������������������������������������������������������������611 pituitary����������������������������������������������������������������������544 prostate��������������������������������������������������������������� 412, 422 retina hereditary��������������������������������������������������������������645 light-induced��������������������������������������������������������651 salivary gland������������������������������������������������������ 148, 151 thymus������������������������������������������������������������������������512 thyroid������������������������������������������������������������������������865 uterus��������������������������������������������������������������������������447 Auricular chondritis����������������������������������������������������������673 Axonopathy�������������������������������������������������������������� 256, 259 Azaserine������������������������������������������� 178, 182, 184, 187, 189 Azoxymethane (AOM)�������������������������������������������� 168, 188

B Bacterial otitis media��������������������������������������������������������674 Balantidium coli��������������������������������������������������������������� 4, 15 BAL fluid, see Bronchoalveolar lavage BALT, see Bronchus-associated lymphoid tissue Bands of Büngner�������������������������������������������������������������261

Barbiturates����������������������������������������������������������������������801 Basal cell carcinoma�����������������������������������514, 519, 525, 749 Basal cell tumors���������������������������������������������������������������525 Basic multicellular unit (BMU)����������������������������������������834 Basophils�������������������� 328, 358, 360, 374, 375, 544, 703, 705 BBB, see Blood-brain barrier β-Catenin�������������������������������������������������������������������������589 Bcl2 protein����������������������������������������������������������������������706 Beagle dogs, spontaneous kidney lesions in������������� 209, 294, 295, 817, 833 Beryllium������������������������������������������� 329, 344–346, 505, 774 Beta cell, see Pancreas BHT, see Butylated hydroxy toluene Bile cholestasis of���������������������������������������������������������������514 Bile duct hyperplasia of������������������������������������������������������ 128, 131 neoplasia of�����������������������������������������������������������������131 Bile salts������������������������������������������������������������� 84, 161, 792 Biliary system bile duct changes induced lesions������������������������������������������������������179 proliferative lesions�����������������������������������������������131 cholestasis����������������������������������������������������������� 117, 514 gallbladder changes�����������������������������������������������������165 Biomarkers bone��������������������������������������������������������������������581–582 for bone toxicity evaluation�����������������������������������������578 See also Renal biomarkers Biotransformation in GI tract�������������������������������������������������������������������138 phase I������������������������������������������������������������������������183 phase II�����������������������������������������������������������������������183 roles of������������������������������������������������������������������������138 See also Metabolism Bisphosphonates����������������������������������������586, 590, 833–835 Black lung (coal worker’s pneumoconiosis)����������������������345 Bladder, see Urinary bladder Bladder cells���������������������������������������������������������������������649 Bleomycin��������������������������������������������������312, 341, 343, 344 Blood animal models and clinical relevance������������������690–692 circulation�������������������������������������������������������������������281 platelet values��������������������������������������������������������������359 red blood cell values�������������������������������������������� 359, 690 Blood-brain barrier (BBB)����������������������� 252, 255, 256, 258, 265, 271, 272, 312, 670, 799, 800, 863 Blood-nerve barrier (BNB)����������������������������������������������260 Blood urea nitrogen (BUN)����������������������220, 234, 242–244, 511, 695, 718, 720, 839 Blood vessels, see Vasculature B-lymphocytes���������������������������������� 358, 366, 509, 727, 812 BM, see Bone marrow BMD, see Bone mineral density β2-Microglobulin (β2-M)�������������������������������� 234, 718, 721

Toxicologic Pathology for Non-Pathologists 895 Index       BMU, see Basic multicellular unit BNB, see Blood-nerve barrier Bone growth plate������������������573, 575, 577, 578, 580–582, 590 histology�������������������������������������������������������������572–576 increase in mass and osteoid spontaneous increases����������������������������������� 584, 585 terminology������������������������������������������ 579, 581–582 joints (see Joints) mineral deposition������������������������������������������������������574 neoplasia chondroma������������������������������������������������������������588 chondrosarcoma����������������������������������������������������588 osteoma��������������������������������������������������������� 588, 589 osteosarcoma�������������������������������������������������587–589 overview����������������������������������������������������������������572 osteonecrosis���������������������������������������������������������������590 osteoporosis������������������������������������������������ 576, 582, 584 regulators��������������������������������������������������������������������576 technical considerations in assessment�����������������������572 Bone marrow (BM) atrophy������������������������������������������������������������������������512 cytologic evaluation of������������������������������������������������324 hematopoiesis and����������������������������������������66, 363, 384, 702, 705, 709, 799 H&E stained��������������������������������������������������������������368 hyperplasia������������������������������������������������������������������512 leukemia and������������������������������������������������������� 710, 758 microenvironment damage to�������������������������������������709 stem cells��������������������������������������������������������������������574 Bone mineral density (BMD)�������������������������������������������579 Bones and joints biomarkers�������������������������������������������579, 581–582, 591 formation of endochondral ossification��������������������� 575, 590, 832 intramembranous ossification�������������������������������575 fractures of atypical������������������������������������������������������������������708 healing of��������������������������������������������������������������584 pathologic����������������������������������������������������� 573, 577 injury, response to background lesions������������������������������������������������144 proliferation of�������������������������������������586, 591, 678, 833 structure of���������������������������������������������������������� 579, 583 woven����������������������������������������������������������������� 575, 586 Bone turnover markers (BTMs)���������������������������������������579 Bouin’s fixative�������������������������������������������254, 255, 404, 632 Bowman’s capsule��������������������������������������203, 204, 227, 228 See also Kidney Brain axonal lesions������������������������������������������������������259–260 demyelination����������������������������������������������������� 801, 808 neoplasia focal gliosis�����������������������������������������������������������274 vacuolation���������������������������������������������������������� 268, 565

oxicologic Pathology for Non-Pathologists 896  ITndex



  

BrdU, see Bromodeoxyuridine Breast cancer�������������������������������������� 285, 462, 754, 819, 823 See also Mammary gland Bromobenzene������������������������������������������������������������������326 Bromocriptine���������������������������������������������������������� 462, 545 Bromodeoxyuridine (BrdU)����������������������������������������������351 Bronchi������������������������������������������������������313, 316, 317, 351 See also Lung Bronchioles������������������������ 313, 317, 319, 320, 322, 328, 338, 346, 348, 880, 883 Bronchoalveolar lavage (BAL)���������������������������������� 352, 353 Bronchus-associated lymphoid tissue (BALT)���������������������357, 366, 786, 811, 812, 814 Brown degeneration, adrenal��������������������������������������������559 Bruch’s membrane�������������������������������������������� 625, 636, 655 BTMs, see Bone turnover markers BUN, see Blood urea nitrogen Burns���������������������������������������������������������������� 532, 638, 655 Bursa������������������������������������������429, 437, 438, 445, 452, 865 Busulfan�������������������������������������������������������������������� 185, 343 Butylated hydroxyanisole (BHA)�������������������������������������163

C Cadmium����������������������������������������� 188, 329, 332, 345, 347, 584, 774, 862–864 Caffeine����������������������������������������������������������������������������547 Calcitonin���������������������������������� 547, 551–553, 555, 582, 726 Calcium ionized�������������������������������������������������253, 266, 267, 538 Calcium channel blockers�������������������������������������������������612 cAMP, see Cyclic adenosine monophosphate Cancer breast������������������������������������������� 285, 462, 754, 819, 823 colon animal models of���������������������������������������������������167 lung in humans�������������������������������������������������������������348 See also Neoplasia/neoplasms Candida albicans��������������������������������������������������������������������4 Captopril��������������������������������������������������������������������������509 Carbendazim������������������������������������������������������������ 417, 421 Carbon disulfide������������������������������������������������������� 641, 650 Carbon monoxide����������������������������������������������������� 334, 673 Carbon nanotubes���������������������������������������������������� 334, 346 Carbon tetrachloride��������������������������������������������������������334 Carcinogenesis�������������������������������������������������� 179, 182, 522 Carcinogenic agents����������������������������������������������������������337 Carcinogenicity testing������������������������������495, 519, 762, 775 Carcinogens chemical��������������������������������������� 152, 179, 182, 188, 522 exocrine pancreas exposure to�������������������������������������763 Carcinoid tumor���������������������������������������������������������������287 Carcinomas sebaceous cell��������������������������������������������������������������522 urothelial���������������������������������������������������������������������240

Cardiac hypertrophy����������������������������������108, 297, 298, 300 Cardiomyocytes action potentials�������������������������������������������������� 281, 288 necrosis of��������������������������������������������293, 298, 299, 301 Cardiotoxicity, see Blood vessels; Heart Cartilage articular, zones of���������������������������������������� 574, 835–836 degeneration of�����������������������������������������������������������586 See also Bones and joints; Ear; Joints Caspases���������������������������������������������� 97, 124, 259, 263, 522 Cast nephropathy��������������������������������������������� 212–213, 238 Cataract�������������� 636, 638–640, 639, 640, 644, 648, 649, 837 Catarrhal otitis media in���������������������������������������������������674 Catecholamines�������������������������������� 284, 293, 304, 430, 539, 556, 558, 562, 703, 729 Cationic amphiphilic drugs (CADS)������������������������ 187, 288 C cell hyperplasia in carcinoma��������������������������������������������551 CCK, see Cholecystokinin CDs, see Collecting ducts Cecum������������������������ 137, 139–142, 164, 168, 169, 173, 191 Cell death mechanisms of apoptosis���������������������������������������������������������������124 necrosis���������������������������������������������������������124–125 programmed�������������������������124, 171, 380, 515, 518, 879 Cell loss, atrophy and�������������������������������������������������� 64, 420 Cell protection������������������������������������������������������������������720 Cell swelling����������������������������������������������379, 601, 699, 882 Cell turnover������������������������������������������������������ 67, 334, 504 Centriacinus������������������������������������������������������������� 317, 320 Cephalosporins��������������������������������������������������������� 214, 510 CGS 14796C�������������������������������������������������������������������643 Chemokines����������������������� 323, 335, 343, 352, 377, 501, 502 Chief cells response to injury of adenomas��������������������������������������������������������������555 carcinomas������������������������������������������������������������555 Chloramphenicol���������������������������������������������� 641, 788, 789 Chloroform����������������������������������������������������������������������867 Chloroquine����������������������� 146, 288, 601, 607, 640, 642, 653 Chlorphentermine���������������������������������������������������� 640, 642 Cholangiocarcinoma������������������������������������������������� 134, 861 Cholangiofibrosis����������������������������������������������������� 131, 880 Cholangioma��������������������������������������������������������������������134 Cholecystokinin (CCK)�����������������������������������165, 176, 181, 184, 186, 187, 189, 194 Cholestasis���������������������������������������������������������������� 117, 514 Cholesteatomas����������������������������������������������������������������674 Cholesterol��������������������������������������� 556–561, 636, 640, 674, 701, 714, 717, 729–731, 839, 881 Chondritis������������������������������������������������������������������������673 Chondroma����������������������������������������������������������������������588 Chondronecrosis���������������������������������������������������������������681 Chondrosarcoma��������������������������������������������������������������588

Choroid��������������������������������������620, 623, 625, 629, 644, 654 Choroid plexus���������������������������272, 274, 275, 512, 524, 799 Chromaffin cells������������������������������������������������������� 558, 562 Chromium�������������������������������������������������������� 337, 510, 717 Chromophobe������������������������������������������������������������������544 Chronic beryllium disease������������������������������������������������245 Chronic obstructive pulmonary disease (COPD)����������������������������������������� 324, 347, 811 Chronic pancreatitis animal models for�������������������������������������������������������189 fibrosis and��������������������������������������������������������� 184, 185 Chronic renal failure����������������������������������223, 235, 237, 555 Ciliary body���������������� 620, 623, 624, 634, 644, 650, 653, 654 Ciliated epithelial cells���������������������������������������������� 318, 324 Cimetidine�����������������������������������������������������������������������601 Cinoxacin�������������������������������������������������������������������������835 Ciprofibrate����������������������������������������������������������������������188 Ciprofloxacin��������������������������������������������������������������������835 Circulatory system������������������������������������������������������������280 See also Blood vessels; Heart Cirrhosis, liver������������������������������������������������������������������724 Cisplatin�����������������������������139, 222, 235–238, 672, 717, 798 CK, see Creatine kinase Clara cell���������������������������������������������������������������������������349 Clear cell change in liver�������������������������������������������������������������129 foci of hepatocellular alteration����������������������������������129 Clitoral glands������������������������������������������������������������������772 Clofibrate�������������������������������������������������������������������������773 Clostridium difficile������������������������������������������������������������ 138 Clusterin����������������������������������������������������234, 243, 718, 720 Coal worker’s pneumoconiosis (black lung)����������������������345 Cobalt����������������������������������������������������������������������� 153, 335 Cocaine�����������������������������������������������������172, 284, 603, 721 Cochlea, see Ear Colchicine�������������������������������������������������������� 210, 608, 611 Colitis����������������������������������������������������������������������� 169, 377 Collagen fibers�������������������������������������������429, 622, 623, 891 Collecting ducts (CDs)�����������������������������202–204, 217, 221, 224, 237, 244, 624, 719, 798, 891 Colon, see Large intestine Complete blood count (CBC)������������������������������������������372 Computed tomography (CT) micro-����������������������������������������������������27, 104, 106, 583 peripheral quantitative���������������������������������������� 582, 599 Congestion lung������������������������������������������������������������������������� 53, 55 spleen����������������������������������������������������������������������������53 Conjunctiva�����������������������������������������������620, 621, 638, 646 Connective tissue fibers����������������������������������������������������633 Constipation����������������������������������������������������� 173, 190, 191 Contact dermatitis��������������������� 375, 497–500, 505, 507, 514 COPD, see Chronic obstructive pulmonary disease Copper����������������������������������������������� 179, 184–188, 863, 864

Toxicologic Pathology for Non-Pathologists 897 Index       Cornea edema of������������������������������������������������������������� 646, 648 subepithelial mineralization��������������������������������699–700 See also Eye Corticosteroids��������������������������������� 167, 184, 221, 556, 571, 605, 639, 643, 704, 716, 729, 730 cartilage effects�����������������������������������������������������������231 cataract induction�������������������������������������������������������639 Corticosterone��������������������418, 556–558, 561, 693, 703, 738 Cortisol�������������������������������546, 556, 557, 561, 702–704, 738 Covalent binding, liver toxicity and����������������������������������325 COX, see Cyclooxygenase COX-2, see Cyclooxygenase-2 Craniopharyngioma����������������������������������������������������������545 Creatine kinase (CK)������������������������� 298, 599, 692, 723, 724 Cristae ampullaris�������������������������������������������������������������685 Crypts��������������������� 17, 20, 140, 164, 166–169, 172, 173, 366 CT, see Computed tomography Cupola������������������������������������������������������������������������������667 Curvilinear body���������������������������������������������������������������643 Cutaneous adnexa������������������������������ 499, 500, 517–519, 522 Cyanide����������������������������������������������������������������������������673 Cyanoketone���������������������������������������������������������������������560 Cyclic adenosine monophosphate (cAMP)����������������������538 Cyclooxygenase (COX)�������������������������������������������� 218, 224 Cyclooxygenase-2 (COX-2), inhibitors����������������������������286 Cyclophosphamide������������������������������������240, 312, 721, 817 Cyclosporine������������������������������184, 187, 189, 237, 238, 291, 517, 717, 797, 798, 811 Cynomolgus monkeys, see Monkeys CYPs, see Cytochromes P450 (CYPs) Cystic degeneration (spongiosis hepatis)����������������� 120, 122, 559, 769, 881 Cytochrome P450 (CYP) enzymes�����������557, 560, 561, 787 Cytopenia����������������������������������������������������������������� 707, 710

D Davidson’s fixative��������������������������� 15, 84, 86, 254, 255, 632 DCT, see Distal convoluted tubule Degenerative joint disease�������������������������������� 586, 587, 882 Demyelination��������������������������������������������������258, 261, 801, 807, 808, 882 Denervation atrophy, drug-induced����������������������������������605 Depigmentation������������������������������������������������������� 642, 827 Dermatomyositis��������������������������������������������������������������606 Dermatopathology, see Skin Dermatotoxicology, see Skin Desferrioxamine���������������������������������������������������������������642 Desmosomes������������������������������������������������������������ 503, 509 Dextran sulfate sodium (DSS) colitis�������������������������������169 Diabetes mellitus (DM)������������������������������������������� 564, 707 Diagnostic pathology/pathologists������������������32, 33, 41, 768 Diaphysis���������������������������� 370, 572, 573, 575, 578, 580, 583 Diarrhea�������������������������������������������� 170, 190, 191, 702, 726

oxicologic Pathology for Non-Pathologists 898  ITndex



  

Diestrus in dogs������������������������������������������������������������������������438 in rats��������������������������������������������������������������������������437 Diethylstilbesterol������������������������������������������������������������864 Differential cell count�������������������������������������������������������709 Diffusion���������������������������������������������������������� 341, 575, 628 Digestive system, see Gastrointestinal tract Digoxin��������������������������������������������������������������������� 290, 721 Dihydrotestosterone (DHT)��������������������������������������������399 Diiodotyrosine (DIT)������������������������������������������������������548 Dimethylbenzα-anthracene (DMBA)��������������������� 180, 182, 190, 783, 787, 819, 821 Diphenylhydantoin�����������������������������������������������������������833 DIPL, see Drug-induced phospholipidosis DISC, see Death-inducing signaling complex Discovery studies��������������������������������������������������������������204 Distal convoluted tubule (DCT)����������������������������� 202, 204, 554, 557 Distribution in PK��������������������������������������������������������������������������119 DIT, see Developmental immunotoxicity testing; Diiodotyrosine DM, see Diabetes mellitus DMBA, see 7,12-Dimethylbenz[a]anthracene DMNM, see 1,4-Dioxido-3-methylquinoxalin-2-yl-N-­ methylnitrone DMS, see Demarcation membrane system DNA, see Deoxyribonucleic acid DNB, see 1,3-Dinitrobenzene DNT, see Developmental neurotoxicity testing Dogs estrous cycle histology of���������������������432, 434, 438, 439 hypospermatogenesis in�������������������������������������� 410, 411 spontaneous kidney lesions in beagle��������������������������206 DOPA, see Dihydroxyphenylalanine Dopamine�������������������109, 268, 275, 287, 289, 290, 419, 442, 444, 445, 454, 460, 462, 545, 558, 562, 823, 824 Dose-response assessment������������������������������������������������414 Downregulation����������������������������������������������������������������797 Doxorubicin������������������������������������������������������235, 285, 303, 611, 650, 721, 833 Doxylamine�������������������������������������������������������������� 149, 150 D-Penicillamine������������������������������������������������������� 229, 606 Drug-induced phospholipidosis (DIPL)��������������������������288 Drusen���������������������������������������������������������������������� 636, 653 DSS, see Dextran sodium sulfate Dual energy X-ray absorptiometry (DXA)��������������� 582, 599 Ductular cells, pancreatic��������������������������������������������������182 Duodenum������������������������ 141, 143, 153, 154, 162, 164–166, 168, 172, 175, 176, 792 DXA, see Dual energy X-ray absorptiometry Dysplasia physeal������������������������������������������������������������������������590 retinal������������������������������������������������������������������ 646, 651

E Ear, auricular chondritis����������������������������������������������������673 Eccrine glands�������������������������������������������������� 489, 522, 525 Echocardiography�������������������������������������������������������������722 ECL cells, see Enterochromaffin-like cells ECM, see Extracellular matrix EDCs, see Endocrine-disrupting compounds Edema, corneal������������������������������������������������� 638, 646, 648 EDTA, see Ethylene diamine tetraacetic acid EEI, see Endocrine-exocrine interface EGF, see Epidermal growth factor EGME, see Ethylene glycol monoethyl ether Elastosis�������������������������������������������������������������������� 449, 883 Embryo������������������������������� 852, 855, 859, 863, 864, 867, 868 Emesis����������������������������������������������� 155, 170, 702, 725, 726 Emetine����������������������������������������������������������������������������608 EMG, see Electromyography EMH, see Extramedullary hematopoiesis Emphysema�����������������������������������������������338, 346, 347, 883 See also Pulmonary emphysema Endochondral ossification�������������������������������� 575, 590, 832 Endocrine-disrupting compounds (EDCs)���������855, 864–866 Endocrine disruption������������447, 457, 567, 819, 856, 864–867 Endocrine-exocrine interface (EEI)���������������������������������185 Endocrine system�������������� 537–567, 827–831, 852, 855, 864 Endogenous hormones�����������������������������������������������������856 Endometriosis����������������������������������������������������������448–449 Endoplasmic reticulum (ER) rough���������������������������������������������������175, 178, 225, 672 smooth, hypertrophy of���������318, 326, 418, 593, 608, 758 Endorphin������������������������������������������������������������������������544 Endothelial cells glomerular, injury to���������������������������������������������������203 Endothelin receptor antagonists������������������������������� 289, 290 Endotoxin����������������������������������������������������������������� 318, 342 Enhanced histopathology��������������������������������� 359, 367, 371 Enteric lymphoid system��������������������������������������������������174 Enteric nervous system��������������������������������������������� 139, 165 Enterochromaffin cell��������������������������������160, 162, 176, 193 Enterochromaffin-like (ECL) cells�������������������������� 160, 162 Enterocytes������������������������������������������������������� 164, 166, 174 Enteroendocrine cells����������������������������������������������� 154, 162 ENU, see N-ethyl-N-nitrosourea Environmental Protection Agency (EPA)���������������� 780, 831 Eosinophilic exocrine pancreas eosinophilic change������������������������175 liver inclusions������������������������������������������������������������137 salivary gland accumulation��������������������������������146–151 Eosinophils��������������������������������163, 329, 338, 358, 360, 366, 372, 375, 509, 600, 703–705 EPA, see Environmental Protection Agency EPCs, see Endothelial precursor cells Epicardium����������������������������������������������������������������� 57, 294 Epidermal growth factor (EGF)�������� 149, 153, 162, 167, 518

Epidermis necrosis of��������������������������������������������������� 499, 505, 512 pustule in��������������������������������������������������������������������504 squamous cell carcinomas of���������������������������������������520 structure and function of���������������������484–488, 491, 662 Epididymis inflammation��������������������������������������������������������������409 phospholipidosis���������������������������������������������������������422 sperm granuloma���������������������������������������� 409, 412, 422 structure����������������������������������������������������������������������421 Epigenetics��������������������������������������������������������������� 751, 790 carcinogenesis and������������������������������������������������������751 Epinephrine���������������������������������������������� 420, 539, 556, 558, 562, 702–704, 703, 704, 707, 729 Epiphysis���������������������������������������������������572–574, 580, 834 EPO, see Erythropoietin ER, see Endoplasmic reticulum Ergotamine�����������������������������������������������������������������������287 Erosions, in gastric mucosa�����������������������������������������������161 ERs, see Estrogen receptors Erythema multiforme�����������������498, 499, 507, 508, 510, 531 Erythroid cells����������������������������������������������������������708–710 Erythropoiesis����������������������������697, 698, 701, 702, 708, 710 Erythropoietin (EPO)�������������������������������697, 702, 707, 710 ES cells, see Embryonic stem cells Esophagus hyperkeratosis�������������������������������������������������������������151 Estrogen receptors (ERs)�������������������������� 421, 443, 447, 460, 461, 754, 816, 821 Estrous cycle, see Female reproductive system ET-1, see Endothelin-1 Ethambutol����������������������������������������������������������������������643 Ethanol�������������������������������������� 18, 87, 89, 91, 184, 186, 189, 368, 520, 632, 646, 801 Ethylene diamine tetraacetic acid (EDTA)�����������������������������������372, 580, 668, 839 Ethylene glycol monoethyl ether (EGME)����������������������������������������� 502–503, 445 Excitotoxicity��������������������������������������������������������������������673 Exogenous hormones����������������������������������������������� 446, 457 Extracellular matrix (ECM)�����������������������������185, 227, 236, 287, 319, 487, 573, 575, 604, 625 Extramedullary hematopoiesis (EMH)��������������������� 66, 363, 383, 384, 702, 705, 788, 813, 835, 884 Eye conjunctivitis��������������������������������������������������������������620 cornea atrophy������������������������������������������������������������������646 dystrophy��������������������������������������������������������������646 hyperplasia������������������������������������������������������������648 keratitis�����������������������������������������������������������������648 neovascularization�������������������������������������������������648 pigmentation��������������������������������������������������������648 vacuolation������������������������������������������������������������632

Toxicologic Pathology for Non-Pathologists 899 Index       Harderian and lacrimal glands hyperplasia and neoplasia�������������������������������������772 inflammation��������������������������������������������������������772 injury response of conjunctiva���������������������������������������������������� 620, 621 cornea�����������������������������������������������������������621–623 eyelids�����������������������������������������������������������620–621 IOP elevation and glaucoma���������������������������������653 lacrimal system�����������������������������������������������������621 lens����������������������������������������������������������������625–626 optic nerve������������������������������������������������������������629 retina�������������������������������������������������������������627–629 RPE�������������������������������������������������������������� 629, 650 uveal tract�������������������������������������������������������������623 vitreous body�������������������������������������������������625–627 lens cataract������������������������������������������������������������������639 physiology�����������������������������������������������������620–629 retina atrophy hereditary��������������������������������������������������������������645 light-induced��������������������������������������������������������651 phospholipidosis���������������������������������������������������642 retinal pigment epithelium changes�������������� 628, 629 structure����������������������������������������������������������������621 technical considerations for analysis�������������������655–656 uveal tract components and changes������������������� 620, 623 Eye drops��������������������������������������������������������������������������638 Eyelids������������������������ 511, 513, 620, 621, 631, 646, 836, 837

F FAH, see Foci of altered hepatocytes Fatty change reversible cell injury and���������������������������������������������183 See also Lipidosis FDA, see Food and Drug Administration FD & C Red No. 3 (erythrosine)�������������������������������������549 Female reproductive system estrous cycle�������������������������������� 432–435, 437–439, 442, 444, 447, 451, 452, 459 introduction to����������������������������������������������������397–399 menstrual cycle����������������������������431–434, 440, 452–454 morphology��������������������������426, 455, 456, 460, 461, 463 structure, function, and cell biology of dog estrous cycle histology�������������������� 434, 438–439 estrous cycle endocrinology����������������������������������434 minipig estrous cycle histology������������� 434, 439–440 rat estrous cycle histology�������������������������������������434 toxicity mechanisms of chemotherapeutic agents��������������������������������������423 hyperprolactinemia������������������������������� 418, 425, 454 sex steroid enzyme alterations�������������������������������442 stress, negative energy balance, and senescence�����449 Feminization, of male mammary glands������������������� 461, 866

oxicologic Pathology for Non-Pathologists 900  ITndex



  

Fenfluramine-phentermine�������������������������������������� 287, 288 Fertility assessment, male�������������������������������������������������426 Fetus�������������������������������������������������� 104, 120, 399, 537, 801 See also Developmental toxicology Fibers, inhaled����������������������������������������������������������333–334 Fiber typing��������������������������������593, 595, 597, 598, 604, 605 Fibroadenomas, of mammary glands��������������������������������460 Fibroblast growth factors (FGFs)����������������������������� 590, 641 Fibroma ovary���������������������������������������������������������������������������446 soft tissue������������������������������������������������������������ 523, 526 Fibro-osseous lesion (FOL)����������������������������� 584–585, 884 Fibroplasia�������������������������������������������������298, 606, 837, 884 Fibrosarcoma���������������������� 231, 523, 526, 589, 757, 762, 877 Fibrosis bone marrow���������������������������������������������������������������145 chronic pancreatitis and�������������������������������������� 184, 185 corneal stroma���������������������������������������������������� 638, 648 definition of����������������������������������������������������������������344 endocardium������������������������������������������������������� 284, 294 following necrosis����������������������������������������������� 183, 273 heart���������������������������������������������������������������������������284 interstitial fibrosis������������������������������� 148, 214, 217, 233, 285, 333, 334, 339, 345 lung���������������������������������������312, 330, 333, 334, 344, 347 animals�������������������������������������������778, 811, 826, 837 humans�����������������������������������������������������������������333 pleura��������������������������������������������������������������������������333 pulmonary etiologic agents of�������������������������������������������������377 injury response of��������������������������������������������������340 pathogenesis of�����������������������������������������������������344 Filtration�����������������������������������203, 206, 208, 225, 226, 238, 624, 713, 718, 721, 736, 787, 793, 796 First pass effect������������������������������������������114, 166, 183, 857 Fixation airway�������������������������������������������������������������������������350 ocular tissue��������������������������������������������������������631–633 testis������������������������������������������������������������������������������15 Flow cytometry, for hematoxicity evaluation���������������������������������������� 373, 705, 709 Fluorescein angiography������������������������������������������� 654, 655 Fluoride�������������������������������������������������������������������� 611, 612 Fluorochrome labels�����������������������������������������������������������28 Fluoro-jade dyes��������������������������������������� 12, 13, 89–90, 253, 258, 259, 263, 264, 270, 807 5-Fluorouracil�������������������������������������������������������������������139 Flutamide�������������������������������������������������������������������������461 Focal glomerulosclerosis���������������������������������������������������226 Focal necrosis��������������������������������������������124, 788, 833, 836 Foci of altered hepatocytes (FAH)��������������������������� 130, 131 Foci of cellular alteration, in hepatocellular neoplasia basophilic type���������������������������������������������������� 128, 129 clear-cell type�������������������������������������������������������������129

eosinophilic type���������������������������������������������������������129 H&E staining for����������������������������������������������� 128, 129 FOL, see Fibro-osseous lesion Follicle-stimulating hormone (FSH)����������������400, 405, 418, 426, 430, 432, 444, 453, 539, 541, 544, 545, 738 Follicular cell����������������������������� 547–551, 773, 828, 830, 865 Follistatin������������������������������������������������������������������ 421, 596 Food and Drug Administration (FDA)��������������������� 20, 252, 272, 495, 526, 629, 630, 667, 780, 869 Forensic pathology/pathologists���������������������������������������165 Forestomach�������������������������������� 66, 152–163, 192, 193, 772 hyperplasia�������������������������������������������������� 159, 162, 163 inflammation������������������������������� 158, 159, 161, 163, 193 neoplasia and clinical relevance����������������������������������772 ulceration�����������������������������������������������66, 159, 161, 162 Formic acid��������������������������������������������������������������� 350, 580 Fovea���������������������������������������������������������627, 628, 634, 655 Fractures, bone healing of��������������������������������������������������������������������583 Free radicals injury, liver toxicity and�����������������������������������������������222 lipid peroxidation initiated by��������������������� 219, 559, 561 FSH, see Follicle-stimulating hormone Fundus���������������������������������������������������������������������� 153, 157 Furosemide��������������������������������������������������������������� 150, 671 Futile redox cycling�����������������������������������������������������������327

G Gabapentin��������������������������������������������������������������� 189, 797 Gallbladder adenomas of������������������������������������������������ 129, 132–133 carcinomas of���������������������������������������������� 129, 132, 133 inflammatory lesions of����������������������������������������������122 neoplasia of���������������������������������� 117, 122, 128, 129, 131 nonneoplastic lesions of��������������������������������������120–132 overview on���������������������������������������������������������113–114 structure, function, and cell biology in����������������114–117 See also Biliary system GALT, see Gut-associated lymphoid tissue Gamma glutamyltransferase (GGT)����������������������� 120, 713, 714, 716–718, 721 Ganglioneuromas�������������������������������������������������������������563 adrenal medulla�����������������������������������������������������������563 Gases, lung absorption of�������������������������������������������������314 Gastric glands����������������������������������������������������������� 158, 162 Gastric mucosa���������������������������157, 158, 160, 161, 163, 193 See also Stomach Gastrin����������������������� 154, 162, 167, 176, 181, 193, 774, 791 Gastrointestinal (GI) tract absorption from�������������������������������������������������� 156, 562 biotransformation in������������������������������������������� 138, 183 epithelial cells of����������������������������������������� 154, 166, 168 sublethally injured�������������������������������������������������187 immune response of����������������������������������������������������138

injury response of constipation�������������������������������������������������� 190, 191 diarrhea������������������������������������������������� 170, 190, 191 growth factors and������������������������������������������������162 hormone responses������������������������������� 154, 162, 165 inflammation���������������������������������������� 139, 173, 193 mucosal response������������154, 158, 161, 162, 168, 172 nervous tissue and motility responses�������������������139 signaling pathways and�����������������������������������������154 vomiting����������������������������������������������������������������190 introduction to����������������������������������������������������137–144 microbiome activities in����������������������������������������������138 nervous tissue of���������������������������������������������������������139 structure and function of��������������������������������������������173 macroscopic and microscopic��������������������������������173 toxicity evaluation of animal models for�����������������������������������������188–189 morphological methods for�����������������������������������139 mutagenicity and carcinogenicity testing��������������193 toxicity mechanisms of agents inducing gastric ulceration���������������� 141, 146, 158, 161, 191 gut microflora and cancer�������������������������������������138 hypoxia�����������������������������������������������������������������171 intestinal neoplasia������������������������������������������������167 microfloral effects, antibiotics and������������������������138 neoplasias and��������������������������������149, 158, 161, 168 GCs, see Germinal centers GDH, see Glutamate dehydrogenase GEM models, see Genetically engineered mouse models Gene expression, toxicants altering�����������������������������������352 Genetically engineered mouse (GEM) models��������������������������������������������� 189, 190, 525 Genomics, for carcinogenesis mechanism prediction��������������������������������������������������������426 Gentamicin����������������� 222, 225, 235, 236, 238, 670, 717, 798 Germ cells degeneration/apoptosis of���������������������������������� 406, 408, 409, 414–417 depletion of������������������������������������������415, 416, 418, 885 development of, dynamics of������������������������������ 400, 403 focal loss of�����������������������������������������������������������������410 Sertoli cells role in metabolic support of���������������������399 sloughing and shedding of���������������������������������� 417, 815 testis���������������������������������������������������������������������� 64, 406 Germinal centers (GCs)����������������������������� 17, 174, 362, 364, 379, 382, 384, 385, 388, 524, 525, 812–814 GFAP, see Glial fibrillary acidic protein GFR, see Glomerular filtration rate GGT, see Gamma glutamyltransferase GH, see Growth hormone Ghrelin�����������������������������������������������������������������������������154 Giant cell, see Testis Gingiva, hyperplasia����������������������������������������� 145, 146, 612

Toxicologic Pathology for Non-Pathologists 901 Index       GI tract, see Gastrointestinal tract Glandular mucosa�����������������������154, 157, 158, 161, 162, 757 Glaucoma��������������������������������������������������������� 637, 639, 653 Glial cells�������������������� 258, 260, 261, 558, 799–801, 885, 888 neural injury reactions of��������������������������������������������265 Glial fibrillary acidic protein (GFAP)��������������������� 253, 258, 261, 265, 266, 807 Glomerular filtration rate (GFR)���������������������208, 225, 736, 787, 793, 796 renal toxicity and abnormalities of������������������������������242 single nephron������������������������������������������������������������237 Glomerulus��������������������������������203, 204, 206, 207, 209, 210, 218, 229, 231, 244, 860, 880, 881, 885 injury mechanisms of basement membrane crosslinks������������� 203, 207, 235 immune-mediated������������������������������������������������225 mesangiolysis��������������������������������������������������������888 podocyte injury��������������������������������������������� 203, 207 injury to nephrotoxicant classification associated with��������������������������������������������������������� 218, 220 See also Kidney GLP-1, see Glucagon-like peptide-1 GLP regulations, see Good Laboratory Practices regulations Glucagon���������������������� 97, 154, 167, 175, 552, 563, 564, 729 Glucagon-like peptide-1 (GLP-1)������������167, 552, 564, 729 Glucocorticoids�������������������������������� 458, 491, 499, 503, 515, 517, 556–558–561, 566, 588–590, 592, 596, 605, 707, 716, 790, 791 Glucuronidation������������������������������������������������������� 789, 801 Glucuronides��������������������������������������������������������������������801 Glutamate dehydrogenase (GDH)������������������� 120, 713–716 Glutaraldehyde�������������������������������30, 83, 102, 205, 233, 633 Glutathione (GSH)������������ 327, 343, 561, 639, 640, 670, 721 cell protection with�����������������������������������������������������720 Glutathione-S-transferase pi (GST-pi)����������������������������130 Glutathione S-transferases (GSTs)�����������������������������������721 Glycogen�������������������������������80, 90, 117, 120–121, 187, 220, 594, 595, 598, 605, 608, 788, 878, 882, 891 Glycol methacrylate (GMA)������������������������������������ 262, 668 GMA, see Glycol methacrylate GnRH, see Gonadotropin-releasing hormone Goblet cells���������������������������������������� 316, 620, 621, 662, 674 hyperplasia lung�����������������������������������������������������������������������316 nasal mucosa������������������������������������������������� 316, 621 Goiter�������������������������������������������������������������������������������549 Gonadotrophs�������������������������������������������������������������������418 Gonadotropin-releasing hormone (GnRH)������������ 405, 417, 418, 424, 437, 447, 541 Gonads������������������������������� 399, 428, 442, 559, 857, 866, 890 Good Laboratory Practices (GLP) regulations�������������� 5, 40, 67, 70, 71, 74, 101, 204, 242, 511, 572 Granular cell tumor�������������������������������������������������� 134, 240

oxicologic Pathology for Non-Pathologists 902  ITndex



  

Granulocytes������������������������������������ 165, 358, 360, 362, 367, 381, 384, 389, 447, 703, 709, 710, 719 Granulocytic leukemia������������������������������������������������������134 Granuloma liver�����������������������������������������������������������������������������788 lung��������������������������������������������������������������������� 329, 505 sperm���������������������������������������������������409, 412, 421, 422 Graves’ disease������������������������������������������������������������������376 Great vessels, transposition of�������������������������������������������282 Ground substance�������������������������������������������������������������487 Growth hormone (GH)��������������������������� 167, 458, 464, 544, 562, 564, 596, 702, 729 Growth plate, bone������������ 573, 575, 577, 578, 580–582, 590, 831–834, 885 GRP, see Gastrin-releasing peptide GSH, see Glutathione GST-Pi, see Glutathione-S-transferase Pi GSTs, see Glutathione S-transferases Guanethidine�������������������������������������������������������������������422 Gut-associated lymphoid tissue (GALT)���������������� 141, 174, 357, 366, 786 Gut microflora������������������������������������������������������������������138

H Hair follicles overview��������������������������������������������������������������522–523 tumors of��������������������������������������������������������������������525 tumors showing differentiation��������������������������� 525, 526 Hair growth�������������������������������������������������������������� 491, 527 Haloperidol�������������������������������������������������������������� 289, 462 Hamsters, spontaneous kidney lesions in��������������������������209 Harderian glands������������������������������������������������������ 621, 631 See also Eye Hazard identification������������������������������������������������ 332, 686 Hazards characterization of�������������������������������������� 423, 451, 464 developmental toxicology identification of identification of����������������������������������������������������������493 in risk assessments���������������������������������������� 463, 774 HCC, see Hepatocellular carcinoma hCG, see Human chorionic gonadotropin H&E, see Hematoxylin and eosin Hearing loss, biotechnology products for�������������������������669 Heart anatomy and physiology�������������������������������������280–281 apoptosis�������������������������������������������������������������284–286 atrial lesions����������������������������������������������������������������285 conduction system of��������������������������������������������������302 drug-induced necrosis, inflammation, and fibrosis����������������������� 288, 289, 291–293, 304 histopathological assessment���������������292, 295, 303–304 hypertrophy��������������������������������� 282, 287, 295, 297–300 inflammation and necrosis models beagle������������������������������������� 289, 290, 294, 295, 304 hamster��������������������������������������������������������� 295, 297

monkey������������������������������������������������� 284, 292–296 mouse����������������������������������������������������������� 296, 297 rat����������������������������������������� 283, 284, 286, 288–291, 293–298, 300 mineralization����������������������������������������������������� 294, 295 myocardial degeneration����������������������282, 293, 299, 304 myocardial infarction������������������������������������������ 286, 299 necrosis��������������������������������270–277, 282, 284, 285, 289, 290, 292, 293, 295, 298, 299, 304, 310 neoplasia�������������������������������������������������������������296–297 phospholipidosis���������������������������������������������������������288 physiology and functional considerations of��������� 280–281 structure and function of cellular and extracellular elements gross and microscopic anatomy�����������������������������288 ventricular myocytes���������������������������������������������304 thrombosis�������������������������������������������286, 290–292, 295 toxic injury response of arrhythmias�����������������������������������������������������������303 cardiac dysfunction�����������������������������������������������280 contraction changes����������������������������������������������281 developmental cardiotoxicities������������������������������283 endocardium���������������������������������������������������������297 neoplasia���������������������������������������������������������������296 valves������������������������������������� 281, 282, 287, 288, 295 toxicity functional evaluation for monitoring arterial blood pressure������������������������289 monitoring myocardial electrical activity��������������281 toxicity mechanisms of���������������������������������������284–289 agents��������������������������������������������������������������������285 functional alterations��������������������������������������������285 xenobiotic interactions������������������������������������������279 toxicity morphologic evaluation for microscopic examination��������������������������������������288 ultrastructural examination�����������������������������������285 valve lesions������������������������������������������������ 292, 294, 295 xenobiotic exposure of������������������������������������������������279 Heart failure cell�������������������������������� 285–287, 297, 300, 724 Heart rate, changes in����������������������������������������������� 303, 304 Heat shock stress��������������������������������������������������������������858 Heinz body anemia�����������������������������������������������������������700 Helicobacter felis��������������������������������������������������������������163 Helicobacter hepaticus�����������������������������������������������������130 Helicobacter pylori 4t������������������������������������������ 163, 754, 774 gastric effects���������������������������������������������� 153, 157, 163 Hemangioma������������������������������������� 134, 240, 297, 757, 766 Hemangiosarcomas��������������������������� 134, 240, 297, 757, 766 Hematopoiesis extramedullary������������������������������ 66, 363, 383, 384, 702, 705, 788, 813, 835, 884 microenvironment in��������������������������������������������������709 suppression of���������������������������������������������������������������66 Hematopoietic stem cells (HSCs)������������������������������������360 Hematopoietic system���������������������������������������������� 707, 708 Hematotoxicity�����������������������������������������������������������������696

Hemoglobin����������������������������������6, 212, 213, 363, 690, 696, 697, 700, 701, 716, 730, 732, 734, 839, 840 oxidation of iron in�����������������������������������������������������701 Hemolytic anemia������������������������������������������������������������375 Hemorrhage adrenal cortex�������������������������������������������������������������559 lung��������������������������������������������������������55, 335, 706, 855 spleen��������������������������������������������������������������������������118 Hemosiderin deposition�������������������������������������������������������������������122 lung�����������������������������������������������������������������������������385 spleen������������������������������������������������������������������ 122, 384 Hepatic microsomal enzymes����������������������������������� 549, 550 Hepatitis 4t��������������������������������������������������������������� 126, 130 See also Liver Hepatoblastomas���������������������������������������������� 131, 133, 758 Hepatocellular adenoma�����������������������������������129, 131–133, 131–133, 757, 861 Hepatocellular carcinoma (HCC)��������������������������� 130, 131, 133, 757, 788 Hepatocellular hyperplasia�����������������������������������������������128 Hepatocellular hypertrophy��������������� 22, 55, 64, 66, 119, 123 Hepatocellular injury, indicators of���������������������������713–716 Hepatocellular neoplasia foci of cellular alteration in basophilic type���������������������������������������������� 128, 129 clear-cell type��������������������������������������������������������129 eosinophilic type���������������������������������������������������129 H&E staining for����������������������������������������� 128, 129 hepatoblastomas and���������������������������������������������������133 Hepatocytes�������������������������������������64, 80, 81, 102, 114–116, 119–121, 123–126, 130, 131, 134, 158, 188, 714, 715, 754, 758, 772, 773, 782, 787, 789 apoptosis of�����������������������������������������������������������������124 cytoplasmic inclusions in��������������������������������������������187 oncotic necrosis of������������������������������������������������������379 See also Liver Hepatomegaly������������������������������������������������������������������119 Hexachlorophene���������������������������������������������� 789, 800, 801 Hibernoma��������������������������������������������������������������� 523, 526 Hilar cell, ovary����������������������������������������������������������������202 Histiocytic lymphoma����������������������� 231, 240, 386, 757, 766 Histiocytic sarcomas��������������������������������� 134, 214, 231, 232, 240, 386, 757, 766 Histiocytoma malignant histiocytoma�������������������������������������� 526, 827 Histochemical stains������������������������14–15, 25, 26, 80, 89–90, 186, 255, 580 Histomorphometry, bone���������������������������������� 104, 578–581 Hodgkin’s lymphoma��������������������������������������������������������760 HOOH, see Hydrogen peroxide Hormones GI tract injury response��������137, 147, 154, 162, 165, 176 male reproductive system������������������������������������426–427

Toxicologic Pathology for Non-Pathologists 903 Index       Host resistance��������������������������������������������������������� 368, 503 H-ras mutations���������������������������������������������������������������821 HSCs, see Hematopoietic stem cells 5-HT3 receptors, see 5-Hydroxytryptamine 3 receptors Human chorionic gonadotropin (hCG)���������������������������420 Hyaline droplets, kidney�������������������� 211, 213–214, 232, 233 Hydralazine������������������������ 229, 264, 284, 290, 378, 509, 606 Hydrocephalus (hydrocephaly)�����������������������������������������886 Hydrogen peroxide (HOOH)���������������������93, 327, 640, 732 Hydrolysis������������������������������������������������������������������������154 Hydronephrosis������������������������� 215–216, 239, 793, 797, 889 Hydropic degeneration/change������������������122, 171, 186, 187 Hydroureter�������������������������������������������������������������� 239, 889 Hydroxychloroquine���������������������������������������������������������642 5-Hydroxytryptamine 3 (5-HT3) receptors���������������������287 Hypercalcemia, cancer-associated���������������������157, 551, 552, 554, 555, 562, 646 Hyperkeratosis���������������������������151–153, 163, 336, 450, 503, 509, 513, 518, 520, 521, 610, 792, 828, 886 Hyperparathyroidism nutritional�������������������������������������������������������������������556 primary�����������������������������������������������������������������������555 secondary���������������������������������������������208, 555, 556, 584 Hyperpigmentation, of uveal tract������������������������������������515 Hyperplasia adrenal cortex����������������������������������������������������� 387, 561 diffuse�����������������������������������������������������������559–561 focal�������������������������������������������������������������� 559, 561 glomerulosa��������������������������������������������������� 556, 557 lipoid���������������������������������������������������������������������559 subcapsular���������������������������������������������������� 559, 560 adrenal medulla diffuse�������������������������������������������������������������������559 focal����������������������������������������������������������������������559 overview����������������������������������������������������������������552 alveolar epithelium�����������������������������������������������������339 articular cartilage������������������������������������������������ 587, 673 bile duct�������������������������������������������������������������� 101, 131 bone marrow����������������������������������������������� 384, 512, 702 colon����������������������������������������������������167–169, 244, 512 compensatory��������������������������������������������������������������773 cornea����������������������������������������������������������������� 638, 648 corneal epithelium������������������������������������������������������638 endometrium dog���������������������������������������������������������������� 431, 438 human��������������������������������������������������� 431, 441, 447 monkeys����������������������������������������������������������������434 rodents�������������������������������������������432, 447, 448, 450 epidermal������������������������������������ 499, 503, 507, 509, 511, 515, 519, 524, 525 esophagus��������������������������������������������������� 145, 151, 152 forestomach����������������������������������������������������������������159 gastric mucosa��������������������������������������������� 157, 160, 193 gingiva���������������������������������������������������������������� 145, 146

oxicologic Pathology for Non-Pathologists 904  ITndex



  

Hyperplasia (cont.) growth plate����������������������������������������������������������������575 islet cells���������������������������������������������������������������������565 kidney renal pelvis����������������������208, 218, 232, 233, 244, 773 renal tubules�����������������������������������101, 207, 221, 512 Leydig cell�������������������������������������������������� 400, 419, 818 liver���������������������������������������������������������������������128–131 liver regenerative hyperplasia������� 160, 168, 180, 336, 673 lung Goblet cell hyperplasia�����������������������������������������316 neuroendocrine hyperplasia����������������������������������318 squamous hyperplasia���������������������������316, 324, 332, 335–337, 348, 349 squamous metaplasia������������� 324, 332, 335, 336, 338 type II cell hyperplasia�������������������������� 339, 340, 344 lymph node atypical follicular hyperplasia������������������������ 550, 768 histiocytic hyperplasia��������������������������� 232, 240, 385 overview�����������������������������������������167, 297, 380, 385 paracortex�����������������17, 364, 365, 370, 371, 811, 813 typical follicular hyperplasia���������������������������������550 mammary gland ductal���������������������� 131, 179, 180, 182, 461, 462, 822 lobular��������������������������������������������������� 457, 459, 462 overview�������������������������������������������������������� 419, 461 organelle���������������������������������������������������������������������171 cell swelling and����������������������������������������������������171 ovary���������������������������������������������������������������������������446 pancreas exocrine pancreas���������������������������������� 541, 563–565 islet cell hyperplasia�������������������������������������� 541, 565 parathyroid gland���������������������������������541, 552, 555, 556 pituitary, diffuse hyperplasia dog���������������������������������������������������������������� 559, 561 intermediate lobe��������������������������������������������������544 rodents������������������������������������������������������������������459 pituitary, focal hyperplasia�������������������������� 459, 559, 561 prostate dog�������������������������������������������������������� 405, 412, 425 human�������������������������������������������������������������������862 mouse�������������������������������������������������������������������409 rat�������������������������������������������������������������������������409 renal tubular����������������������������������������������������������������101 RPE����������������������������������������������������������������������������652 salivary duct�������������������������������������������������������� 148, 149 salivary gland���������������������������������������145, 146, 148–149 sebaceous cell��������������������������������������������������������������525 small intestine����������������������������������������������������� 167, 169 spleen lymphoid���������������������������������������������������������������524 plasma cell������������������������������������������������������������385 squamous cell���������������������������������������519, 520, 610, 612 thymus�������������������������������������������������������� 384, 512, 524

thyroid�������������������������������������������������547, 549, 550, 773 diffuse�������������������������������������������������������������������549 focal������������������������������������������������������ 550–552, 555 overview����������������������������������������������������������������547 urothelial����������������������������������������������������� 208, 218, 797 vascular��������������������������������������������������������������� 297, 551 Hyperprolactinemia������������������������������������������ 418, 425, 454 Hypersensitivity immune system and�������������������������������������������� 328, 374 in respiratory tract������������������������������������������������������329 vasculitis���������������������������������������������������������������������509 Hyperstimulation models��������������������������184, 189, 373, 374 Hyperthyroidism������������������������������������������������������ 167, 724 Hypertrophy adrenal, ACTH stimulation and���������������������������������560 adrenal gland���������������������������������������������� 387, 559–561 appearance of����������������������������������������������������������������64 cardiac��������������������������������������������������108, 297–298, 300 exocrine pancreas������������������������� 146, 149, 167–170, 180 gastric mucosa�������������������������������������������������������������162 heart����������������������������������������������������������� 297–298, 300 kidney juxtaglomerular apparatus�����������������������������202–204 renal tubules������������������������������������������ 237, 512, 796 liver�����������������������������������������������������������������������������123 muscle�������������������������������������������������������������������������604 organelle���������������������������������������������������������������������604 phospholipidosis and��������������������������������������������������167 pituitary����������������������������������������������������������������������547 renal, PCT and�����������������������������������������������������������560 RPE����������������������������������������������������������������������������652 salivary gland������������������������������������������������������ 148, 149 small intestine�����������������������������������������������������167–168 thymus������������������������������������������������������������������������512 thyroid������������������������������������������������������������������������865 vascular�������������������������������� 280, 281, 287, 289, 296–298 Hypoparathyroidism���������������������������������������������������������555 Hypoplasia�������������������������� 410, 411, 413, 414, 817, 818, 822 Hypospermatogenesis, in dogs��������������������������������� 410, 511 Hypothalamic-pituitary-thyroid axis���������������� 539, 548, 827 Hypothalamus, adenohypophysis regulation in����������������545 Hypothyroidism����������������������������������������499, 515, 816, 827 Hypoxia�������������������������������������171, 219, 285, 304, 318, 338, 558, 697, 702, 788, 800, 802 GI tract injury and������������������������������������������������������171

I IARC, see International Agency of Research on Cancer Iba1, see Ionized calcium-binding adaptor molecule 1 Ibuprofen��������������������������������������������������������������������������717 ICA, see Iridocorneal angle Idiosyncratic reactions������������������������������������������������������700 IGFs, see Insulin-like growth factors Ileum��������������������������������� 140–142, 164–166, 172, 173, 792

Immune system allergy, hypersensitivity in�������������������������������������������373 autophagy and���������������������������������������������������� 259, 274 DCs role in�����������������������������������������������������������������365 injury response of autoimmune diseases and hypersensitivity reactions�������������������������������������������������� 373, 374 lymphoid organ structure and physiology�����������356–366 T-and B-lymphocytes in compartments of�������� 358, 366 toxicity evaluation of biomarkers in������������������������������������������������ 368, 390 morphologic����������������������������������������������������������356 toxicity evaluation of���������������������������������������������������390 toxicity mechanisms of antigen-specific��������������������������������������������� 374, 376 direct���������������������������������������������������������������������379 indirect������������������������������������������������������������������378 See also specific organs Incus���������������������������������������������������������������������������������662 Indirect toxicity, immune system and�������������������������������378 Industrial (regulatory) toxicologic pathology overview of���������������������������������������������������������������������5 Infections, necrosis complicated by�����������������������������������172 Inflammation adnexa�������������������������������������������������������������������������518 autoimmune diseases and�������������������������������������������382 bone����������������������������������������������������������������������������586 cornea����������������������������������������������������������������� 644, 648 frustrated macrophages leading to GI tract injury and���������������������������������������������� 139, 145 immune-mediated vascular�����������������������������������������291 islet cell�����������������������������������������������������������������������565 joint����������������������������������������������������������������������������587 large intestine ulceration and��������������������������������������171 in mammary glands����������������������������������������������������409 from necrosis��������������������������������������������������������������295 pulmonary�������������������������������������������������� 332, 342–343 renal carcinogenesis and chronic���������������������������������210 stomach ulceration and�����������������������������������������������161 uveal tract�������������������������������������������������������������������644 Information, data compared to������������������������������� 48, 57–58 Inhalation asbestos��������������������������������������������������������������� 333, 345 exposure studies, for respiratory tract��������������������������312 fibers���������������������������������������������������������������������������333 manure������������������������������������������������������������������������343 particles������������������������������������������������������314, 315, 317, 330–333, 345 toxicology�������������������������������������������������������������������316 INHAND, see International Harmonization of Nomenclature and Diagnostic Criteria Inhibin������������������������������������������������������������������������������401 Innate immunity, of liver���������������������������������� 360, 379, 736 Inner ear injury responses and mechanisms of���������������������������669

Toxicologic Pathology for Non-Pathologists 905 Index       ototoxicant-induced morphologic changes�����������������671 specific ototoxicants����������������������������������������������������670 Insulin�����������������������������������������97, 175, 176, 272, 458, 538, 563–566, 582, 596, 729, 750, 760, 764 Insulin-like growth factors (IGFs)�����������������������������������611 Integumentary system����������������������������������������������483–532 See also Skin International Agency of Research on Cancer (IARC) International Harmonization of Nomenclature and Diagnostic Criteria (INHAND)��������������� 32, 57, 64, 68, 134, 143, 241, 348, 500, 577, 598, 630, 768, 769, 870, 875 Interstitial fluid, regulation of��������������������������� 341, 401, 425 Interstitial pneumonia����������������������������������������������342–345 Intestine, see Large intestine; Small intestine Intimal hyperplasia�����������������������������������������������������������295 Intracytoplasmic inclusions�������������������������������������� 554, 607 Intramembranous ossification������������������������������������������575 Intraocular pressure (IOP)�������������������������623, 643, 653, 654 Intravascular macrophage (IVM)�������������������������������������322 Intravascular perfusion�����������������������������������������������������268 Investigative toxicologic pathology/ pathologists�������������194 In vitro methods for adrenal cortex evaluation�������������������������������238–239 for blood vessel toxicity evaluation�����������������������������302 for respiratory tract testing�����������������������������������������334 Involution���������������������������������������������������������������� 359, 384, 387, 545, 550 Iodoacetate�����������������������������������������������������������������������642 Ionized calcium-binding adaptor molecule 1 (Iba1)���������267 IOP, see Intraocular pressure Iopanoic acid��������������������������������������������������������������������549 4-Ipomeanol����������������������������������������������������� 326, 339, 342 Iridocorneal angle (ICA)���������������������������624, 644, 653, 654 Iris����������������������������������������������������� 620, 623, 644, 653, 654 See also Eye Iron�������������������������������������� 15, 106, 122, 163, 168, 333, 345, 363, 610, 670, 698, 700–702, 708 kidney pigmentation���������������������������������������������������213 liver pigmentation�����������������������������������������������122–123 Irradiation, parathyroid gland tumor development and���������������������������������������������555 Islets of Langerhans (islets) amyloidosis�����������������������������������������������������������������565 hyperplasia of��������������������������������������������������������������565 inflammation of����������������������������������������������������������565 neoplasias of���������������������������������������������������������������565 Isolated vascular muscle strips Isoniazid����������������������������������������������������������� 509, 519, 715 Isopropylamine hydrochloride Isoproterenol�������������������������������������������� 108, 150, 181, 284, 298, 301, 721 IUPHAR, see International Harmonization of Nomenclature and Diagnostic Criteria IVM, see Intravascular macrophage

oxicologic Pathology for Non-Pathologists 906  ITndex



  

J Jejunum��������������������������������������141, 142, 164, 165, 172, 792 JGA, see Juxtaglomerular apparatus Joints anatomy and histology����������������������������������������572–576 cartilage degeneration arthritis�����������������������������������������������������������������592 atrophy������������������������������������������������������������������585 drug-induced degeneration��������������������������� 588, 590 spontaneous degeneration�����������������������������586–588 hyperplasia of articular cartilage�������������������������573–575 technical considerations in analysis�������������������� 580–584, see Bones and joints Juxtaglomerular apparatus ( JGA)����������������������������� 202, 203 See also Kidney

K Kainic acid�������������������������������������������������������� 263, 641, 801 Kanamycin������������������������������������������������������������������������670 Karnovsky’s fixative����������������������������������������������������������633 Karyolysis����������������������������������������������������������������� 222, 379 Karyorrhexis������������������������������������������������������������� 222, 379 KCs, see Kupffer cells Keratinocytes�����������������������������187, 484–486, 491, 499, 500, 503, 505, 509, 510, 512–514 necrosis of����������������������������������������������������������� 512, 513 Keratitis�������������������������������������������������������������������� 646, 648 Keratoacanthomas�������������������������������������522, 525, 752, 753 Ketoconazole��������������������������������������������������������������������560 Kidney amyloidosis���������������������������������������������������������209–211 anatomy��������������������������������������������������������������202–204 apoptosis in, drugs inducing���������������������������������������222 blood supply����������������������������������������������� 202–203, 210 biomarkers of, evaluation of novel����������������������������������������������������� 234, 242, 243 Bowman’s capsule changes���������������������������������� 203, 204 carcinogenesis of, mechanisms of genotoxic carcinogens and������������������������������������764 lysosomal enzyme release and�������������������������������213 nongenotoxic carcinogens and������������������������������207 oxidative stress/chronic inflammation����������� 222, 226 rat CPN exacerbation and neoplasms����������� 206, 207 carcinogenic potential testing for���������208, 210, 215, 240 crystal deposition������������������������������������������������ 214, 239 drug-induced injury to, from altered cardiac function208 function of������������������������������������������������������������������202 glomerulonephritis dog������������������������������������������������������������������������229 human�����������������������������������������������������������227–229 monkey������������������������������������������������� 213, 226, 229 mouse�������������������������������������������������������������������231 rat�������������������������������������������������������������������������233

glomerulosclerosis dog������������������������������������������������������������������������555 mouse�������������������������������������������������������������������235 rat����������������������������������������������������������������� 235, 238 glomerulus mineralization�����������������������������������������������211–212 vacuolation������������������������������������������������������������226 hypertrophy of������������������������������������������������������������237 hypoxia of�������������������������������������������������������������������219 infarction��������������������������������������������������������������������202 injury response of glomerulus injury, inflammatory�������������������217–218 hyperplasia and neoplasia�����������������������������230–233 mineralization�����������������������������������������������211–212 morphologic response�������������������������������������������219 physiologic, molecular, and biochemical responses������������������������������������������������� 208, 210 renal papillary necrosis������������������������������������������224 renal tubule injuries����������������������������������������������234 interstitial fibrosis��������������������������������������� 214, 217, 233 interstitial nephritis����������������������������������������������������887 interstitium��������������������������202, 206, 209–211, 214, 217, 220, 224, 244 ischemia of�������������������������������������������215, 223, 224, 237 juxtaglomerular apparatus structure����������������������������������������������������������������204 metabolic activity of����������������������������������������������������202 mineralization overview����������������������������������������������������������������211 neoplasia, epithelial neoplasms adenoma���������������������������������������������������������������877 carcinoma�������������������������������������������������������������877 mesenchymal tumors��������������������������������������������877 nephroblastoma����������������������������������������������������231 neoplasia, overview���������������������������������������������230–233 nephron enzyme distribution����������������������������������������������202 glomerulus������������������������������������������������������������202 renal tubule��������������������������������������������������� 219, 221 papillary inflammation and necrosis������������������� 218, 224 pigment droplets���������������������������������������������������������213 PTH action and���������������������������������������������������������211 renal pelvis dilatation���������������������������������������������������������������797 hyperplasia�������������� 208, 218, 232, 233, 244, 773, 797 structure����������������������������������������������������������������204 renal tubules cast nephropathy������������������������������������������ 206, 212, 879, 881 cystic change�������������������������������������������������214–215 dilatation������������������������������������������������������� 225, 793 hyaline droplets���������������������������������������������213–214 hyperplasia���������������������������������������������������� 207, 223 hypertrophy����������������������������������������������������������237

necrosis, histopathology�����������������211, 213, 218, 222 vacuolation����������������������218, 220, 225, 226, 238, 241 spontaneous lesions of in beagle dogs�������������������������������������������������������209 CPN�������������������������������������������������������������206–209 in hamsters�����������������������������������������������������������209 in mice������������������������������������������������������������������229 in monkeys������������������������������������������������������������229 in rabbits������������������������������������������������������� 202, 229 in rats������������������������������������������������������������ 202, 230 structure of����������������������������������������������������������202–204 toxicity evaluation of GFR abnormalities��������������������������������������� 229, 235 tubular function abnormalities������������������������������213 toxicity mechanisms of immune-mediated injury��������������������������������������225 renal papilla injuries����������������������������������������������224 tubules of, injuries to degeneration and necrosis�����������������������������218–220 tubulointerstitial disease���������������������������������������235 urinary excretion of xenobiotics����������������������������������242 vascular changes����������������������������������������������������������244 weight changes������������������������������������������� 204, 211, 229 See also Renal biomarkers Kidney injury molecule-1 (KIM-1)������������������������� 229, 234, 695, 718, 719 KIM-1, see Kidney injury molecule-1 Kupffer cells (KCs)������������������������������������������� 115, 116, 128

L Laboratory animal studies, for developmental toxicity������������������������������������������������������������867 Lacrimal glands�����������������������������������������148, 620, 621, 638 See also Eye Lacrimal system�������������������������������������������������������� 621, 640 Lactation��������������������������� 164, 167, 168, 456–458, 461, 545, 795, 819, 823, 854 Lamina cribrosa (LC)����������������������������������������������� 629, 645 Lamina propria��������������������������154, 155, 158, 164, 165, 168, 172–174, 239, 315, 335, 674, 676, 677, 680 Langerhans cells (LCs)������������������������������������� 485, 486, 510 Large granular lymphocytic leukemia����������������������� 134, 240 Large intestine anatomy��������������������������������������������������������������164–166 drug-induced changes animals�������������������������������������������������� 164, 167, 170 humans��������������������������������������������������������� 166, 167 function of����������������������������������������������������������164–166 histology����������������������������������������������������� 164–166, 169 hyperplasia����������������������������������������������������������167–168 hypoxia of�������������������������������������������������������������������171 infection bacteria�����������������������������������������������������������������166 inflammation���������������������������������������������� 168, 170, 173

Toxicologic Pathology for Non-Pathologists 907 Index       injury, response to cecal enlargement�������������������������������������������������166 proliferative response��������������������������������������������172 ulceration and inflammation���������������������������������173 microscopic lesions of�������������������������������������������������173 neoplasia adenocarcinoma��������������������������������������������169–170 adenoma������������������������������������������������������� 168, 169 classification����������������������������������������������������������168 inflammation role��������������������������������� 168, 170, 173 overview�������������������������������������������������������� 168, 169 neoplasia of��������������������������������������������������������� 168, 169 structure of����������������������������������������������������������164–166 ulceration������������������������������������������������������������171–173 Larynx anatomy and histology���������������������������������������� 313, 316 anatomy of������������������������������������������������������������������316 injury, response to�������������������������������������������������������335 neoplasia���������������������������������������������������������������������336 toxicity testing for�������������������������������������������������������350 Laxatives, colonic hyperplasia�������������������������������������������171 LC, see Lamina cribrosa LCs, see Langerhans cells Leiomyoma mesovarial�������������������������������������������������������������������446 soft tissue��������������������������������������������������������������������757 Leiomyosarcoma����������������������������������������������� 170, 240, 757 Lens capsule rupture of��������������������������������������� 644, 649, 650 cataract of�������������������������������������������������������������������639 compounds causing�����������������������������������������������640 injury response of��������������������������������������������������������648 structure and function of������������������������������������ 625, 626 toxicity mechanisms of��������������������������������������� 639, 640 See also Eye Leukemia��������������������������������������������������������������������������758 classification overview�������������������������134, 386, 710, 758 erythroid leukemia������������������������������������������������������710 granulocytic leukemia�������������������������������������������������710 induction by anticancer therapies��������������� 223, 373–374 large granular lymphocytic leukemia������������������ 134, 240 lymphocytic leukemia�������������������������������������������������134 myeloblastic leukemia����������������������������������������� 703, 709 virus induction in rodents��������������������130, 180, 386, 761 Leukocyte infiltrates������������������������������������������������� 295, 644 Leydig cells atrophy of������������������������������������������������������������� 19, 418 hyperplasia of������������������������������������������������������ 419, 818 morphologic changes to��������������������������������������418–419 tumors of��������������������������������������������������������������������762 See also Testis LFB, see Luxol fast blue LH, see Luteinizing hormone Lidocaine������������������������������������������������������������������ 638, 639

oxicologic Pathology for Non-Pathologists 908  ITndex



  

Limiting ridge�������������������������������������������140, 155, 158, 159 Lipase������������������������������������������������ 146, 154, 175, 790, 792 Lipid-lowering drugs��������������������������������������������������������731 Lipid metabolism, as liver toxicity cellular target����������������������������������������������� 121, 731, 792 Lipidosis���������������������������������������������������������������������������121 microvesicular�������������������������������������������������������������121 Lipid peroxidation free radicals initiating��������������������������������� 219, 327, 599 injury��������������������������������������������������������������������������324 Lipofuscin cardiac�������������������������������������������������������������������������275 kidney�������������������������������������������������������������������������213 liver pigmentation�����������������������������������������������122–123 spleen��������������������������������������������������������������������������385 Lipofuscin pigment��������������������������������������������������122–123 Lipoma soft tissue������������������������������������������������������������522–523 Liposarcoma�������������������������������������� 231, 522–523, 526, 757 Lipoxygenase��������������������������������������������������������������������337 Liquefactive necrosis���������������������������������������������������������653 Lithium������������������������������������������������������������ 221, 549, 601 Liver acinus of�������������������������������������������������������������� 114, 115 age and sex differences����������117, 118, 122, 128, 129, 131 amyloid deposition�����������������������������������������������������209 cirrhosis�������������������������������������������������������������� 130, 724 clear cell change����������������������������������������������������������129 cytochromes P450����������������327, 339, 546, 671, 783, 789 drug crystals����������������������������������������������������������������132 environmental factors influencing function����������������138 eosinophilic inclusions������������������������������������������������240 with fatty change��������������������������������������������������������121 foci of hepatocellular alteration������������������ 128, 129, 132 hepatic neoplasia due to toxicant injury of cholangiocarcinomas���������������������������������������������134 hepatocellular neoplasias���������������������������������������129 Kupffer cell sarcomas��������������������������������������������115 stellate cell���������������������������������������������������� 115, 134 hepatocellular adenoma������������������������������ 129, 131–133 hepatocellular carcinoma���������������������������� 130, 131, 133 hepatocyte function����������������������������114–116, 119–121, 123–125, 128, 130, 134 hepatocytes in��������������������� 114–116, 119, 121, 123–125, 128, 130, 134 hepatomegaly and�������������������������������������������������������119 histophysiology and compartments of����������������115–117 human hepatotoxicity��������������������������������� 125, 127, 131 hyperplasia of������������������������������������������������������128–131 hypertrophy and hyperplasia enzyme inducers���������������������������������������������������123 epidermal growth factor induction������������������������149 overview�����������������������������������������119, 123, 128, 131 peroxisomal proliferation������������������������������ 123, 773 hypertrophy of���������������������������������������������������� 119, 123

immunological mechanisms of toxic injury to inflammation chronic hepatitis���������������������������������������������������127 drug-induced������������������������������������������������ 116, 121 spontaneous������������117, 118, 122, 124, 128, 132–134 lobules of������������������������������������������������������������114–117 metaplastic foci of pancreatic tissue����������������������������177 mitochondrial changes������������������������������������������������121 morphology and histology���������� 113, 114, 117–121, 124, 127–129, 131–133 necrosis bridging�������������������������������������������������������� 125, 126 centrilobular�������������������������������������������������� 124, 125 focal�������������������������������������������������������������� 124, 126 periportal������������������������������������������������������124–126 single cell��������������������������������������������������������������124 zonal���������������������������������������������������������������������124 nonneoplastic responses to toxicant injury of bile duct hyperplasia���������������������������������������������131 cholangiofibrosis���������������������������������������������������131 cholestasis�������������������������������������������������������������117 infiltrations and pigments�������������������������������������126 phospholipidosis�������������������������������������������121–122 nonparenchymal cell injury in Kupffer cells�������������������������������������������������� 115, 116 sinusoidal endothelial cells������������������������������������116 stellate cells��������������������������������������������������� 115, 134 oral absorption into����������������������������������������������������119 phospholipidosis�������������������������������������������������120–122 pigmentation������������������������������������������������������122–123 preclinical toxicity study relevance to humans������������118 proliferative lesion classification���������������������������������131 regenerative hyperplasia����������������������������������������������128 safety assessment����������������� 113, 117, 120, 123–125, 129 sinusoidal dilatation������������������������������������ 115–117, 123 spongiosis hepatitis�����������������������������������������������������122 steatosis��������������������������������������������������������������� 117, 121 structure, function, physiology and cell biology in������������������������������������������������114–117 techniques for analysis������������������������������������������������120 tissue sampling��������������������������������������������������� 120, 124 vascular changes������������������������������������������ 117, 127–128 weight changes������������������������������������������� 114, 119, 123 See also Hepatocellular degeneration and necrosis Liver mass (liver weight) assessment��������������������������������119 Liver toxicity cellular targets of cytoplasm������������������������������������������������������120–122 lipid metabolism���������������������������������������������������121 mitochondria��������������������������������������������������������121 nucleus������������������������������������������������������������������121 mechanisms of covalent binding���������������������������������������������������325 GSH���������������������������������������������������������������������325 metabolic activation����������������������������������������������325

morphology and microscopic evaluation for������������������������������������121 Liver weight (liver mass) assessment��������������������������������119 Lobules liver���������������������������������������������������������������������114–117 mammary glands������������������������������������������������ 400, 457 Longitudinal bone growth�������������������������577, 578, 590, 832 Loop diuretics�������������������������������������������663, 670, 671, 685 Loop of Henle���������������������������������������������������������� 202, 223 Lower urinary tract toxicity evaluation of special�������������������������������������������������������������������239 urinalysis���������������������������������������������������������������240 Lungs aerosol absorption in���������������������������������������������������349 anatomy and histology����������������������������������������313–323 blood vessels hyperplasia��������������������� 324, 330, 332, 334–336, 338 hypertrophy�������������������������������������������������� 324, 334 vasculitis�������������������������������������������������������� 290, 291 cancer of in animals�������������������������������������������������������������348 in humans�������������������������������������������������������������348 congestion�������������������������������������������������������������������335 emphysema������������������������������������������338, 344, 346, 347 eosinophilic inclusions������������������������������������������������343 fibrosis drug induction, animals�������������������������������� 316, 329 drug induction, humans�������������������������������� 344, 345 pleura��������������������������������������������������������������������333 gas absorption in���������������������������������������������������������320 alveolar parenchyma������������������������������������� 320, 321 gas-exchange regions of�����������������������313, 314, 319–321 respiratory bronchioles�������������������������� 313, 319, 320 granuloma��������������������������������������������������� 329, 334, 345 hemorrhage�����������������������������������������������������������������335 hyperplasia Goblet cell hyperplasia�����������������������������������������316 neuroendocrine hyperplasia����������������������������������318 squamous hyperplasia����������������������������������� 316, 324 squamous metaplasia������������� 324, 332, 335, 336, 338 type II cell hyperplasia�������������������321, 339, 340, 344 inflammation drug induction������������������������������������������������������329 spontaneous����������������������������������������������������������335 lymphoid system���������������������������������������������������������344 macrophage accumulation����������� 329, 331, 333, 342, 344 mice with tumors in�������������������������������������������� 316, 326 neoplasia hamster�����������������������������������������������������������������337 mouse�������������������������������������������������������������������754 overview��������������������������������� 746, 748, 751, 753–755 rat�������������������������������������������������������������������������337 safety assessment������������������������������������������ 764, 765 strain A mouse pulmonary tumor bioassay�����������348

Toxicologic Pathology for Non-Pathologists 909 Index       phospholipidosis etiology�����������������������������������������������������������������167 morphology���������������������������������������������������� 31, 167 overview���������������������������������������������������� 30, 31, 167 safety assessment����������������������������������������������������30 pigmentation��������������������������������������������������������������345 pleura������������������������������������322, 323, 333, 334, 341, 345 pulmonary edema���������������� 325, 327, 328, 340–342, 353 structural evaluation��������������������� 311–313, 319, 335, 352 Lupus-like syndromes������������������������������������������������������229 Luteinizing hormone (LH)����������������������401, 405, 417–419, 426, 427, 430, 432, 442, 446, 453, 454, 462, 539, 541, 544, 545, 738 Luteotrophs����������������������������������������������������������������������445 Luxol fast blue (LFB)���������������������������������������253, 260, 267, 805, 806, 808 Lymphatics��������������������������������154, 165, 203, 281, 323, 330, 339, 341, 348, 364–366, 593, 609, 623, 747 Lymph nodes areas drained by����������������������������������������������������������385 assessment������������������������������������������������������������������366 atrophy���������������������������������������������������������������� 384, 385 HEVs in���������������������������������������������������������������������365 histopathology in��������������������������������������������������������356 histophysiology and compartments of������������������������359 hyperplasia atypical follicular hyperplasia������������������ 17, 365, 814 histiocytic hyperplasia����������������������������������� 385, 386 overview������������������������������������������������ 364, 367, 369 paracortex����������������������������������������������� 17, 364, 813 safety assessment����������������������������������������� 356, 367, 379, 380, 391 sinus histiocytosis�������������������������������������������������������385 structure and physiology of���������������������������������356–366 T-and B-lymphocytes in������������������������������������ 358, 366 Lymphocyte depletion pattern�����������������������������������������729 Lymphocyte predominance����������������������������������������������377 Lymphocytic leukemia�����������������������������������������������������134 Lymphocytic lymphoma���������������������������������������������������386 Lymphoid depletion������������������������������������������� 34, 385, 704 Lymphoid organs autoimmune diseases and alterations to������������� 359, 373, 374, 376 structure and physiology of���������������������������������356–366 weight and gross pathology alterations of aging and��������������������������������������������������������������384 stress and��������������������������������������������������������������384 Lymphomas��������������������������� 2, 134, 214, 231, 232, 240, 386, 710, 754, 757, 760, 766, 774, 877 classification overview�������������������������������� 386, 758, 877 induction by anticancer therapies��������������� 223, 373–374 lymphocytic lymphoma����������������������������������������������386 virus induction in rodents�����������������������������������������������4 Lysosomes������������������������������������������������ 121, 213, 220, 234, 607, 608, 773

oxicologic Pathology for Non-Pathologists 910  ITndex



  

M Macrophages alveolar�������������������������������������27, 31, 312, 321, 322, 324, 330, 331, 333, 334, 343 infiltrates of����������������������������������������������������������330 inflammation and autoimmune disease from frustrated��������������������������������������������������������376 intravascular����������������������������������������������������������������322 necrosis and infiltration of���������������������������������� 282, 382 Macula����������������������������������������������� 627, 628, 645, 655, 665 Macular degeneration, age-related���������������������������� 636, 655 Magnesium����������������� 179, 211, 239, 554, 690, 691, 735, 836 Magnetic resonance imaging (MRI)������������������� 26–28, 104, 106–107, 584, 599 Malabsorption, intestinal������������������������������������������ 727, 728 Male fertility���������������������������������������������������������������������398 Male reproductive system injury response of background pathology������������������������������������������406 immaturity and peripuberty��������������������������406–414 organ weight changes������������������������������������424–425 recovery and reversibility in����������������������������������423 stress and body weight loss�����������������������������������424 introduction to����������������������������������������������������397–399 structure, function, and cell biology of accessory sex organs����������������������������������������������405 embryonic development��������������������������������399–400 hormonal regulation���������������������������������������������405 postnatal development of reproductive tract���������������������������������������������������������399–400 Sertoli cell division and maturation����������������������400 Sertoli cell regulation of protein secretion������������401 testis��������������������������������������������������������������400–402 toxicity evaluation of fertility assessment������������������������������������������������426 fixation of testis����������������������������������������������������404 hormone analysis������������������������������������������426–427 morphologic evaluation����������������������������������������402 organ weights������������������������������������������������424–425 special techniques�������������������������������������������������403 sperm assessment��������������������������������������������������426 toxicity mechanisms of morphologic patterns to different injuries������������402 See also Prostate; Testis; specific organs Malignant hyperthermia������������������������������������������� 529, 531 Malignant melanoma����������������������������������������������� 514, 519 Malleus�����������������������������������������������������������������������������662 MALT, see Mucosa-associated lymphoid tissue Mammary glands development of bud and ductal tree formation�������������������������������455 ductal elongation��������������������������������������������������455 lobuloalveolar differentiation��������������������������������458 pregnancy and lactation����������������������������������������459 species variation in������������������������������������������������455

feminization of male���������������������������������������������������461 fibroadenomas of��������������������������������������������������������460 hyperplasia of���������������������������������������������� 459, 461–462 injury response of morphologic response����������������������������������� 461, 464 lobules of������������������������������������������������������������457–459 morphologic evaluation of���������������������������������� 461, 464 neoplasia of��������������������������������������������������������� 462, 463 structure and function of������������������������������������456–458 toxicity evaluation of animal studies�������������������������������������������������������455 whole mount preparations of��������������������������������������463 See also Breast cancer Manganese������������������������������������������������������� 106, 184, 339 Manure, inhalation of�������������������������������������������������������343 MAPK, see Mitogen-activated protein kinase Matrix metalloproteinases������������������������������������������������833 Maximum tolerated dose (MTD)������������������������������������764 M cells���������������������������������������������������������������������� 174, 366 MCT, see Monocrotaline MCTP, see Metabolite dehydromonocrotaline MDA, see Malondialdehyde Megakaryocytes (MKs)�����������������������������702, 705, 706, 708 cytoplasmic maturation and migration of�������������������705 development of�����������������������������������������������������������706 progenitors������������������������������������������������������������������706 Megakaryopoiesis�������������������������������������������������������������707 Melamine����������������������������������������������������������������� 216, 221 Melanin pigment������������������������������ 274, 485, 486, 494, 514, 623, 635, 648, 653 Melanocytes����������������������������������������������������� 146, 485, 544 Melanocyte-stimulating hormone (MSH)�����������������������544 Melanoma����������������������������������514, 519, 523, 827, 852, 861 Membranous labyrinth�����������������������������������������������������663 Meningioma������������������������������������������������������������� 749, 758 Menstrual cycle, of monkeys���������������������������������������������434 Mercuric chloride����������������������������������������������������� 633, 798 Mercury������������������������������ 222, 329, 633, 672, 717, 801, 863 Merkel cells����������������������������������������������������������������������485 Mesangiolysis�������������������������������������������������������������������888 Mesenchymal cells������������������������������ 97, 287, 296, 297, 399, 429, 456, 517, 526, 584, 598, 611, 882, 884 Mesenchymal proliferative lesions������������������������������������240 Mesothelioma��������������������������������������������333, 345, 348, 758 Metabolic activation cell injury and�������������������������������������������������������������202 liver toxicity and���������������������������������������������������������114 respiratory tract toxicity and�������������������������������325–328 Metabolism neurotransmitter reduced��������������������������������������������593 thyroid follicular cells and�������������������������������������������549 xenobiotic disposition and phase I���������������������������������������������������������� 166, 183 phase II��������������������������������������������������������� 166, 183 Metabolite dehydromonocrotaline (MCTP)��������������������327 Metalloproteins����������������������������������������������������������������833

Metallothionein (MT)�����������������������������������������������������189 Metamyelocytes����������������������������������������������������������������709 Metaphysis��������������������������������572, 573, 575, 578, 580, 581, 583, 590, 833–835 impaired osteoclast formation in��������������������������������835 Metaplasia������������������������� 148, 150, 168–169, 183, 188, 324, 332, 335, 336, 338, 423, 441, 447, 448, 649, 674, 758, 759, 888 Metestrus in dogs������������������������������������������������������������������������433 in rats��������������������������������������������������������������������������433 Methanol������������������������������������������������������������������������ 6, 87 Methemoglobin�����������������������������������������191, 701, 732, 734 Methicillin������������������������������������������������������������������������218 3-Methylfuran�������������������������������������������������� 326, 334, 339 3-Methylindole (3MI)�������������������������������326, 334, 337, 341 Methyl mercury������������������������������������������������ 672, 801, 863 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridone (MPTP)����������������������������������������������������������263 Metronidazole������������������������������������������������������������������519 Mice lung tumors in�������������������������������������������� 332, 348, 349 mammary gland development in��������������������������������455 mammary gland injury in�������������������������������������������480 reproductive system background pathology in������������436 sexual immaturity and peripuberty of�������������������������406 spermatogenic cycle in������������������������������������������������404 spontaneous kidney lesions in�������������������������������������206 See also Genetically engineered mouse models Microbiomes���������������������� 138, 139, 191, 369, 483, 489, 491 Microbiota������������������������������������������������������������������������138 Micro-CT������������������������������������������� 27, 104, 106, 581, 583 Microflora, gut������������������������������������������������������������������138 Microglia����������������������������� 97, 253, 258, 259, 261, 265–267, 807, 885, 888 Microgliosis��������������������������������261, 266, 274, 275, 807, 888 Microliths�������������������������������������������������������������������������665 Microphthalmia����������������������������������������������������������������646 microRNA�������������������������������������������������������� 300, 301, 599 Middle ear bacterial otitis media in�����������������������������������������������674 catarrhal otitis media in����������������������������������������������674 injury responses and mechanisms of�������������������670–673 squamous cyst in���������������������������������������������������������674 xenobiotics injected into tympanic bulla and������������� 675, 677–679 xenobiotics injected into tympanic membrane and�������������������������������������������������� 675, 678, 680 Midzonal necrosis�������������������������������������������������������������125 Mifepristone (RU486)������������������������������������������������������450 Mineralization����������������������������122, 148, 294, 440, 795, 888 bones���������������������������������������������������������� 574, 590, 591 corneal subepithelial������������������������������������������� 646, 647 renal������������������������������������������������������������ 157, 211–212 Mineralocorticoids������������������������������������������� 556, 559, 560

Toxicologic Pathology for Non-Pathologists 911 Index       Minipigs estrous cycle in, histology of���������������������������������������433 reproductive system background pathology in������������398 sexual immaturity and peripuberty of�������������������������406 thymus of, stress-related changes to����������������������������704 Minoxidil������������������������������������284, 285, 290, 296, 300, 517 MIT, see Monoiodotyrosine Mitochondria������������� 204, 284, 311, 323, 594, 595, 608, 715 toxicity altered function of������������������������������������������600 Mitochondria, liver changes���������������������������������������������114 Mitochondrial permeability transition (MPT)�����������������284 Mitogen-activated protein kinase (MAPK)���������������������522 MKs, see Megakaryocytes MMTV, see Mouse mammary tumor virus Modeling, PD�������������������������������������������������������������������236 Monkeys estrous cycle in, histology of���������������������������������������434 menstrual cycle of�������������������������������������������������������434 reproductive system background pathology in������������398 uterus endometrial hyperplasia�������������������������������� 434, 450 Monocrotaline (MCT)��������������������������������������������� 327, 341 Monocytes���������������������������������126, 146, 266, 330, 340, 344, 358, 360, 372, 386, 389, 485, 574, 703–705, 729 Monoiodotyrosine (MIT)������������������������������������������������548 Morgagnian globules��������������������������������������������������������649 Morphine��������������������������������������������������������� 170, 640, 837 Morphologic development, normal human�������������� 812, 828 Morphometry��������������� 80, 101, 104, 388, 783, 804, 805, 809 Motility, of GI tract����������������������������������������������������������139 Motor units�������������������������������������������������������������� 593, 605 Mouse mammary tumor virus (MMTV)�������������������������460 MPT, see Mitochondrial permeability transition MRI, see Magnetic resonance imaging MT, see Metallothionein MTD, see Maximum tolerated dose Mucin������������������������������������������������ 147, 148, 150, 165, 654 Mucosa-associated lymphoid tissue (MALT)����������������� 357, 361–363, 365–366, 368–371, 386 structure and physiology of���������������������������������365–366 T-and B-lymphocytes in��������������������������������������������366 Mucosa, gastric cytotoxicity and damage to cytotoxic agents and heavy metals causing������������191 NSAID inducing��������������������������������������������������161 radiomimetic agents causing���������������������������������173 epithelial cells of proliferation of���������������������������������������������� 334, 335 erosions in����������������������������������������������������������� 161, 172 injury response of colon�������������������������������141, 142, 164, 168, 169, 171 small intestine�������������������������������������������������������164 stomach������������������141, 144, 153–158, 160, 161, 163 Mucosal immunity��������������������������������������������������� 174, 363 Mucus������������������� 27, 312, 315, 317, 336–338, 347, 376, 888

oxicologic Pathology for Non-Pathologists 912  ITndex



  

Müllerian ducts�������������������������������������������������������� 399, 429 Multinucleated syncytial cells�������������������������������������������593 Muscle spindles����������������������������������������������������������������593 Muscularis mucosae��������������������������� 154, 155, 159, 165, 172 Mustard gas (sulfur mustard)��������������������������������������������337 Mutagenicity testing, for GI tract evaluation�������������������193 Mycoplasma pulmonis����������������������������������������������������� 2, 854 Mycotoxins��������������������������������������������������������������� 172, 183 Myelin edema����������������������������������������������������������������� 268, 802 primary demyelination and�����������������������������������������261 secondary demyelination and��������������������������������������261 Myelinopathy(ies)��������������������������������������������� 259, 260, 800 Myeloblasts��������������������������������������������������������������� 703, 709 Myelopoiesis���������������������������������������������������������������������710 Myelosuppression����������������������������������������������������� 702, 706 Myenteric plexus���������������������������������������������� 139, 152, 154 Myoblasts�������������������������������������������������������������������������602 Myocardial degeneration���������������������������282, 293, 299, 304 Myocardial infarction with toxic relations������������������������������286, 299, 723, 858 Myocardium������������������������������108, 281, 284, 286, 288–290, 293, 294, 297, 300–302–304, 723 Myocytes�������������� 64, 286, 293, 298, 303, 512, 593, 596-598, 600–609, 721–724, 858 Myofibers������������285, 293, 298, 299, 593, 597, 601, 602, 608 Myofibrils����������������������������������������������������������������� 297, 593 Myofibroblasts������������������������������18, 227, 319, 321, 322, 502 Myofilament degeneration��������������������������������������� 593, 608 Myoglobin���������������������������������298, 300, 594, 595, 599, 603, 608, 723, 724, 732, 734 Myopathies, corticosteroids inducing�������������������������������605 Myotonia��������������������������������������������������������������������������601 Myotoxicity, see Skeletal muscle Myotubes��������������������������������������������������������������������������602

N N-Acetyl-α-(D)-glucosaminidase (NAG)��������������� 718, 721 NAG, see N-Acetyl-α-(D)-glucosaminidase (NAG) Nalidixic acid��������������������������������������������������������������������835 NALT, see Nasal-associated lymphoid tissue (NALT) Nanofibers������������������������������������������������������������������������334 Naphthalene�������������������������������������� 318, 334, 339, 360, 641 Nasal-associated lymphoid tissue (NALT)�������������� 357, 366, 811, 812, 814 Nasal cavity����������� 48, 144, 314–316, 336, 337, 350, 765, 892 Nasal epithelium����������������������������������������������� 315, 316, 336 Nasal mucosa���������������������������������������������������� 315, 328, 334 Nasal septum������������������������������������������������������������ 314, 335 Nasal vestibule������������������������������������������������������������������315 National Cancer Institute (NCI)��������������������������������������875 National Toxicology Program (NTP)���������������134, 167, 231, 585, 587, 588, 609, 611, 761, 819 Natural killer (NK) cells������������������������������������������� 117, 358

NBF, see Neutral buffered formalin (NBF) NCI, see National Cancer Institute (NCI) Necrotic cells���������������������������������������������������� 223, 335, 382 Neomycin����������������������������������������������������������������� 510, 670 Nephritis���������������������������������������������������������� 245, 885, 887 Nephroblastoma���������������������������������������������������������������231 N-ethyl-N-nitrosourea�����������������������������������������������������153 Neural retina���������������������������������������������������������������������650 Neurohypophysis��������������������������������������������������������������544 Neuromuscular blockade, drug-induced���������������������������601 Neuronophagia�����������������������������������������������������������������899 Neurotransmitters��������������������������������������259, 265, 268, 593 Neutral buffered formalin (NBF)���������������������83–85, 87, 91, 93, 96, 368, 369, 632, 633 Neutropenia����������������������������������������������������������������������710 Neutrophil gelatinase-associated lipocalin (NGAL)������������������������� 229, 234, 242, 243, 673, 718–720, 869 Nickel��������������������������������������������������������������� 329, 337, 510 Nicotine�������������������������������������������������������������������� 184, 562 Nifedipine���������������������������������������������������������������� 184, 562 Nipple������������������������������������������������ 455, 456, 459, 511, 819 Nissl stains������������������������������������������������������������������������263 Nitric oxide��������������������������������������������������������������� 343, 515 Nitrosamines���������������������������������������������153, 189, 337, 862 “No-effect” outcome�����������������������������������������������������������46 Norepinephrine������������������ 176, 268, 539, 556, 558, 562, 703 Nose������������������� 313–315, 334, 350, 508, 609, 610, 630, 706 Nuclear receptors (NRs)���������������������������������������������������539 Nutritional hyperparathyroidism��������������������������������������556

O Obstructive nephropathy���������������������������221, 689, 798, 891 Ocular fixatives�����������������������������������������������������������������632 Off-target effects������������������������������������������������������ 287, 391 Olfactory epithelium (OE)���������������� 312, 315, 326, 334–336 Oligodendroglioma����������������������������������������������������������758 Omeprazole�������������������������������������������������������������� 162, 591 Oncogenes���������������������������������������������������������������� 349, 751 Ophthalmic diagnostic and functional tests���������������������653 Opioid peptides����������������������������������������������������������������558 Optical coherence tomography (OCT)����������������������� 91, 98, 205, 369, 654, 655 Oral cavity������������������137, 144–147, 151–153, 163, 316, 612 Organogenesis�������������������������������������������������� 399, 858, 867 Organohalides�������������������������������������������������� 670, 672–673 Organophosphates���������������������������������������������������� 189, 864 Ossicles���������������������������������������������� 662, 663, 667, 668, 678 Osteoarthritis (OA)�������������������������������������������������� 586, 592 Osteoblast������������������������������������93, 554, 574, 576, 584, 588, 589, 591, 834, 889 Osteoclast������������������ 554, 574, 576, 582, 584–586, 589–591, 678, 823, 834, 835, 884 Osteocytes�������������������������������������������������������� 574, 588, 591

Osteoid, increased��������������������������������������������� 586, 590, 591 Osteoma����������������������������������������������������������� 588, 589, 757 Osteonecrosis����������������������������������������������������������� 590, 681 Osteons����������������������������������������������������������������������������574 Osteoporosis����������������������� 576, 582, 584, 592, 612, 823, 834 Osteosarcoma(s)����������������������������������������587–589, 757, 877 Osteosclerosis�������������������������������������������������������������������678 Otic capsule������������������������������������������������������ 664, 678, 681 Otitis media����������������������������������������������������������������������674 Otolith membrane������������������������������������������������������������665 Ototoxicity������������������������������������������������666–672, 685, 686 Oval cell, hyperplasia/proliferation�������������������������� 128, 131, 662, 663, 681 Oval window���������������������������������������������������� 662, 663, 681 Oxidant stress����������������������������������������������������������� 333, 334 Oxyphil cells���������������������������������������������������������������������553 Ozone���������������������������������312, 318, 324, 329, 334–336, 338

P p53, 771 Pancreatic stellate cells (PSCs)��������������������������������� 176, 185 Paneth cell, small intestine������������������������������������������������366 Pannus������������������������������������������������������������������������������587 Papilla�������������������������������� 202, 204, 205, 208, 210, 211, 224, 488, 489, 517, 882, 891 Papillomas���������������������������������������� 145, 162, 519–522, 749, 753, 756, 757, 771 Parathyroid cysts���������������������������������������������������������������554 Parathyroid hormone (PTH)���������������������������157, 211, 538, 551–554, 556, 582, 585, 588, 589, 610, 726 Parietal cell������������������������������������������������������� 154, 162, 791 Pars distalis������������������������������������������������������� 543, 544, 546 Pars plana��������������������������������������������������623, 625, 644, 650 Pars plicata�����������������������������������������������������������������������623 Pathology reports���������������������������������� 34, 68, 259, 261, 746 Pathology working groups (PWGs)�����������������������������45–76 PCNA, see Proliferating cell nuclear antigen (PCNA) Peer review����������������������������������������������������45–76, 101, 768 Pemphigus�������������������������������������������������������� 499, 508, 509 Penicillamine������������������������������������������������������������ 509, 606 Penicillin����������������������������������������������������229, 329, 373, 508 Pentamidine���������������������������������������������������������������������565 Perfusion, intravascular�����������������������������������������������������268 Pergolide���������������������������������������������������������������������������287 Periarteriolar lymphoid sheath (PALS)�������������������� 364, 388 Pericardium����������������������������������������������������������������������295 Pericytes������������������������������������������������������������������� 272, 445 Peripheral quantitative CT (pQCT)���������������� 582, 583, 599 Periportal necrosis���������������������������������������������������� 125, 126 Permeability transition�����������������������������������������������������284 Permeability transition (PT) pores�����������������������������������284 Peroxisome, proliferation in liver������������������������������ 188, 287 Peroxisome proliferator-activated receptor (PPAR) agonists�����������������������������������������������������������297

Toxicologic Pathology for Non-Pathologists 913 Index       Persistent fetal vasculature (PFV)������������������������������������645 Peyer’s patches���������������������������������������������36, 165, 174, 366 Pharmacology, exaggerated���������������� 283, 356, 374, 392, 761 Phase II metabolism���������������������������������������������������������165 Phenacetin������������������������������������������������������������������������326 Phenobarbital (PB)���������������������������� 550, 671, 783, 789, 790 Phenobarbitone������������������������������������������������ 123, 772, 773 Phenothiazines��������������������������������������������������������� 643, 653 Phenytoin����������������������������������������������������������������� 146, 606 Pheochromocytoma�����������������������������������552, 562, 563, 768 Phosphorus������������������������ 126, 211, 551, 552, 554–556, 582, 690, 691, 701, 718, 726–728 Phosphotungstic acid hematoxylin (PTAH)������������ 598, 608 Photoallergy����������������������������������������������������� 498, 513–514 Photocarcinogenicity���������������������������������������� 513–514, 526 Photogenotoxicity���������������������������������������������������� 513, 514 Photoreceptor nuclei displacement (PDN)����������������������651 Photosafety testing, for skin toxicity���������������������������������526 Phototoxicity��������������494–495, 513–514, 635, 643, 651, 652 Physeal dysplasia��������������������������������������������������������������590 Pigment deposition������������������������������������������������������ 65, 67 Pinna������������������������������������������661, 662, 667, 673, 676, 836 Platelet-derived growth factor (PDGF)���������������������������286 Platelet, drug response���������������������������������������������� 699, 702 Pneumonitis����������������������������������������������329, 342, 375, 377 Polychlorinated biphenyls��������������������������������� 560, 816, 861 Polymerase chain reaction (PCR)����������������������� 97, 261, 701 Polymyositis����������������������������������������������������������������������606 Positron emission tomography (PET)�������103, 109–110, 584 Potency, of drugs���������������������������������������������������������������867 Pregabalin�������������������������������������������������������������������������297 Pregnancy����������������������������������124, 164, 383, 430, 431, 434, 435, 437, 438, 449, 457, 459, 461, 557, 819 Pregnenolone��������������������������������������������������������������������557 Primary demyelination�����������������������������������������������������261 Primary hyperparathyroidism�������������������������������������������555 Procainamide���������������������������������������������229, 376, 509, 606 Prolactin (PRL)�������������������������������� 417, 419, 423, 430, 437, 444, 445, 448, 449, 453, 454, 458, 460–462, 544, 546, 562, 738, 824, 832 Proliferating cell nuclear antigen (PCNA)����������������� 24, 351 Propylthiouracil����������������������������������������������������������������373 Prostaglandins (PGs)���������� 161, 224, 286, 337, 420, 517, 641 Protein synthesis����������������� 148, 209, 515, 600, 608, 640, 672 Proteinuria����������������������������������������� 205, 208, 229, 733, 734 Psoralen����������������������������������������������������������������������������514 Ptaquiloside����������������������������������������������������������������������753 Pulmonary edema���������������325, 327, 328, 340–342, 353, 855 Pulmonary emphysema��������������������������������������������346–347 Pulmonary function tests���������������������������������� 344, 351–352 Pulmonary inflammation���������������������������������� 332, 342–343 Purkinje fibers������������������������������������������������������������������281 Puromycin�������������������������������������������������������� 187, 225, 227 Pustule�������������������������������� 497, 499, 504, 509, 518, 675, 891

oxicologic Pathology for Non-Pathologists 914  ITndex



  

Pyknosis����������������������������������������������������284, 379, 591, 601 Pylorus������������������������������������������������������������������������������141

Q QA, see Quality Assurance QT prolongation������������������������������������������������������ 288, 289 Quality Assurance (QA)����������������������������������������������� 46, 74

R Radiation therapy cataract induction�������������������������������������������������������639 leukemia/lymphoma induction��������������������������� 134, 386 oocyte/follicular degeneration induction��������������������453 Radiomimetics�������������������������������������������������� 172, 173, 639 Reactive metabolites����������������������������������������� 183, 325–327 Reactive oxygen species���������������������������� 286, 323, 324, 327, 333, 379, 522, 670, 672, 673, 754 Receptor antagonism������������������������������������������������ 422, 460 Red blood cell, drug response��������������������������� 55, 67, 89, 90, 213, 270, 313, 319, 359, 690, 699–701, 716, 839 Red pulp, see Spleen Regenerative hyperplasia������������������� 160, 168, 180, 336, 673 Regression, tumor�������������������������������������������������������������104 Renal artery branches�������������������������������������������������������202 Renal mesenchymal tumor (RMT)����������������������������������231 Renal osteodystrophy��������������������������������������������������������584 Renal pelvis, see Kidney Renal tubule, see Kidney Reporting, pathology findings�������������������������������� 5, 45, 694 Reserpine���������������������������������������������������������� 150, 462, 562 Reticulum cell sarcoma type A�����������������������������������������877 Retina, see Eye Rhabdomyosarcoma����������������������������������523, 526, 608, 757 Rhinitis������������������������������������������������������328, 335, 508, 760 RU486, see Mifepristone S Salicylates�������������������������������������������������������������������������672 Salinomycin����������������������������������������������������������������������562 Salivary glands anatomy�������������������������������������������������������������� 146, 147 atrophy���������������������������������������������������������������� 147, 148 dog���������������������������������������������������������������������� 152, 153 drug-induced dysfunction�������������������������������������������146 duct hyperplasia and metaplasia���������������������������������147 eosinophil accumulation������������������������������������� 158, 163 hyperplasia����������������������������149, 151, 152, 159, 160, 167 hypertrophy����������������������������������������������������������������167 inflammation��������������������������������������������������������������148 necrosis�����������������������������������������������������������������������150 neoplasia���������������������������������������������������������������������149 primates����������������������������������������������������������������������148 rodents���������������������������������������������������������������� 147, 149 Salmonella typhimurium������������������������������������������������������4 Sarcolemma��������������������������������������� 593, 598, 602, 604, 608

Sarcoma spleen������������������������������������������������������������������ 363, 813 uterine stromal sarcoma��������������������������������������430–431 Sarcomere�����������������������������������297, 593, 598, 600, 601, 604 Sarcoplasmic reticulum��������������������������������������������� 285, 593 Satellite cells����������������������������������������������������� 261, 593, 602 Schlemm’s canal����������������������������������������������������������������624 Schwannoma���������������������������������������������������� 170, 297, 758 Sclera�����������������������������������������254, 620, 623–625, 629, 634, 640, 650, 653, 837 Scoliosis����������������������������������������������������������������������������863 SDH, see Serum sorbitol dehydrogenase (SDH) Sebaceous adenoma����������������������������������������������������������525 Sebaceous gland, drug-induced changes���������������������������515 Sebaceous glands�����������������������488, 512, 515–521, 525, 620, 662, 672, 676, 677, 772 Sebaceous squamous carcinoma����������������������������������������145 Secondary effects��������������������������51, 383, 387, 421, 422, 527 Secretin������������������������������������������������������������� 154, 165, 176 Secretory epithelial cells���������������������������������������������������318 Selective Estrogen Receptor Modulators (SERMs)������������������������������������������������ 443, 461 Semicircular canals������������������������������������������� 663, 665–667 Seminiferous tubule������������� 64, 399–404, 406, 408, 411, 413, 414, 418, 420, 421, 423, 425, 784, 817, 818, 892 SERMs, see Selective Estrogen Receptor Modulators Sertoli cell������������������������� 399–401–403, 405, 414–416–418, 423, 446, 815–817, see Ovary; Testis Serum creatinine (sCr)������������������������������205, 234, 243, 718 Serum sickness�����������������������������������������������������������������375 Serum sorbitol dehydrogenase (SDH)�������119, 120, 713–716 Severity grading������������������������32–34, 69, 117, 121, 123, 125 Short bowel syndrome������������������������������������������������������138 Sialodacryoadenitis virus���������������������������103, 109–110, 584 Sinoatrial (SA) node���������������������������������������������������������281 Sinus histiocytosis, lymph node����������������������������������������385 Sinusoidal endothelial cell injury��������������������������������������115 Skeletal muscle atrophy�������������������������������������������������������� 599, 604–605 fiber types����������������������������������������������������������� 597, 605 focal inflammation implant induction�������������������������������������������������160 infection����������������������������������������������������������������498 parenteral preparations�����������������������������������������173 spontaneous��������������������������������������������������599–600 histological techniques����������������������������������������592–596 hypertrophy��������������������������������������������������������604–605 inflammation��������������������������������������������������������������606 diffuse and necrotizing inflammation�������������������386 overview����������������������������������������������������������������606 mineralization��������������������������������������������� 122, 211, 294 neoplasia���������������������������������������������������������������������608 phospholipidosis���������������������������������������������������������607 vacuolation����������������������������������������������������������607–608

Skin alopecia���������������������������������491, 497, 515, 517, 518, 527 amyloid deposits������������������������������������������������� 209, 210 atrophy������������������������������������������������������������������������826 drug reaction prevalence���������������������������������������������500 elastosis�����������������������������������������������������������������������499 hair follicle������������488, 489, 492, 499, 500, 502, 504, 512, 515–518, 522, 523, 525–527–529, 592, 662, 752, 824–827, 859 hyperpigmentation�����������������������������������������������������515 hyperplasia������������������������������������������������������������������503 hypopigmentation�������������������������������������������������������213 immune cells���������������������������������������������������������������213 inflammation contact dermatitis��������������������������497, 505, 507, 514 implanted biomaterials��������������������������������� 504, 631 injection site�����������������������������������523, 524, 606, 607 irritancy�������������������������������������������������������� 493, 495 photoallergy�������������������������������������������������� 513, 514 phototoxicity�����������������������������������������494, 513–514, 635, 651, 652 spontaneous����������������������������������������������������������587 topical drug induction�������������������������������������������493 mineralization in subcutaneous and soft tissues������������������������������������������������������ 269, 522 necrosis�����������������������������������������������������������������������507 neoplasia animal models����������������������������������������������� 167, 189 basal cell carcinoma��������������� 514, 519, 523, 525, 749 classification and diagnosis���������������������������230–233 keratoacanthoma����������������������������523, 525, 752, 753 melanomas������������������������������������������������������������827 overview�������������������������������������������������������� 746, 756 sebaceous adenoma�����������������������������������������������523 sebaceous squamous carcinoma������������� 514, 520, 523 squamous cell carcinoma����������������149, 169, 753, 757 squamous papilloma���������������������������������������������757 safety assessment��������������������������������������������������������492 sebaceous gland changes����������������������488, 512, 515, 517 species differences����������������������������������������������� 405, 774 subcutaneous neoplasms canine histiocytoma����������������������������������������������526 classification����������������������������������������������������������526 fibroma�����������������������������������������������������������������526 fibrosarcoma���������������������������������������������������������526 hibernoma�������������������������������������������������������������526 histiocytic sarcoma���������������������������������������� 757, 766 injected and implanted substance induction���������504 leiomyoma������������������������������������������������������������757 leiomyosarcoma����������������������������������������������������757 lipoma�������������������������������������������������������������������757 liposarcoma�����������������������������������������������������������757 malignant histiocytoma����������������������������������������526 miscellaneous neoplasms����������������������� 411, 412, 549 pleomorphic sarcoma������������������������������������ 523, 526

Toxicologic Pathology for Non-Pathologists 915 Index       rhabdomyosarcoma������������������������������� 523, 526, 608 species differences�������������������������������������������������524 systemic drug induction����������������������������������������526 vascular lesions and safety assessment�������������������283 Small intestine atrophy������������������������������������������������������������������������166 hyperplasia������������������������������������������������������������������167 hypertrophy����������������������������������������������������������������167 infection bacteria���������������������������������������������������������������������4 metazoan������������������������������������������������������������������4 protozoan parasites���������������������������������������������������4 viruses�����������������������������������������������������������������������4 inflammation��������������������������������������������������������������164 lipidosis�����������������������������������������������������������������������121 phospholipidosis���������������������������������������������������������121 polyps�������������������������������������������������������������������������160 species comparison������������������������������������� 403, 434, 530 structural and histochemical characteristics����������������165 techniques for analysis������������������������������������������������187 ulceration��������������������������������������������������������������������172 Smooth endoplasmic reticulum��������� 318, 326, 418, 593, 758 Smooth muscle cell apoptosis���������������������������������������������������������������������488 vasculature������������������������������������������������������������������509 Smooth muscle cells����������������������������������291, 430, 592, 890 Sodium�������������������������������� 99, 106, 166, 168, 171, 286, 287, 300, 549, 557, 611, 632, 640, 654, 663, 725, 726, 773, 833, 847 Sodium iodide symporter (NIS)���������������������������������������549 Solid-state carcinogenesis�����������������������������������������������������7 Somatostatin (SST)��������������������154, 176, 544, 563, 564, 596 Spermatid, head retention���������������������������������������� 404, 407 Spermatogenesis���������������� 397, 400, 403–407, 409, 410, 412, 414, 416–418, 423–425, 428, 441, 814, 879 Spermatogenic cycle������������������������� 400–403, 410, 416, 418, 423, 424, 815 Sperm granuloma����������������������������������������������������� 409, 421 Spheroids������������������������������������������������������������������ 259, 275 Spinal cord neoplasia����������������������������������������������������� 295, 296, 765 Spiral limbus, loss of cellularity in������������������������������������683 Spleen amyloid deposition�����������������������������������������������������295 anatomy and histology������������������������������������������������364 atrophy������������������������������������������������������������������������384 congestion�������������������������������������������������������������������363 extramedullary hematopoiesis�������������������������������������363 fibrosis������������������������������������������������������������������������364 foam cell accumulation��������������������������������������� 167, 704 functions���������������������������������������������������������������������364 granuloma�������������������������������������������������������������������409 hyperplasia lymphoid�������������������������������������������������������364–366 plasma cell������������������������������������������������������������364

oxicologic Pathology for Non-Pathologists 916  ITndex



  

Spleen (cont.) neoplasia���������������������������������������������������������������������378 phospholipidosis���������������������������������������������������������416 pigmentation��������������������������������������������������������������385 technical considerations in analysis�����������������������������367 Spongiosis hepatis������������������������������������������������������������122 Squamous carcinoma colon���������������������������������������������������������������������������164 prostate��������������������������������������������������������������� 399, 405 skin�����������������������������������������������������������������������������161 Squamous epithelium (SE)�������������������������������145, 146, 151, 155, 159, 161, 163, 168, 315–317, 335, 431, 443, 447, 456, 462, 662, 674, 677, 758, 886, 892 Squamous hyperplasia lung�����������������������������������������������������������������������������165 nasal mucosa���������������������������������������������������������������163 Squamous metaplasia cervix������������������������������������������������������������������ 447, 448 lung�����������������������������������������������������������������������������460 nasal mucosa���������������������������������������������������������������447 thyroid������������������������������������������������������������������������384 Squamous papilloma nasal mucosa���������������������������������������������������������������152 skin������������������������������������������������������������� 523, 526, 757 Staging, of testis���������������������������������������������������������������402 Stapes��������������������������������������������������������������� 662, 663, 681 Statins��������������������������������������������������������184, 595, 603, 731 Steatosis�������������������������������������������������������������������� 117, 121 Stellate cell, liver����������������������������������������115, 134, 176, 185 Stereocilia�����������������������������������������������������������������663–666 Stereology, for neurotoxicity evaluation����������������������������252 Stevens-Johnson syndrome�������������������������������498, 499, 507, 508, 510, 512 Stomach adenoma mouse����������������������������������������������������������� 159, 160 overview����������������������������������������������������������������160 rat�������������������������������������������������������������������������151 carcinoid tumor�������������������������������������������������� 159, 162 carcinoma histopathology������������������������������������������������������142 nitrosative compounds������������������������������������������153 drug-induced inflammation and ulceration����������������155 dysplasia����������������������������������������������������������������������162 endocrine cells���������������������������������������������������� 175, 176 epithelial morphology and physiology������������������������153 gastric mucosa atrophy������������������������������������������������������������������158 cell kinetics�����������������������������������������������������������157 hyperplasia�������������������������������������159, 160, 167–168 hypertrophy��������������������������������������������������167–168 mucus layer�����������������������������������������������������������317 hepatic metaplasia�������������������������������������������������������717 intestinal metaplasia������������������������������������������� 168, 169 metaplasia�����������������������������������������������������������168–169

mineralization����������������������������������������������������� 157, 158 motility patterns���������������������������������������������������������156 mucus depletion�������������������������������������������������� 312, 315 spontaneous changes in laboratory animals�������� 280, 383 See also Forestomach Stratum basale (SB)�������������������������������������������������� 484, 485 Stratum corneum (SC)����������������������������� 484, 488, 499, 500, 503, 518, 528–530 Stratum granulosum (SG)����������������� 484, 485, 488, 489, 503 Stratum lucidum (SL)���������������������������������������������� 485, 665 Stratum spinosum (SS)��������������������������������������������� 484, 485 Streptozotocin�������������������������������������������������� 238, 564, 640 Styrene������������������������������������������������������318, 339, 670, 671 Subcutis���������������484, 487, 498, 499, 520, 523, 526, 527, 587 Submucosa����������������� 139, 151, 154, 157–159, 161, 164, 165, 168, 169, 172–174, 239, 317, 323, 337, 366, 674 Submucosal plexus���������������������������������������������������� 139, 154 Substance P����������������������������������������������������������������������268 Succinic dehydrogenase (SDH)������������������������119, 120, 636, 695, 713–716 Sulfonamides���������������������������������������������������� 221, 510, 549 Surfactants����������������������������������312, 318, 322, 329, 343, 638 Swainsonine���������������������������������������������������������������������187 Sweat glands�������������������������������������� 488, 489, 515, 517, 518 Synchronization, cell��������������������������������������������������������826 Syncytial cells, multinucleated������������������������������������������593 Synovial membrane�������������������������������������������������� 574, 882 Synovial sarcomas�������������������������������������������������������������589 Synthetic vitreous fibers���������������������������������������������������333 Systemic lupus erythematosus (SLE)����������������������� 375, 509

T Tamoxifen����������������������������������������������������������������� 448, 826 Tapetum lucidum��������������������������������������������� 625, 641–643 Target cells, reporter cells or���������������������������������������������554 T cell large intestine��������������������������������������������� 164–166, 366 small intestine��������������������������������������������� 164–166, 366 thymus depletion������������������������������������������������358–359 Teeth discoloration���������������������������������������������������������������611 drug-induced changes����������������������������������������� 421, 426 rodent features�������������������������������������120–121, 144, 610 Telogen effluvium������������������������������ 488, 490, 517, 825, 826 Tendonitis������������������ 575, 577, 583, 592, 597, 599, 623, 836 Tendons������������������������������������������������������������ 592, 623, 836 Teratoma��������������������������������������������������������������������������758 Terminal end bud (TEB)�������������������������� 455, 463, 783, 784, 819–822, 825 Testis atrophy�������������������������������������������������407, 410, 413, 414 drug-induced changes animals������������������������������������������������������������������422 humans��������������������������������������������������������� 421, 423 germ cells����������������������400, 401, 410–412, 414, 814, 815

giant cell���������������������������������������������������������������������418 hormonal regulation���������������������������������������������������405 Leydig cell atrophy�������������������������������������������412–414, 418, 419 hyperplasia������������������������������������������������������������419 overview��������������������������������������������������������418–419 necrosis�����������������������������������������������������������������������418 neoplasia Leydig cell tumors������������������������������������������������419 mesothelioma��������������������������������������������������������758 overview�������������������������������������������������������� 462, 463 Sertoli cell overview��������������������������������������������������������416–417 phospholipidosis���������������������������������������������������416 vacuolation������������������������������������������������������������416 spermatid head retention������������������������������������407–408 spermatogenesis�������������������������������������������������� 403, 405 spontaneous changes���������������������������������������������������412 structure����������������������������������������������������������������������401 techniques for analysis������������������������������������������������400 toxicity mechanisms��������������������������������������������600–608 weight����������������������������������������������������������������� 604, 605 Testosterone����������������������������� 399–401, 405, 417–419, 422, 424, 426, 427, 456, 460, 596, 738, 814, 815, 817, 820, 822, 835 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)��������������� 188, 774, 784, 821, 822, 868 Tetracycline����������������������������������������������������������������������581 TGF-β, see Transforming growth factor-beta Thick ascending limb (TAL)���������������������������� 203, 204, 223 Thioguanine���������������������������������������������������������������������562 Thiouracil�������������������������������������������������������������������������373 Threshold, multifactorial��������������������������������������������������595 Thrombopoiesis����������������������������������������������������������������707 Thrombosis, heart��������������������������������������286, 291, 292, 295 Thromboxane A2�������������������������������������������������������������286 Thymocytes�������������������������������������������������������������� 358, 378 Thymus anatomy and histology���������������������������������������� 364, 369 atrophy�������������������������������������������������������� 359, 384, 387 hyperplasia���������������������������������������������������������� 384, 512 hypertrophy�������������������������������������������������������� 512, 554 lymphocyte depletion�������������������������������������������������729 Thyroglobulin�����������������������������������������������������������547–549 Thyroid-binding globulin (TBG)���������������������������� 548, 550 Thyroid gland������������������������28, 82, 376, 541–543, 546–554, 783, 786, 827–831, 859 atrophy���������������������������������������������������������������� 552, 865 C cell hyperplasia in carcinoma����������������������� 551, 552, 769 medullary carcinoma���������������������������������������������552 overview��������������������������������������������������������551–552 hyperplasia diffuse�����������������������������������������������������������549–552 drug induction������������������������������������������������������548

Toxicologic Pathology for Non-Pathologists 917 Index       focal�������������������������������������������������������������� 550, 551 overview��������������������������������������������������������547–556 hypertrophy����������������������������������������������������������������554 inflammation animal models�������������������������������������������������������792 human�������������������������������������������������������������������548 neoplasia���������������������������������������������������������������������552 pigmentation������������������������������������������������������ 213, 514 squamous metaplasia������������������������������������������ 441, 447 structure����������������������������������������������������������������������547 Thyroid hormone����������������������������� 515, 547–550, 566, 596, 816, 827, 831, 865 Thyroid-stimulating hormone (TSH)��������������376, 539, 541, 544–550, 566, 754, 773, 827, 828, 831 Thyrotropin-releasing hormone (TRH)����������� 541, 548, 549 Thyroxine (T4)��������������������������������� 167, 539, 546, 548, 550, 702, 790, 791, 831 Tongue, lesions�������������������������� 137, 144–147, 592, 600, 790 Total bilirubin (TBILI)������������� 692, 713, 714, 716–717, 729 Toxic benzene metabolites����������498, 499, 507, 508, 510, 512 Toxic exposure, routes of���������������������������������������������������335 Toxicokinetics (TK)��������������������������������� 119, 194, 202, 492, 511, 512, 635, 669, 693, 698, 709, 737, 782 Tp53���������������������������������������������������������������������������������190 Trabecular meshwork (TM)�����������������������������624, 637, 643, 653, 675, 837 Trachea���������������������������������������313, 316, 317, 543, 547, 855 anatomy and histology����������������������������������������313–314 neoplasia���������������������������������������������������������������������313 Training, for toxicologic pathology���������������������������������������3 Transforming growth factor-α (TGF-α)������������������ 287, 590 Transforming growth factor-beta (TGF-β)����������������������146 Transitional epithelium (TE)����������������������������������� 315, 335 Transmission electron microscopy (TEM)������������������ 30, 31, 102, 171, 172, 233, 261, 262, 288, 598, 634, 672 Trastuzumab���������������������������������������������������������������������285 TRH, see Thyrotropin-releasing hormone Trichothecenes�����������������������������������������������������������������172 Triiodothyronine (T3)�������������������������������539, 546, 548, 831 Trimethyltin������������������������������������������������������������� 652, 672 Triparanol�������������������������������������������������������������������������640 Troponins����������� 284, 298–301, 593, 599, 695, 721–723, 725 TSH, see Thyroid-stimulating hormone T-tubules������������������������������������������������������������������ 285, 593 Tubulointerstitial disease��������������������������������������������������235 Tumorigenesis�������������������������������������������519, 522, 783, 852 Tumor necrosis factor-alpha (TNF-α)������������� 324, 723, 750 Tumor regression��������������������������������������������������������������827 Tumor suppressor genes, as carcinogenesis mechanism������������������������������������������������������190 Tunica muscularis������������������������������ 152, 154, 158, 161, 164 Tunica serosa������������������������������������������������������������ 164, 165 Turbinates�����������������������������������314, 315, 335, 336, 588, 610 Type 1 diabetes mellitus (T1DM)���������������������������� 564, 565 Type 2 diabetes mellitus (T2DM)���������������������������� 564, 565

oxicologic Pathology for Non-Pathologists 918  ITndex



  

Type II astrocytes�������������������������������������������������������������893 Type II cell hyperplasia������������������������������������ 339, 340, 344 Tyrosine kinase inhibitor (TKs)�����������������������185, 186, 243, 286, 515, 611, 833 Tyzzer’s disease�����������������������������������������������������������������173

U Ulcers, of duodenum���������������������������������������������������������162 Ultraviolet (UV) radiation������������������������������������������������638 Upregulation���������������������������������������������������������������������146 Urethra������������������������������� 239, 240, 399, 404, 405, 889, 891 Urinalysis���������������������������������������79, 89, 110, 205, 234, 240, 243, 244, 717, 731, 732 Urinary bladder inflammation��������������������������������������������������������������239 mineralization����������������������������������������������������� 211, 258 neoplasia clinical relevance of animal models����������������� 33, 208 mesenchymal tumors��������������������������������������������240 urothelial carcinoma���������������������������������������������240 urothelial papilloma����������������������������������������������240 overview����������������������������������������������������������������������239 urothelial hyperplasia atypical hyperplasia��������������������������������������� 232, 459 diffuse hyperplasia���������������������������������������� 240, 459 Urothelial cells������������������������������������������������������������������889 Urticaria����������������������������������������������������������� 498, 507, 508 Uterus adenomyosis����������������������������������������������������������������448 atrophy������������������������������������������������������������������������448 cyclical changes�����������������������������������������������������������451 decidual reaction���������������������������������������������������������448 endometrial hyperplasia dog������������������������������������������������������������������������448 human�������������������������������������������������������������������439 rodents������������������������������������������������������������������450 neoplasia carcinoma�������������������������������������������������������������446 leiomyoma������������������������������������������������������������446 mesenchymal neoplasms���������������������������������������451 overview�������������������������������������������������������� 431, 446 polyps�������������������������������������������������������������������431 stromal sarcoma����������������������������������������������������432 Uveitis������������������������������������������������������������������������������650

V Vacuolation brain���������������������������������������������������������������������������268 choroid plexus�������������������������������������������������������������272 cornea�������������������������������������������������������������������������495 islet cells���������������������������������������������������������������������563 kidney glomerulus�������������������������������������202, 203, 205, 238 renal tubules�������������������������������������������������� 203, 225

muscle�������������������������������������������������������������������������269 myocardial degeneration������������������������������������� 282, 293 Sertoli cell�������������������������������������������������������������������416 Vagina cyclical changes�����������������������������������������������������������444 inflammation��������������������������������������������������������������447 neoplasia���������������������������������������������������������������������462 Vaginal opening�����������������������������������������434–436, 785, 836 Vagus nerve����������������������������������������������������������������������273 Valve disease, see Heart Valvulopathies������������������������������������������������������������������287 Vascular endothelial growth factor (VEGF)������������ 226, 286, 445, 502, 641, 832, 833 Vascular leak syndrome, interleukin-2 induction���������339, 343 Vascular permeability���������������������������������������� 106, 339, 648 Vasculature amyloid deposition�����������������������������������������������������295 aortic inflammation, necrosis, and aneurysm��������������282 apoptosis���������������������������������������������������������������������124 brain lesions����������������������������������������������������������������270 corneal neovascularization�������������������������� 638, 644, 653 histology������������������������������������������������������������� 165, 227 hyperplasia������������������������������������������������������������������551 hypertrophy����������������������������������������������������������������758 Inflammation (see Vasculitis) kidney anatomy����������������������������������������������������������������202 changes�����������������������������������������������������������������207 liver changes������������������������������������������������������� 114, 672 overview����������������������������������������������������������������������645 plexiform lesions������������������������������������������������� 627, 628 pulmonary blood vessels hyperplasia������������������������������������������������������������292 hypertrophy����������������������������������������������������������295 vasculitis����������������������������������������������������������������290 Vasculitis�����������������������������228, 230, 290, 291, 305, 383, 386, 498, 508, 509, 606, 706 animals overview����������������������������������������������������������������228 humans, classification and etiology�����������������������������230 polyarteritis dog����������������������������������������� 183, 295, 296, 386, 420 rodents���������������������������������������������������������� 183, 386 pulmonary blood vessels���������������������������������������������280 treatment-related necrosis and vasculitis dog������������������������������������������������������������������������641 mouse����������������������������������������������������������� 640, 643 primates����������������������������������������������������������������641 rat�������������������������������������������������������������������������641 Vas deferens������������������������������������������������������399, 404–405, 420–422, 426 Vasodilators������������������������������������������������������284–286, 290, 304, 305 Ventricles���������������������������������� 107, 281–283, 723–724, 750, 799, 800, 803, 886

Verapamil�������������������������������������������������������������������������289 Vestibular apparatus������������������������������������������ 661–663, 665 Vestibular system������������������������������������������������������ 670, 685 See also Ear Vestibule����������������������������������������������������315, 663, 665, 681 Veterinary medicine, safety assessment�������������������������������32 Vigabatrin����������������������������������������������������������������� 653, 802 Villi��������������������������������������������������������������������31, 142, 164, 166, 168, 791 Vincristine�������������������������������������������������������� 286, 519, 608 Visual streaks��������������������������������������������������������������������628 Vitamin D�������������������������������������������������491, 552, 554, 726 Vomiting������������������������������������������������������������������� 190, 726

W Warfarin������������������������������������������������������������������� 641, 833 White pulp, see Spleen Wnt signaling�������������������������������������������������������������������100

Toxicologic Pathology for Non-Pathologists 919 Index       Wolffian ducts���������������������������������������������������������� 399, 429 Woven bone������������������������������������������������������ 575, 586, 588

X Xylitol�������������������������������������������������������������������������������562 X-zone, adrenal�����������������������������������������������������������������557

Y Yolk sac������������������������������������������������������������� 266, 399, 428

Z Zidovudine��������������������������������������������������������������� 286, 608 Zinc�������������������������������������������152, 162, 184, 186, 187, 312, 329, 643, 863, 864 Zygote������������������������������������������������������������������������������402 Zymbal’s gland������������������������������������������������� 662, 673, 772 Zymogens�������������������������������������������������������������������������184