Glutathione (1990) [1 ed.]
 9781138105645, 9780203713372, 9781351364393, 9781351364386, 9781351364409, 9781138558939

Table of contents :

1. Publications on Glutathione, 1983-1987, A Bibliometric Study 2. Determination of Tissue Glutathione 3. Manipulation of Liver Glutathione Status-A Double-Edged Sword 4. Compartmentation of Cellular Glutathione in Mitochondrial and Cytosolic Pools 5. Hormonal Influence of GSH Content in Isolated Hepatocytes 6. Glutathione Transport and Its Significance in Oxidative Stress 7. Glutathione and Alcohol. Glutathione in Prokaryotes 8. Biosynthesis and Regulation of g-Glutamyl Transpeptidase 9. The Role of g-Glutamyl Transpeptidase (g GTPase) in Mammary Tissue 10. Structure, Mechanism, Functions, and Regulatory Properties of Glutathione Reductase 11. Glutathione Transferase in Human Tumors and Human Cancer Cell Lines 12. The Formation of Disulphide Bonds in the Synthesis of Secretory Proteins: Properties and Role of Protein Disulfide-Isomerase 13. The Glyoxalase System: Towards Functional Characterization and a Role in Disease Processes 14. Glutaredoxin: Structure and Function 15. Glutathione and Protein Function 16. Role of Glutathione in the Regulation of Protein Synthesis and Degradation in Eukaryotes 17. Aging and Increased Oxidation of the Sulfur Pool 18. Glutathione Metabolism in the Mammalian Ocular Lens 19. Role of Glutathione in the Aging Process of the Lens 20. Renal Handling of Glutathione 21. Glutathione in Ischemia and Reperfusion-Induced Tissue Injury 22. Free Radicals and Thiol Compounds (The Role of Glutathione against Free Radical Toxicity) 23. N-Acetylcysteine Stereoisomers as in vivo Probes of the Role of Glutathione in Drug Detoxification 24. Glutathione Levels in Human Hepatocytes Exposed to Paracetamol 25. Biological Implications of the Nucleophilic Addition of Glutathione to Quinoid Compounds 26. Glutathione and Hepatobiliary Transport of Xenobiotics 27. The Role of Glutathione in the Enzymatic Deiodination of Thyroid Hormone 28. Glutathione in Pineal Mechanisms and Functions 29. Nutritional Significance of Glutathione 30. The Potential Benefits of Dietary Glutathione on Immune Function and Other Practical Implications 31. Hereditary Disorders in Glutathione Metabolism. Index.

Citation preview

Glutathione : Metabolism and Physiological Functions Editor

José Vina, M.D., Ph.D. Professor and Chairman Department of Physiology University of Valencia Valencia, Spain

First published 1990 by CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 Reissued 2018 by CRC Press © 1990 by Taylor & Francis CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access ( or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organiza-tion that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. A Library of Congress record exists under LC control number: 89070796 Publisher’s Note The publisher has gone to great lengths to ensure the quality of this reprint but points out that some imperfections in the original copies may be apparent. Disclaimer The publisher has made every effort to trace copyright holders and welcomes correspondence from those they have been unable to contact. ISBN 13: 978-1-138-10564-5 (hbk) ISBN 13: 978-0-203-71337-2 (ebk) Visit the Taylor & Francis Web site at and the CRC Press Web site at

PREFACE In the last two decades our knowledge of the functions of glutathione in cell metabolism has been constantly increasing. Bibliometric studies show a growing interest in glutathione by the scientific community. There is strong evidence that this peptide has a prominent role in several cell functions ranging from antioxidant defense to regulation of metabolic pathways or of hormonal action. Furthermore, methods for the accurate measurement of glutathione levels, specially oxidized glutathione, have appeared recently and have led us to revise old concepts regarding glutathione status of cells. On the other hand, it is possible to modify the glutathione levels in cells. This has advantages and shortcomings: if used well it may help us to understand and to treat important diseases. However, knowledge of the side effects of changing the glutathione status of cells is also of primary importance. In the past years the role of glutathione in areas such as detoxication of xenobiotics or maintenance of cell structure and function was established. However, more recently, a prominent role of glutathione in fields such as nutrition, aging, and immunology is becoming fully recognized. It is obvious that a good knowledge of the physiological properties of glutathione will help the clinician to understand the physiopathology and therapeutics of glutathione-related diseases. Thus, understanding the metabolism and physiological functions of glutathione and related enzymes may be of great importance to the investigators or the students in various areas of scientific knowledge and also to the professionals that note that glutathione becomes relevant in their fields of interest and who want to keep up with the changing scene in their areas of expertise. The aim of this book is to provide not only well-established thought but also up-to-date information that will help those interested in the metabolism and properties of glutathione.

THE EDITOR José Vina is Professor and Chairman of the Department of Physiology, University of Valencia. Dr. Vina received his M.D. and his Ph.D. (cum laude) in 1978, both at the University of Valencia. He carried out postgraduate research at the Metabolic Research Laboratory, University of Oxford, under the direction of Sir Hans A. Krebs. Part of his research was supported by a FEBS fellowship awarded to Dr. Vina. Dr. Vina is a member of the American Physiological Society, the Biochemical Society, the Spanish Society of Physiological Sciences, and the Spanish Biochemical Society. He has received the Boehringer Prize for biochemical research. Dr. Vina is the author of over 60 scientific papers. His current major research interests relate to the role of sulfur amino acids and glutathione in cell functions, especially the aging process.

CONTRIBUTORS Theo Akerboom, Ph.D. Institute of Physiological Chemistry I University of Düsseldorf Düsseldorf, West Germany Luigi Atzori, M.D. Department of Toxicology Karolinska Institute Stockholm, Sweden Joseph V. Bannister, D.Phil. Professor Biotechnology Centre Cranfield Institute of Technology Cranfield, England William H. Bannister, D.Phil. Professor Department of Biomedical Sciences University of Malta Msida, Malta J. A. Bárcena, Ph.D. Department of Biochemistry and Molecular Biology University of Cordoba Cordoba, Spain R. Barouki, M.D. U-99 INSERM Henri Mondor Hospital Creteil, France Craig R. Baumrucker, Ph.D. Associate Professor Department of Dairy and Animal Science The Pennsylvania State University University Park, Pennsylvania Jeffrey B. Blumberg, Ph.D. Professor School of Nutrition and Associate Director/Senior Scientist USDA Human Nutrition Research Center Tufts University Boston, Massachusetts

J. A. Bocanegra, Ph.D. Department of Biochemistry and Molecular Biology University of Cordoba Cordoba, Spain Anders Brunmark, Ph.D. Research Associate Department of Pathology II University of Linköping Linköping, Sweden Enrique Cadenas, Ph.D. Associate Professor Institute for Toxicology University of Southern California Los Angeles, California P. G. Campbell, Ph.D. The Allegheny-Singer Research Institute Pittsburgh, Pennsylvania José V. Castell, Ph.D. Professor of Biochemistry Experimental Hepatology Unit Centro de Investigacion del Hospital la Fe Servicio Valenciano de Salud Valencia, Spain George B. Corcoran, Ph.D. Associate Professor Toxicology Program College of Pharmacy University of New Mexico Albuquerque, New Mexico Ian A. Cotgreave, Ph.D. Department of Toxicology Karolinska Institute Stockholm, Sweden Norman P. Curthoys, Ph.D. Professor and Chairman Department of Biochemistry Colorado State University Fort Collins, Colorado

D. Delmulle, M.D. Laboratoire de Biochimie Toxicologique et Cancérologique Catholic University of Louvain Brussels, Belgium

Robert B. Freedman, D.Phil. Professor of Biochemistry Biological Laboratory University of Kent Canterbury, England

Carmine Di Ilio, M.Sc., Ph.D. Faculty of Medicine Institute of Biochemical Sciences University of Chieti Chieti, Italy

Tadayasu Furukawa, Ph.D. President Nutri-Quest, Inc. Chesterfield, Missouri

Teresa Donato, Ph.D. Assistant Researcher Experimental Hepatology Unit Centro de Investigacion del Hospital la Fe Servicio Valenciano de Salud Valencia, Spain A. Esteller, Ph.D. Professor Department of Physiology and Pharmacology University of Salamanca Salamanca, Spain Jose M. Estrela, M.D., Ph.D. Professor Department of Physiology School of Pharmacy University of Valencia Valencia, Spain Robert C. Fahey, Ph.D. Professor Department of Chemistry University of California at San Diego La Jolla, California Giorgio Federici, M.D., Ph.D. Professor Department of Biology University of Rome Rome, Italy J. Florindo, M.D. Department of Biochemistry and Molecular Biology University of Cordoba Cordoba, Spain

Dimitrios Galaris, Ph.D. Institute for Toxicology School of Pharmacy University of Southern California Los Angeles, California C. Garcia-Alfonso, Ph.D. Department of Biochemistry and Molecular Biology University of Cordoba Cordoba, Spain F. Goethals, Ph.D. Laboratoire de Biochimie Toxicologique et Cancérologique Catholic University of Louvain Brussels, Belgium Maria J. Gómez-Lechón, Ph.D. Associate Researcher Experimental Hepatology Unit Centro de Investigacion del Hospital la Fe Servicio Valenciano de Salud Valencia, Spain J. Gonzalez, M.D., Ph.D. Professor and Chairman Department of Physiology, Pharmacology, and Toxicology University of León León, Spain G. Guellaën, Ph.D. U-99 INSERM Henri Mondor Hospital Creteil, France

Consuelo Guerri, Ph.D. Instituto de Investigaciones Citólogicas de la Caja de Ahorros de Valencia Valencia, Spain

Agne Larsson, M.D., Ph.D. Professor Department of Pediatrics Uppsala University Uppsala, Sweden

Darryl L. Hadsell Department of Dairy and Animal Science The Pennsylvania State University University Park, Pennsylvania

Juan López-Barea, Ph.D. Professor Department of Biochemistry and Molecular Biology University of Cordoba Cordoba, Spain

J. Hanoune, M.D., Ph.D. U-99 INSERM Henri Mondor Hospital Creteil, France Otto Hockwin, Dr.rer.nat. Professor Department of Experimental Ophthalmology University of Bonn Bonn, Federal Republic of Germany Arne Holmgren, M.D., Ph.D. Professor Department of Physiological Chemistry Karolinska Institute Stockholm, Sweden Inge Korte, Dr.rer.nat. Department of Experimental Ophthalmology University of Bonn Bonn, Federal Republic of Germany Andries Sj. Koster, Ph.D. Assistant Professor Department of Pharmacology Faculty of Pharmacy University of Utrecht Ultrecht, The Netherlands

A. López-Ruiz, Ph.D. Department of Biochemistry and Molecular Biology University of Cordoba Cordoba, Spain E. Martinez-Galisteo, Ph.D. Department of Biochemistry and Molecular Biology University of Cordoba Cordoba, Spain Simin N. Meydani, D.V.M., Ph.D. Associate Professor Nutritional Immunology and Toxicology Laboratory USDA Human Nutrition Research Center Tufts University Boston, Massachusetts

Y. Laperche, Ph.D. U-99 INSERM Henri Mondor Hospital Creteil, France

Jaime Miquel, Ph.D. Research Associate Linus Pauling Institute of Science and Medicine Palo Alto, California and Associate Professor Department of Neurochemistry Faculty of Medicine University School of Medicine Alicante, Spain

Amparo Larrauri, Ph.D. Experimental Hepatology Unit Centro de Investigacion del Hospital la Fe Servicio Valenciano de Salud Valencia, Spain

Peter Moldéus, Ph.D. Professor Department of Toxicology Karolinska Institute Stockholm, Sweden

M. L. Munoz, M.D. Center for Medical Documentation and Informatics Valencia, Spain Gerald L. Newton, B.A. Staff Research Associate Department of Chemistry University of California at San Diego La Jolla, California Federico V. Pallardo, M.D., Ph.D. Postdoctoral Fellow Department of Physiology University of Valencia Valencia, Spain J. Peinado, Ph.D. Department of Biochemistry and Molecular Biology University of Cordoba Cordoba, Spain W. B. Quay, Ph.D. Department of Molecular and Cell Biology University of California Berkeley, California and Bio-Research Laboratory Napa, California William B. Rathbun, Ph.D. Associate Professor Department of Ophthalmology University of Minnesota Minneapolis, Minnesota Frank A. M. Redegeld, Ph.D. Department of Pharmacology Faculty of Pharmacy University of Utrecht Utrecht, The Netherlands M. Roberfroid Professor Laboratoire de Biochimie Toxicologique et Cancérologique Catholic University of Louvain Brussels, Belgium

Franscisco J. Romero, M.D., Ph.D. Assistant Professor Department of Physiology Faculty of Medicine University of Valencia Valencia, Spain Guillermo T. Sáez, M.D., Ph.D. Assistant Professor Department of Biochemistry and Molecular Biology School of Medicine University of Valencia Valencia, Spain and Biotechnology Centre Cranfield Institute of Technology Cranfield, England Russell Scaduto, Jr., Ph.D. Department Cellular and Molecular Physiology The Milton S. Hershey Medical Center Hershey, Pennsylvania Helmut Sies Professor Institute of Physiological Chemistry I University of Düsseldorf Düsseldorf, Federal Republic of Germany Noriko Tateishi, Ph.D. Assistant Professor Department of Oncology Biomedical Research Center Osaka University Medical School Osaka, Japan M. L. Terrada, M.D., Ph.D. Professor of Medical Documentation and Director The Center for Medical Documentation and Informatics University of Valencia Valencia, Spain Paul J. Thornalley, Ph.D. Lecturer Department of Chemistry and Biological Chemistry University of Essex Colchester, England

V. Thybaud, Ph.D. Laboratoire de Biochimie Toxicologique et Cancérologique Catholic University of Louvain Brussels, Belgium

Hans Weber, Ph.D. Senior Medical Writer Syntex Laboratories, Inc. Palo Alto, California

Gisa Tiegs, Ph.D. Faculty of Biology University of Konstanz Konstanz, West Germany

Marianne Weis, M.Sc. Department of Toxicology Karolinska Institute Stockholm, Sweden

Wout P. van Bennekom, Ph.D. Assistant Professor Department of Pharmaceutical Analysis Faculty of Pharmacy University of Utrecht Utrecht, The Netherlands

Albrecht Wendel, Ph.D. Professor Faculty of Biology University of Konstanz Konstanz, West Germany

Luis A. Videla, M.Sc. Professor Department of Biological Sciences Faculty of Medicine University of Chile Santiago, Chile

Christoph Werner, Ph.D. Faculty of Biology University of Konstanz Konstanz, West Germany

Theo J. Visser, Ph.D. Professor Department of Internal Medicine III and Clinical Endocrinology Erasmus University Medical School Rotterdam, The Netherlands

Bradley K. Wong, Ph.D. Senior Scientist Department of Pharmacokinetics/Drug Metabolism Parke-Davis Pharmaceutical Research Ann Arbor, Michigan

DEDICATION To my wife Pilar and to our children Pepe, Tomás, and Maria-Aurora

TABLE OF CONTENTS Chapter 1 Publications on Glutathione, 1983 to 1987. A Bibliometric S tudy.................................... M. L. Terrada and M. L. Munoz Chapter 2 Determination of Tissue Glutathione.................................................................................. Frank A. M. Redegeld, Andries Sj. Koster, and Wout P. van Bennekom



Chapter 3 Manipulation of Liver Glutathione Status — A Double-Edged Sword............................ . 21 Albrecht Wendel, Gisa Tiegs, and Christoph Werner Chapter 4 Compartmentation of Cellular Glutathione in Mitochondrial and Cytosolic Pools..................................................................................................................... 29 Francisco J. Romero and Dimitrios Galaris Chapter 5 Hormonal Influence of GSH Content in Isolated Hepatocytes.......................................... 39 F. Goethals, V. Thybaud, D. Delmulle, and M. Roberfroid Chapter 6 Glutathione Transport and Its Significance in Oxidative Stress........................................ 45 Theo Akerboom and Helmut Sies Chapter 7 Glutathione and Alcohol....................................................................................................... 57 Luis A. Videla and Consuelo Guerri Chapter 8 Glutathione and Prokaryotes................................................................................................ 69 Gerald L. Newton and Robert C. Fahey Chapter 9 Biosynthesis and Regulation of 7 -Glutamyl Transpeptidase............................................. 79 Y. Laperche, G. Guellaën, R. Barouki, and J. Hanoune Chapter 10 The Role of 7 -Glutamyl Transpeptidase (7 GTPase) in Mammary Tissue........................ 93 Darryl L. Hadsell, C. R. Baumrucker, and P. G. Campbell Chapter 11 Structure, Mechanism, Functions, and Regulatory Properties of Glutathione Reductase........................................................................................................ 105 J. López-Barea, J. A. Bárcena, J. A. Bocanegra, J. Florindo, C. García-Alfonso, A. López-Ruiz, £ . Martinez-Galisteo, and J. Peinado Chapter 12 Glutathione Transferase in Human Tumors and Human Cancer Cell Lines................... 117 Carmine Di Ilio and Giorgio Federici

Chapter 13 The Formation of Disulfide Bonds in the Synthesis of Secretory Proteins: Properties and Role of Protein Disulfide-Isomerase........................................................125 Robert B. Freedman Chapter 14 The Glyoxalase System: Towards Functional Characterization and a Role in Disease Processes.............................................................................................................. 135 Paul J. Thornalley Chapter 15 Glutaredoxin: Structure and Function......................................................................... — 145 Arne Holmgren Chapter 16 Glutathione and Protein Function...................................................................................... 155 Ian A. Cotgreave, Marianne Weis, Luigi Atzori, and Peter Moldéus Chapter 17 Role of Glutathione in the Regulation of Protein Synthesis and Degradation in Eukaryotes................................................................................................177 Jose M. Estrela and Federico V. Pallardo Chapter 18 Aging and Increased Oxidation of the Sulfur P o o l......................................................... 187 Jaime Miquel and Hans Weber Chapter 19 Glutathione Metabolism in the Mammalian Ocular L ens................................................193 William B. Rathbun Chapter 20 Role of Glutathione in the Aging Process of the Lens.....................................................207 Otto Hockwin and Inge Korte Chapter 21 Renal Handling of Glutathione......................................................................................... 217 Norman P. Curthoys Chapter 22 Glutathione in Ischemia and Reperfusion-Induced Tissue Injury.....................................227 Russell C. Scaduto Jr. Chapter 23 Free Radicals and Thiol Compounds (The Role of Glutathione Against Free Radical Toxicity)........................................................................................................237 Guillermo T. Sáez, William H. Bannister, and Joseph V. Bannister Chapter 24 N-Acetylcysteine Stereoisomers as In Vivo Probes of the Role of Glutathione in Drug Detoxification...................................................................................255 Bradley K. Wong and George B. Corcoran

Chapter 25 Glutathione Levels in Human Hepatocytes Exposed to Paracetamol...............................263 José V. Castell, Amparo Larrauri, Teresa Donato, and Maria J. Gómez-Lechón Chapter 26 Biological Implications of the Nucleophilic Addition of Glutathione to Quinoid Compounds........................................................................................................... 279 Anders Brunmark and Enrique Cadenas Chapter 27 Glutathione and Hepatobiliary Transport of Xenobiotics................................................ 295 J. Gonzalez and A. Esteller Chapter 28 The Role of Glutathione in the Enzymatic Deiodination of Thyroid Hormone...............317 Theo J. Visser Chapter 29 Glutathione in Pineal Mechanisms and Functions........................................................... 335 W. Q. Quay Chapter 30 Nutritional Significance of Glutathione............................................................................. 341 Noriko Tateishi Chapter 31 The Potential Benefits of Dietary Glutathione on Immune Function and Other Practical Implications...............................................................................................351 Tadayasu Furukawa, Simin N. Meydani, and Jeffrey B. Blumberg Chapter 32 Hereditary Disorders in Glutathione Metabolism............................................................. 359 Agne Larsson Index................................................................................................................................... 367

1 Chapter 1


Publications on glutathione are disseminated within the international scientific com­ munity which uses the English language as lingua franca through bibliographic databases and indices, two of which are exclusively biomedical: the American Index Medicus/MEDLARS and the Dutch Excerpta Medica/EMBASE. From 1983 to 1987, these two databases and the French PASCAL indexed a similar number of publications on the subject (Table 1). In order to know the structure of this literature on glutathione, we turn to the Index Med/cws/MEDLARS mainly due to the precise semantic specification of contents as provided by its thesaurus which is referred to as Medical Subject Headings. Foremost, we should insist once again that the area covered by the Index Medicus/ MEDLARS does not, as in the case of the rest of Western bibliographical databases, cor­ respond in a balanced way to the international distribution of biomedical publications. In general terms, it should be remembered that (1) the Index Medicus/MEDLARS presents a marked bias in favor of English-speaking countries, particularly the U.S., (2) it satisfactorily reports on production from West Germany, the Netherlands, and the Scandinavian countries, but considerably less so in the case of Latin Europe and above all, the East Block countries, and (3) it is of very little use for Soviet and Japanese production. 1,2 In spite of these limitations, its contents undoubtedly reflect the biomedical literature disseminated within the international scientific community which uses the English language as lingua franca. The annual distribution of indexed publications on glutathione, according to this database for the mentioned 1983 to 1987 period, is as follows (Table 2). It should be mentioned that the relatively low number of publications corresponding to 1987 is a result of the delay with which certain journals appear or are indexed. The great majority of these publications are articles (2731 = 99.7%) that appeared in 508 journals (Table 3). This distribution fits Bradford’s law of scattering, 3 4 whereby “ if scientific journals are arranged in order of decreasing productivity of articles on a given subject, they may be divided into a nucleus of periodicals more particularly devoted to the subject, and several groups or zones containing the same number of articles as the nucleus, when the number of periodicals in the nucleus and succeeding zones is l:n:n 2 . . . ” (Table 4). According to this table, the Bradford nucleus consists of a single journal (the American Biochemical Pharmacology), the second zone has three journals (the American Journal of Biological Chemistry, Applied Pharmacology, and Biochemical and Biophysical Research Communications), and so on. The distribution according to countries for those journals included in the first seven zones is as follows (Table 5). On the other hand, it is worth knowing the distribution of articles on glutathione according to country of origin. This information is supplied in 2522 cases, i.e., 92.07% of all articles indexed in the Index Medicus/MEDLARS (Table 6 ). As mentioned above, the bias of the coverage of Index Medicus/MEDLARS explains in part the high figure corresponding to the U.S., the low figure for the Soviet Union, and the absence of countries such as China. In this sense, the fact that Japan holds second place is of considerable significance. The same distribution with countries grouped in terms of geographical area is given below (Table 7).


Glutathione: Metabolism and Physiological Functions TABLE 1 Number of Publications on Glutathione Indexed from 1983 to 1987 Excerpta Medica/EMBASE Index Medi'cws/MEDLARS PASCAL

2307 2739 2225

TABLE 2 Annual Distribution of Publications Year

Number of publications

1983 1984 1985 1986 1987

453 560 559 661 506



% 16.53 20.44 20.40 24.13 18.47

Almost half of this literature comes from 54 institutions with 10 or more publications each (Table 8 ). These 54 institutions belong in turn to 12 countries (Table 9). The 2739 publications on glutathione indexed by the Index Medicus/MEDLARS cor­ responded to 5528 authors. Their distribution fit (r = 0.9817) Lotka’s productivity law5 whereby regardless of the scientific discipline involved the number of authors of n publi­ cations is inversely proportional to n2 (Table 10). According to Lotka’s law, the productivity index of an author is the logarithm of the number of his/her publications. Thus, in terms of this index, we can group the authors of literature on glutathione into three productivity levels (Table 11). Through the classical studies by Price,6 it is known that this productivity index is not correlated to the Platz visibility index (logarithm of the number of personal citations in the scientific community). This can also be seen in the case of the 43 large producers of publications on glutathione by noting the number of citations they have received during the period from 1974 to 1987 according to SCISEARCH (Table 12). The sole consequence deduced from this comparison is that all large-productivity authors of publications about glutathione are cited by the international scientific community according to three visibility levels (Table 13). Finally, we will mention the distribution of publications on glutathione according to the number of authors (Table 14). The mode of this distribution (3) and mean authors per publication (3.26) are indicators that the study of a scientific subject is strongly institutionalized. 7

3 TABLE 3 Distribution According to Journals Journals Biochemical Pharmacology Journal of Biological Chemistry Toxicology and Applied Pharmacology Biochemical and Biophysical Research Communications Biochimica et Biophysica Acta Cancer Research Chemical and Biological Interactions Journal of Pharmacology and Experimental Therapeutics Carcinogenesis Archives of Biochemistry and Biophysics Drug Metabolism and Disposition Biochemical Journal Toxicology Letters International Journal of Radiation Oncology, Biology, and Physiology Comparative Biochemistry and Physiology Toxicology International Journal o f Radiation Biology Research Communications in Chemical Pathology and Pharmacology Archives of Toxicology FEBS Letters Mutation Research Current Eye Research European Journal of Biochemistry Journal of Toxicology and Environmental Health Experimental Eye Research Advances in Experimental Medicine and Biology Journal of Clinical Investigation Molecular Pharmacology Xenobiotica American Journal of Physiology Fundamentals in Applied Toxicology Methods in Enzymology Analytical Biochemistry Life Sciences Proceedings of the National Academy of Sciences of the U.S.A. Radiation Research Biochemistry Cancer Letters Experientia Journal Applied Toxicology Mechanisms o f Ageing and Development Proceedings of Clinical Biological Research Acta Pharmacologica et Toxicologica Blood Journal of Inorganic Biochemistry Journal of Nutrition Endocrinology Journal of Chromatography British Journal of Cancer Bulletin of Environmental Contamination and Toxicology Drug Chemistry and Toxicology Journal of Bacteriology Biomedica Biochimica Acta British Journal of Radiology Diabetes International Journal of Biochemistry

Number of articles 205 97 82 72 71 69 51 49 44 42 41 39 39 38 37 37 32 32 30 26 24 23 23 23 22 21 21 21 21 20 19 19 18 18 18 18 17 16 16 16 16 16 14 14 13 13 12 12 11 11 11 11 10 10 10 10

Glutathione: Metabolism and Physiological Functions


TABLE 3 (continued) Distribution According to Journals Number of articles



Prostaglandins 6 journals with journals with 10 journals with 7 journals with 20 journals with 13 journals with 26 journals with 39 journals with 81 journals with 249

9 8

7 6

5 4 3 2 1

54 80 49

articles each articles each articles each articles each articles each articles each articles each articles each articles each


65 104 117 162 249 2731


TABLE 4 Distribution of Journals According to Bradford Zones Number of articles



205 251 240 205 256 233 247 277 289 279 249


248.27 ± 26.19

1 2

3 4 5 6

7 8

9 10

Number of journals



3 4 5

3 1.33 1.25


1 .6

1.37 1.45


16 32 59


1.84 2.03 2.07



1.79 ± 0.50

TABLE 5 Distribution According to Countries of the Journals Included in the First Seven Bradford Distribution Zones Zones


1i 2 3 4 5 6 7

1i 3 3 3 4 7 11




West Germany




1 1 1 —




— 1 1 3 2 7

1 1 2 —




11 3 4 6 8 11 16




5 TABLE 6 Distribution of Publications According to Country of Origin Countries

Number of publications

U.S. Japan U.K. West Germany Italy Sweden Canada France Netherlands Australia India Spain Finland Israel Norway East Germany Belgium Chile Hungary Poland Argentina New Zealand Switzerland Turkey Mexico Nigeria Austria Czechoslovakia Jamaica Denmark Jordan Brazil Bulgaria Soviet Union Taiwan Yugoslavia Egypt Hong Kong Iraq Kenya Libya South Africa Venezuela

1234 180 176 154 128 123 81 58 55 43 41




19 19 19 17 16 16 16 13 12 12 12

9 6 6

% 48.93 7.14 6.98 6 .1 1

5.06 4.88 3.21 2.30 2.18 1.70 1.62 0.83 0.75 0.75 0.75 0.67 0.63 0.63 0.63 0.52 0.48 0.48 0.48 0.36 0.24 0.24

5 4 4 3 3

0 .2 0


0.08 0.08 0.08 0.08 0.08 0.04 0.04 0.04 0.04 0.04 0.04 0.04

2 2 2 2 1 1 1 1 1 1 1

0.16 0.16 0 .1 2 0 .1 2


Glutathione: Metabolism and Physiological Functions TABLE 7 Distribution of Publications According to Geographical Areas of Origin Number of publications


U.S. Other American English-speaking countries Latin America Western Europe Soviet Union Eastern Europe Japan Other Eastern Asiatic countries Arabic countries Israel Black Africa South Africa Australia and New Zealand

1234 85 37 798

48.93 3.37 1.47 31.64 0.08 2.14 7.14 1.74 0.24 0.75 0.27 0.04 2.18



Geographical areas


54 180 44 6

19 7 1


TABLE 8 Institutions with Ten or More Publications Country

Institution Karolinska Institute University of California University of Texas National Cancer Institute North Carolina Research Triangle Institute University of Duesseldorf University of Minnesota Veterans Administration Medical Center University of Luebeck Institut National de la Santé et de la Recherche Médicale Cornell University Medical Col­ lege Oregon State University University of Rochester John Hopkins University Baylor College of Medicine University of Nebraska University of Tuebingen Vanderbilt University Columbia University Medical Research Center Emory University Mount Vernon Hospital State University of New York University of Santiago University of Michigan University of Turin Industrial Toxicology Research Center

Number of publications


Sweden U.S. U.S. U.S. U.S.

80 75 63 47 43

3.17 2.97 2.50

West Germany U.S. U.S.

Cumulative percentage

1 .8 6

3.17 6.14 8.64 10.50


1 2 .2 0

34 33 33

1.35 1.31 1.31

13.55 14.86 16.17

West Germany France

30 27

1 .1 2


17.29 18.36





U.S. U.S. U.S. U.S. U.S. West Germany U.S. U.S. U.K. U.S. U.S. U.S. Chile U.S. Italy India

23 23

0.91 0.91 0.87 0.83 0.83 0.71 0.71 0.67 0.67 0.63 0.63 0.63 0.63 0.63 0.63 0.59

20.26 21.17 22.04 22.87 23.70 24.41 25.12 25.79 26.46 27.09 27.72 28.35 28.98 29.61 30.24 30.83

22 21 21

18 18 17 17 16 16 16 16 16 16 15

7 TABLE 8 (continued) Institutions with Ten or More Publications Institution National Heart, Lung, and Blood Institute Oakland University Duke University Medical Center Kansas State University Rockefeller University University of Florida University of Illinois University of Kansas Medical Center University of Toronto Wadsworth Veterans Administra­ tion Medical Center Kyoto University University of Genova University of Stockholm University of Washington National Center for Toxic Re­ search University of Arizona University of Leiden University of London Washington State University Harvard Medical School University of Louisville University of Massachusetts University of Sidney Case Western Reserve University Mount Sinai School of Medicine Tokyo College of Pharmacy University of Wisconsin Total


Number of publications


Cumulative percentage





U.S. U.S. U.S. U.S. U.S. U.S. U.S.

15 14 14 14 14 14 14

0.59 0.56 0.56 0.56 0.56 0.56 0.56

32.01 32.57 33.13 33.69 34.25 34.81 35.37

Canada U.S.

14 14

0.56 0.56

36.93 37.49

Japan Italy Sweden U.S. U.S.

13 13 13 13

0.52 0.52 0.52 0.52 0.48

38.01 38.53 39.05 39.56 40.05

0.48 0.48 0.48 0.48 0.44 0.44 0.44 0.44 0.40 0.40 0.40 0.40

40.53 41.01 41.49 41.97 42.41 42.85 43.29 43.73 44.13 44.53 44.93 45.33

U.S. Netherlands U.K. U.S. U.S. U.S. U.S. Australia U.S. U.S. Japan U.S.


12 12 12 12 11 11 11 11 10 10 10 10


Note: Percentage of the total number of publications (2522) where the country of origin is indicated.


Glutathione: Metabolism and Physiological Functions TABLE 9 Distribution According to Country of Origin of the Institutions with Ten or More Publications Number of institutions


Number of publications

U.S. West Germany Italy Japan Sweden U.K. Australia Canada Chile France India Netherlands

37 3 29




2 2 2

768 82 1.15 23 93 29





14 16 27 15



1 1

Percentage of publications 30.45 3.25 0.92 3.69 1.15 0.44 0.56 0.64 1.07 0.64 0.48

Note: Percentage of the total number of publications (2522) where country of origin is indicated.

TABLE 10 Distribution of Publications According to Authors Number of articles 1 2

3 4 5

Number of authors 4096 747 314 149 68







23 16






5 3 3

13 15 16 17

1 1





23 24 28 29 31 32 34



1 1 1 1 1 1


9 TABLE 11 Distribution of Authors According to Productivity Level Productivity level

Productivity index ip)

Number of authors


P > 1 > p >

43 1389 4096 5528

0.78 25.13 74.09

Large producers Medium producers Low producers Total




TABLE 12 Comparison of the Productivity and Visibility Indices of the Large Producers Number of publications on glutathione (MEDLARS)

Productivity index

Number of citations (SCISEARCH)

Visibility index

34 32 31 29

1.53 1.50 1.49 1.46

1038 852 2661 646

3.02 2.93 3.42 2.81

TABLE 13 Distribution of Large Producers According to Visibility Level Visibility level

Visibility index (p)

Number of authors


Great visibility Medium visibility Small visibility

P> 3 3 > p > 2 2 > p > 1

4 32 7

9.30 74.42 16.28



TABLE 14 Distribution of Publication According to the Number of Authors Number of authors (n)

Number of publication with (n) authors


230 726 780 488 279 164 41

1 2

3 4 5 6









% 8.39 26.50 28.47 17.81 10.18 5.98 1.49 0.80 0.36 0.18


Glutathione: Metabolism and Physiological Functions

REFERENCES 1. Braun, T ., Glänzel, W ., and Schubert, A ., ScientometricsIndicators, World Scientific, Singapore, 1985, chap. 4. 2. Terrada, M. L., La Literatura Médica Espanola Contemporânea. Estúdio Estadistico y Sociométrico, Centro de Documentación e Informática Médica, Valencia, Spain, 1973, chap. 1 to 4. 3. Bradford, S. C., Documentation, Crosby Lockwood, London, 1948, 154. 4. Mikhailov, A. I., Cherny, A. I., and Giliarevskii, R. S., Scientific Communications and Informatics, Information Resources Press, Arlington, VA, 1984, 162. 5. López Pinero, J. M ., El Análisis Estadistico y Sociométrico de la Literatura Científica, Centro de Doc­ umentación e Informática Médica, Valencia, Spain, 1972, 49. 6 . Price, D. J. S., Little Science, Big Science, Columbia University Press, New York, 1965, chap. 2. 7. Terrada, M. L. et al., Bibliometria de la producción y ei consumo de literatura médica en Espana, 1973—1977, Centro de Documentación e Informática Médica, Valencia, Spain, 1981, 157.

11 Chapter 2

DETERMINATION OF TISSUE GLUTATHIONE Frank A. M. Redegeld, Andries Sj. Koster, and Wout P. van Bennekom


Introduction............................................................................................................... 12


Sample Handling....................................................................................................... 12


Overview of Methods for the Determination of Glutathione.............................. .. 13 A. Spectrophotometric M ethods....................................................................... 13 B. High-Performance Liquid Chromatography M ethods........................... ..... 14 C. Flow-Injection Analysis of Glutathione....................................................... 14 1. Principles of Glutathione Determination by FlowInjection Analysis............................................................................. 14 2. Properties of the Flow-Injection Assay of Glutathione.................. 17


General Comments................................................................................................... 17


Future Developments............................................................................................... 18

Acknowledgments................................................................................................................ 18 References............................................................................................................................. 18


Glutathione: Metabolism and Physiological Functions

I. INTRODUCTION The tripeptide glutathione (L-7 -glutamyl-L-cysteinylglycine) participates in numerous cellular functions. Glutathione is involved, for example, in the synthesis of proteins, 12 regulation of enzyme activity, 2,3 amino acid transport, 1,2 and the catabolism of reactive oxygen species. 3,4 Glutathione also serves as a coenzyme in the synthesis of endogenous compounds (e.g., leukotrienes) and the detoxification of exogenous compounds. 1,4 Perturbations of the glutathione status as a consequence of genetic defects, physiological reactions, pathological causes, or by the action of chemical compounds are generally reflected by a lowering of the concentration of the reduced form of glutathione (GSH), which can be accompanied by an increase in the levels of the oxidized form (GSSG). Therefore, monitoring of glutathione levels has been of growing importance in a wide variety of biological and medicinal fields. 1,4 A great number of methods for the determination of glutathione has been described in the literature. These methods involve stoichiometric reactions, recycling reactions, and chromatographic procedures with various ways of detection. In this chapter a brief overview of currently used methods will be described. Furthermore, a new method using flow-injection analysis will be delineated.

II. SAMPLE HANDLING Accurate determination of glutathione in biological samples is largely dependent on a proper sample treatment. Generally, the GSSG concentration is very low compared to the GSH concentration. 5 For instance, rat liver GSH and GSSG levels range from 2.0 to 7.0 and from 0.007 to 0.084 jxmol/g wet weight, 5 respectively. Autoxidation of GSH during sample preparation can give erroneously high GSSG levels. Improper sample treatment can give rise to either an under- or overestimation of the glutathione levels in tissue. The major causes which introduce artifacts in the analysis of GSH and GSSG are summarized in Table 1. In order to prevent artifactual GSH oxidation during sample handling, GSH can be trapped with suitable agents such as N-ethylmaleimide (NEM) , 5,8,19 2-vinylpyridine (2-VP) , 14 or iodoacetic acid (IAA) . 15,16 NEM is preferred because of its rapid reaction rate (completion within 1 min), in contrast to 2-VP (20 to 60 min) or IAA (5 to 15 min). Alkylation with NEM can be achieved on ice, whereas derivatization with 2-VP or IAA occurs at room temperature. Precipitation of proteins by acid treatment and GSH trapping with NEM can be accom­ plished simultaneously, thereby quenching metabolism of GSSG and autoxidation of GSH instantaneously. Trichloroacetic acid, metaphosphoric acid, sulfosalic acid, picric acid, and perchloric acid are used to precipitate proteins. Although it is reported to produce massive GSH oxidation in erythrocyte extracts, 3 perchloric acid was found to be an acceptable precipitant. 5,17 Perchloric acid can be removed as a precipitate of the potassium salt at neutral pH. This is advantageous because the presence of acids in the samples may disturb sequential chromatographic or enzymatic procedures.5 25 In enzymatic assays using glutathione reductase, excess NEM must be removed from the samples because of the inhibitory effect of NEM on the glutathione reductase activity. 14 The GSSG reductase activity is not affected by 2-VP , 14 but the use of 2-VP can lead to erroneously high GSSG concentrations5 because of the reaction conditions described above. Extraction with organic solvents , 10,13 chromatographic procedures , 5 or alkaline hydrolysis12,18 are used to remove or inactivate NEM. Extraction or chromatography is laborious and time-consuming and can introduce other inhibitory compounds10 into the sample. Alkaline hydrolysis, 12 as modified by Redegeld et al, 18 allows rapid and simple removal of NEM and is advantageous when large numbers of samples must be handled.

13 TABLE 1 Factors Affecting Glutathione Concentrations during Sample Work-Up Glutathione form




Too high Too low


Too high Too low

Reduction of GSSG, reduction of thiol esters of GSH 2 Autoxidation (pH, metal ions, temperature, acid) , 6-8 metabolism 7 -glutamyltranspeptidase (7 -GT) (kidney, intestine) , 2 mixed disulfide forma­ tion, 2 artifactual GSH binding9 Instability of GSH-AT-ethylmaleimide adduct, 10,11 autoxidation óf GSH6 8 Reduction of GSSG, hydrolysis (local extreme alkalization) , 13 metabo­ lism by 7 -GT (kidney, intestine) 2

TABLE 2 Spectrophotometric Methods for the Determination of Glutathione Reagent or enzyme/cofactor

Reacting group(s)



Chemical Reactions 5,5 '-Dithiobis(2-nitrobenzoic acid) o-Phthalaldehyde

-SH -SH/-SSprimary amines

Nonspecific -SH reaction Nonspecific, erroneously high GSSG .and GSH levels

19, 20, 32 2 1 , 22

Enzymatic Reactions Glyoxylase I/methylglyoxal Maleylpyruvic acid isomerase/ maleylpyruvate Formaldehyde dehydrogenase/ NAD+/formaldehyde GSSG reductase/NADPH GSSG reductase/DTNB GSH-S-transferase/l-Cl-2,4 dinitrobenzene/o-dinitrobenzene


Specific for GSH Specific for GSH

5, 23 24


Specific for GSH



Specific for GSSG Specific for GSH and GSSG Specific for GSH

5 , 10, 13, 26 27, 28


III. OVERVIEW OF METHODS FOR THE DETERMINATION OF GLUTATHIONE A. SPECTROPHOTOMETRIC METHODS Several spectrophotometric methods for the determination of glutathione have been described. Some involve the direct reaction of glutathione to form a chromophore or fluorophore. Others are based on the enzymatic conversion of glutatione (Table 2). In general, the chemical methods that are based on the reactivity of the sulfhydryl group are not specific for GSH. The method using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) measures total nonprotein thiols. Determination of glutathione with o-phthalaldehyde (OPA) can be affected in the presence of amino acids and other thiols, resulting in an under- 29 or overestimation30 of GSH levels. GSSG concentrations are overestimated when using the OPA procedure. 31 The use of enzymatic analysis of glutathione enhances the specificity of the determi­ nation. However, only the DTNB-based enzymatic method is suitable for the estimation of both GSH and GSSG levels in biological samples. 13 This recycling assay (Figure 1) offers a high sensitivity. Since both GSH and GSSG are reacting in the recycling assay, separate analyses have to be made to determine “ total” glutathione (GSH 4- GSSG) and GSSG levels. (GSH is trapped by NEM5,613 or 2-VP14 prior to the analysis.) Physiological and artificial affectors


Glutathione: Metabolism and Physiological Functions

FIGURE 1. Enzymatic recycling reaction of GSH with DTNB. The thiolate anion (TNB) has a significant absorbance at 412 nm (c = 13,600 M

of the GSSG reductase activity can cause nonlinearity32’34 of the reaction and results in a lack of sample proportionality. Calibration with internal standards30,33 or assaying under modified conditions, 10 whereby the enzymatic reaction is no longer rate limiting, is proposed to overcome this drawback. B. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHODS A wide variety of high-performance liquid chromatography (HPLC) methods for the determination of glutathione in biological samples is described. Fluorometric, electrochem­ ical, and UV detection have been used. Fluorometric and UV detection of glutathione require pre- or postcolumn derivatization. A selection of the currently used HPLC methods is given in Table 3. Some disadvantages are connected with the use of HPLC. Precolumn derivatization procedures might affect the GSH/GSSG ratio because of long incubation times or heating of the samples. Special precautions must be taken to prevent autoxidation of GSH during the derivatization period. 16 Separation of the thiols requires frequent gradient elution and the use of special columns which results in long analysis times. Although some of these HPLC methods are only suitable for the analysis of GSH, most HPLC procedures allow the simultaneous measurement of GSH and GSSG. However, to ensure accurate GSSG analysis,45 it is necessary to prevent autoxidation of GSH by entrap­ ment. Thus, samples should be analyzed twice. The use of electrochemical detectors requires care and skill. The sensitivity of many electrodes decreases rapidly.46' 50 Cleaning or regenerating the electrodes is often necessary which makes this method not very useful for routine analysis of glutathione. In general, HPLC analysis of glutathione needs expensive and sophisticated equipment. Several advantages, such as the potential to analyze metabolites of glutathione, 15 37’40’41 the avoidance of interference by other thiols in the glutathione analysis, and a commonly high sensitivity are offered by HPLC methods. C. FLOW-INJECTION ANALYSIS OF GLUTATHIONE Flow injection analysis (FIA) involves selective conversion of a compound during trans­ port in a carrier stream to a suitable flow-through detector. The fixed geometrical arrange­ ments and constant flow rates in FIA make this system useful for kinetic assays. 52,53 The recycling reaction of GSH with DTNB, catalyzed by GSSG reductase, 13 (Figure 1) was adapted recently to FIA. This resulted in a sensitive and specific assay for glutathione. 1. Principles of Glutathione Determination by Flow-injection Analysis The DTNB-GSSG reductase recycling reaction assay for glutathione is carried out in the flow-injection system outlined in Figure 2. A peristaltic pump delivers the carrier (a potassium-phosphate buffer) and the reagent solutions. The reagent solutions and carrier are combined in a mixing device and are propelled through a tube filled with glass beads, i.e., a single bead string reactor (SBSR), to the flow­ through UV dectector which is set at 412 nm. After a sample is injected into the system, the

TABLE 3 Some HPLC Methods for the Determination of Glutathione in Biological Samples Detection Fluorometric

UV detection



Detection limit

Retention time (min)

7-F-2,1,3-benzoxadiazole 4-sulfonate (SDD-F)




GSH: —9





GSH: - 1 0 — 15

AT-chloro-5-dimethyIamino-naphthalene- 1 -sulfonamide o-Phthalaldehyde



GSH: 200 pmol

GSH: - 1 0






Iodoacetic acid/l-F-2,4 dinitrobenzene



GSH and GSSG: 250 pmol GSH and GSSG: 10 pmol GSH: 50 pmol GSSG: 25 pmol

GSH: - 1 0 GSSG: - 1 6 GSH: -1 2 .5 GSSG: - 3 3 GSH: - 1 6 .7 GSSG:—18.9



amino acids



Pre Post


GSH 3) was obtained. This high sensitivity of FIA is competing with that of other more expensive and time-consuming methods for the determination of glutathione (Table 3). Because the assay responds to both GSH and GSSG, separate analyses have to be performed for the estimation of the concentration of GSH. GSH concentrations can be obtained by the difference between the “ total” glutathione (GSH + GSSG) concentration and the concentration of GSSG. Simultaneous analysis of glutathione levels in hepatocytes by FIA and the cuvette-batch method13 yielded an excellent correlation between both methods. No amplification of the signals of cysteine, cystine, and dithiothreitol (DTT) was mea­ sured. However, high concentrations of DTT or cysteine can interfere with glutathione analysis because of their stoichiometric reaction with DTNB. Such interference can easily be checked with FIA by measuring in the nonenzymatic mode (without GSSG reductase and reduced nicotinamide adenine dinucleotide phosphate, NADPH). In conclusion, FIA features a relatively cheap method for the assay of glutathione, being simple, rapid (sample throughput of 30 h _1), highly reproducible, and very sensitive. In combination with an autosampler it is very suitable for the accurate analysis of glutathione in large numbers of samples.

IV. GENERAL COMMENTS At this moment a wide variety of methods for the determination of glutathione is available. Which method is most suitable for the assay of glutathione is dependent on several factors. (1) The use of a selective method is required when samples containing glutathione-


Glutathione: Metabolism and Physiological Functions

related compounds (e.g., metabolites of glutathione) have to be assayed for glutathione. (2 ) The use of sensitive methods is demanded for the determination of glutathione in small tissue samples or when tissue concentrations of glutathione (particularly GSSG) are very low. (3) Simple and rapid methods are preferred for the processing of large numbers of samples (e.g., routine analysis). The described enzymatic assay of glutathione, involving GSSG reductase-catalyzed cycling of GSH with DTNB, is in general a feasible method. Using the cuvette-batch method13 (simple and cheap), FIA18 (rapid, sensitive, and easy to automate) or HPLC45 (avoiding interference of high concentrations of, for example, cysteine and DTT) generally results in accurate analysis of GSH and GSSG.

V. FUTURE DEVELOPMENTS For the analysis in biological samples, rapid, sensitive, and above all simple methods that are selective for GSH and GSSG should be developed. The development of a glutathionespecific electrode is under investigation56 in our laboratory. Such an electrode could be valuable for the monitoring of the glutathione status in vivo or for on-line monitoring of glutathione levels during experiments. The development of specific labels (e.g., fluorescent labels) for glutathione could enable intracellular glutathione to be measured (e.g., in single cells) using videomicroscopy.

ACKNOWLEDGMENTS The authors thank Dr. M. A. J. van Opstal for the critical reading of the manuscript and E. Heidema for preparing the manuscript.

REFERENCES 1. Kosower, N. S. and Kosower, E. M ., The glutathione status of cells, Int. Rev. Cytol., 54, 109, 1978. 2. Meister, A. and Anderson, M. E ., Glutathione, Annu. Rev. Biochem., 52, 711, 1983. 3. Larsson, A., Orrenius, S., Holmgren, A., and Mannervik, B., Functions of Glutathione, Raven Press, New York, 1983. 4. Sies, H. and Wendel, A., Functions of Glutathione in Liver and Kidney, Springer-Verlag, Berlin, 1978. 5. Akerboom, T. P. M. and Sies, H ., Assay of glutathione, glutathione disulfide, and glutathione mixed disulfides in biological samples, in Methods in Enzymology, Vol. 77, Jakoby, W. B., Ed., Academic Press, New York, 1981, 373. 6 . Sies, H. and Akerboom, T. P. M ., Glutathione disulfide (GSSG) efflux from cells and tissues, in Methods in Enzymology, Vol. 105, Packer, L., Ed., Academic Press, New York, 1984, 445. 7. Anderson, M. E ., Tissue glutathione, in CRC Handbook of Methods for Oxygen Radical Research, Greenwald, R. A., Ed., CRC Press, Boca Raton, FL, 1985, 317. 8 . Srivastava, S. and Beutler, E., Accurate measurement of oxidized glutathione content of human, rabbit, and rat red blood cells and tissues, Anal. Biochem., 25, 70, 1968. 9. Anderson, M. E. and Meister, A ., Dynamic state of glutathione in blood plasma, J. Biol. Chem., 255, 9530, 1980. 10. Eyer, P. and Podhradsky, D ., Evaluation of the micromethod for determination of glutathione using enzymatic cycling and Ellman’s reagent, Anal. Biochem., 153, 57, 1986. 11. Beutler, E., Srivastava, S. K ., and West, C., The reversibility of N-ethylmaleimide (NEM) alkylation of red cell glutathione, Biochem. Biophys. Res. Commun., 38, 341, 1970. 12. Sacchetta, P ., Di Cola, D ., and Federici, G ., Alkaline hydrolysis of iV-ethylmaleimide allows a rapid assay of glutathione disulfide in biological samples, Anal. Biochem., 154, 205, 1986. 13. Tietze, F ., Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissue, Anal. Biochem., 27, 502, 1969.

19 14. Griffith, O. W ., Determination of glutathione and glutathione disulfide using glutathione reductase and 2vinylpyridine, Anal. Biochem., 106, 207, 1980. 15. Reed, D. J ., Babson, J. R ., Beatty, P. W ., Brodie, A. E., Ellis, W. W ., and Potter, D. W ., Highperformance liquid chromatography analysis of nanomole levels of glutathione, glutathione disulfide, and related thiols and disulfides, Anal. Biochem., 106, 55, 1980. 16. Farris, M. W. and Reed, D. J., High-performance liquid chromatography of thiols and disulfides: dinitrophenol derivatives, in Methods in Enzymology, Vol. 143, Jakoby, W. B. and Griffith, O. W., Eds., Academic Press, New York, 1987, 101. 17. Jocelyn, P. C., Spectrophotometric assay of thiols, in Methods in Enzymology, Vol. 143, Jakoby, W. B. and Griffith, O. W., Eds., Academic Press, New York, 1987, 44. 18. Redegeld, F. A. M ., Van Opstal, M. A. J., Houdkamp, E ., and Van Bennekom, W. P ., Determination of glutathione in biological material by flow-injection analysis using an enzymatic recycling reaction, Anal. Biochem., 174, 489, 1988. 19. Ellman, G. L ., Tissue sulfhydryl groups, Arch. Biochem. Biophys., 82, 70, 1959. 20. Jocelyn, P. C., Biochemistry o f the SH Group, Academic Press, New York, 1972. 21. Cohn, V. H. and Lyle, J., A fluorimetric assay for glutathione, Anal. Biochem., 14, 434, 1966. 22. Hissin, P. J. and Hilf, R ., A fluorimetric method for determination of oxidized and reduced glutathione in tissues, Anal. Biochem., 74, 214, 1976. 23. Bernt, E. and Bergmeyer, H. U ., Glutathione, in Methods in Enzymatic Analysis, Vol. 4, Bergmeyer, H. U. Ed., Academic Press, New York, 1974, 1643. 24. Lack, L. and Smith, M ., An enzymatic determination of reduced glutathione, Anal. Biochem., 8, 217, 1964. 25. Koivusalo, M. and Uotila, L ., Enzymatic method for the quantitative determination of reduced glutathione, Anal. Biochem., 59, 34, 1974. 26. Owens, C. W. I. and Belcher, R. V., A colorimetric micromethod for the determination of glutathione, Biochem. J., 94, 705, 1965. 27. Asaoka, K. and Takahashi, K ., An enzymatic assay of reduced glutathione using glutathione-S-aryltransferase with O-dinitro-benzene as a substrate, J. Biochem., 90, 1237, 1981. 28. Davies, M. H ., Birt, D. F ., and Schnell, R. C., Direct enzymatic assay for reduced and oxidized glutathione, J. Pharmacol. Methods, 12, 191, 1984. 29. Scaduto, R. C., Dithiothreitol and amino acids interfere with the fluorometric determination of glutathione with orthophthalaldehyde, Anal. Biochem., 174, 265, 1988. 30. Brigelius, R., Muckel, C. C., Akerboom, T. P. M ., and Sies, H ., Identification and quantitation of glutathione in hepatic protein mixed disulfides and its relationship to glutathione disulfide, Biochem. Phar­ m acol, 32, 2529, 1983. 31. Beutler, E. and West, C., Comment concerning a fluorimetric assay for glutathione, Anal. Biochem., 81, 458, 1977. 32. Oshino, N. and Chance, B ., Properties of glutathione release observed during reduction of organic hy­ droperoxide, déméthylation of aminopyrine and oxidation of some substances in perfused rat liver, and their implications for the physiological function of catalase, Biochem. J., 162, 509, 1977. 33. Bartoli, G. M. and Sies, H ., Reduced and oxidized glutathione efflux from liver, FEBS Lett., 8 6 , 89, 1978. 34. Häberle, D ., Wahlländer, A ., and Sies, H., Assessment of the kidney function in maintenance of plasma glutathione concentration and redox state in anaesthetized rats, FEBS Lett., 108, 335, 1979. 35. Imai, K. and Toyo’oka, T., Fluorimetric assay of thiols with fluorobenzoxadiazoles, in Methods in Enzymology, Vol. 143, Jakoby, W. B. and Griffith, O. W., Eds., Academic Press, New York, 1987, 67. 36. Newton, G. L ., Dorian, R., and Fahey, R. C ., Analysis of biological thiols: derivatization with monobromobimane and separation by reverse-phase high-performance liquid chromatography, Anal. Biochem., 114, 383, 1981. 37. Fahey, R. C. and Newton, G. L ., Determination of low-molecular-weight thiols using monobromobimane fluorescent labeling and high-performance liquid chromatography, in Methods in Enzymology, Vol. 143, Jakoby, W. B. and Griffith, O. W., Eds., Academic Press, New York, 1987, 85. 38. Murayama, K. and Kinoshita, T ., Determination of glutathione on high performance liquid chromatog­ raphy using N-chlorodansylamide (NCDA), Anal. Lett., 14, 1221, 1981. 39. Nakamura, H. and Tamura, Z., Fluorometric determination of thiols by liquid chromatography with postcolumn derivatization, Anal. Chem., 53, 2190, 1981. 40. Nakamura, H. and Tamura, Z ., Simultaneous fluorometric determination of thiols and disulfides by liquid chromatography with modified postcolumn derivatization, Anal. Chem., 54, 1951, 1982. 41. Keller, D. A. and Menzel, D. B., Picomole analysis of glutathione, glutathione disulfide, glutathione Ssulfonate, and cysteine-S-sulfonate by high-performance liquid chromatography, Anal. Biochem., 151, 418, 1985. 42. Tabor, C. W. and Tabor, H ., An automated ion-exchange assay for glutathione, Anal. Biochem., 78, 542, 1977.


Glutathione: Metabolism and Physiological Functions 43. Reeve, J., Kuhlenkamp, J., and Kaplowitz, N., Estimation of glutathione in rat liver by reversed-phase high-performance liquid chromatography: separation from cysteine and 7 -glutamyIcysteine, J. Chromatogr., 194, 424, 1980. 44. Beales, D ., Finch, R., McLean, A. E. M ., Smith, M ., and Wilson, J. D., Determination of penicillamine and other thiols by combined high-performance liquid chromatography and post-column reaction with Ellman’s reagent: application to human urine, J. Chromatogr., 226, 498, 1981. 45. Alpert, A. J. and Gilbert, H. F., Detection of oxidized and reduced glutathione with a recycling postcolumn reaction, Anal. Biochem., 144, 553, 1985. 46. DeMaster, E. G ., Shirota, F. N., Redfern, B., Goon, D. J. W ., and Nagasawa, H. T ., Analysis of hepatic reduced glutathione, cysteine and homocysteine by cation-exchange high-performance liquid chro­ matography with electrochemical detection, J. Chromatogr., 308, 83, 1984. 47. DeMaster, E. G. and Redfern, B., High-performance liquid chromatography of hepatic thiols with elec­ trochemical detection, in Methods in Enzymology, Vol. 143, Jakoby, W. B. and Griffith, O. W., Eds., Academic Press, New York, 1987, 110. 48. Allison, L. A. and Shoup, R. E., Dual electrode liquid chromatography detector for thiols and disulfides, Anal. Chem., 55, 8 , 1983. 49. Lunte, S. M. and Kissinger, P. T ., Detection of thiols and disulfides in liver samples using liquid chromatography/electrochemistry, J. Liq. Chromatogr., 8 , 691, 1985. 50. Carro-Ciampi, G ., Hunt, P. G ., Turner, C. J., and Wells, P. G ., A high-performance liquid chro­ matographic assay for reduced and oxidised glutathione in embryonic, neonatal, and adult tissue using a porous graphite electrochemical detector, J. Pharmacol. Methods, 19, 75, 1988. 51. Debets, A. J. J., Van de Straat, R., Voogt, W. H ., Vos, H ., Vermeulen, N. P. E ., and Frei, R. W ., Simultaneous determination of glutathione, glutathione disulphide, paracetamol and its sulphur containing metabolites using HPLC and electrochemical detection with on-line generated bromine, J. Pharm. Biomed. A nal, 6 , 329, 1988. 52. Ruzicka, J. and Hansen, E. H ., Flow Injection Analysis, John Wiley & Sons, New York, 1981. 53. Valcarcel, M. and Luque de Castro, M. D., Flow Injection Analysis — Principles and Applications, John Wiley & Sons, Chichester, England, 1987. 54. Carlberg, J. and Mannervik, B ., Purification and characterization of the flavoenzyme glutathione reductase from rat liver, J. Biol. Chem., 250, 5475, 1975. 55. Pihl, A., Eldjarn, L ., and Bremer, J., On the mode of X-ray protective agents. III. The enzymatic reduction of disulfides, J. Biol. Chem., 227, 339, 1957. 56. Hoogvliet, J. C., Van de Mark, E. J., and Van Bennekom, W. P ., personal communication, 1989.

21 Chapter 3





Endogenous Influences Affecting the Liver Glutathione Content..........................22


Exogenous Manipulation of Hepatic Glutathione...... ............................................ 23 A. Enhancement of Hepatic Glutathione..........................................................23 B. Depletion of Liver Glutathione....................................................................25 1. Potentiation of Hepatotoxicity Following GSH Depletion.............25 2. Protection against Hepatotoxicity Following Glutathione Depletion......................................................................................... 25


Summary.................................................................................................................. 26

References............................................................................................................................. 27


Glutathione: Metabolism and Physiological Functions

I. INTRODUCTION Glutathione is considered to represent the primary soluble intracellular low molecular weight compound providing a continuously maintained integrity of structures essential for living cells. Nature has developed a sophisticated enzymatic machinery for utilizing the chemical properties of this simple tripeptide in many different ways in addition to the spontaneous chemical reactions of glutathione (GSH) with reactive and hence potentially harmful endogenous or exogenous compounds. If we neglect the enzymology of the glutamyl moiety in this context, the enzymatic defense system encompasses the glutathione peroxi­ dases, the glutathione transferases, and various ancillary enzymes involved in the synthesis or regeneration of substrates or cofactors. The GSH peroxidase (GSH-Px) system consists of a set of enzymes which work in different cell compartments in a mutually supplementary way. The “ classical” GSH-Px, the selenoenzyme, 1 resides in the rodent liver in the cytosolic and the mitochondrial space. 2 It reduces H20 2 at a high rate as well as a wide variety of organic hydroperoxides including free fatty acid hydroperoxides. 3 However, it does not act upon esterified hydroperoxy fatty acids, e.g., peroxidized phospholipid.4 This task is accomplished by the newly discovered selenoenzyme, phospholipid hydroperoxide GSH-Px (PH-GSH-Px), which preferentially acts upon interfacial substrates in liposomal form . 5 An exciting feature of this activity is a synergism by vitamin E . 6 However, the rate constant of PH-GSH-Px for H20 2 is very low. These two GSH peroxidases, which both contain selenocysteine, show a different requirement for dietary selenium in rodents.7 Incidentally, some isoenzymes of the GSH transferase family exhibit GSH-Px activity towards some organic hydroperoxides but not with H20 2. This property resulted in the term “ non-selenium-dependent GSH-Px” (non-Se-GSH-Px) . 8 The general physiological signif­ icance of this in vitro-tstablished enzyme activity is only marginally investigated. In some organs, e.g., the mouse liver, this enzyme activity seems to play a subordinate role .9 However, this statement must not be generalized for other organs or species. The uncontested primary role of the GSH transferases consists of the conjugation of either endogenous metabolites (e.g., eicosanoids) or foreign compounds without or following biotransformation to the tripeptide to yield premercapturic acids which are further processed in the kidney. This enzymatic reaction links the concentration and availability of hepatic GSH directly to the detoxication potential of the organ. It is a common feature of all GSH-dependent enzymes introduced so far that they consume GSH in order to serve their protective function. Therefore, GSH depletion is considered to enhance the risk for cellular injury while fortification of the GSH system may provide additional protection. It is the purpose of this article to discuss that experimental evidence corroborates the latter statement. However, data are presented which show that GSH depletion may exacerbate certain exogenous hepatotoxic challenges but definitively protects against endogenously triggered inflammatory liver injury.

II. ENDOGENOUS INFLUENCES AFFECTING THE LIVER GLUTATHIONE CONTENT The hepatic glutathione content undergoes a marked diurnal fluctuation in various species with a maximum in the postresorption phase and a nadir after the sleeping period. This means that in nocturnal rodents the liver GSH content amounts to about 60 nmol/mg protein in the morning and falls to 40 nmol/mg protein by evening. 10 Artificial reversal of the feeding habit of mice reverses the diurnal rhythm. Starvation for more than 24 h abolishes it and leads to a drop of the hepatic GSH to 50% of the fed state. Upon refeeding, a rebound effect is observed which leads transiently to highly enhanced GSH levels in the liver, provided

23 that the diet is sufficient in sulfur-containing amino acids. 11 On the other hand, excessive methionine or cysteine in the diet only moderately increases the hepatic GSH levels on a long time scale, 12 indicating that the upper margin of intrahepatic GSH is efficiently regulated. In the fasted state, animals are more susceptible to hepatotoxins than in the fed or refed state. It is up to the reader to extrapolate the optimal time for imposing a GSH-consuming ethanol load (refer to chapter 7) to the human liver.

III. EXOGENOUS MANIPULATION OF HEPATIC GLUTATHIONE A. ENHANCEMENT OF HEPATIC GLUTATHIONE There are two primary causes which limit efforts aimed to enhance the hepatic GSH content by exogenous means. (1) Extracellular GSH has an extremely short half-life in the circulation. It is 1.6 min in man13 while in the mouse 1.9 min was determined. 13 (2) The organ is impermeable to the tripeptide in its reduced as well as in its oxidized form . 14 Nevertheless, several approaches succeeded in raising intrahepatic GSH. When starved mice received intravenous injections of liposomally entrapped GSH, their hepatic GSH level increased twofold within 2 h . 16 These animals were fully protected against highly hepatotoxic doses of acetaminophen. 17 Interestingly, injections of soluble free GSH resulted in a similar hepatic GSH increase with similar pharmacokinetics but rendered no protection against acetaminophen. A similar result was obtained in this model when acet­ ylcysteine was administered in order to supply a GSH synthesis precursor. This observation indicates that the known metabolic zonation of intrahepatic GSH18 plays a pivotal role in the protection potential of the tripeptide. A different approach used the permeability of monomethyl or -ethyl GSH esters to increase the liver GSH content and to render protection against acetaminophen. 19 Obviously, the feedback inhibition of GSH biosynthesis was overcome with these GSH esters indicating that they were first transported and then hydrolyzed intracellularly. When rodents are fed high doses of phenolic antioxidants such as butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT), an initial depletion of hepatic GSH is observed which is then followed by an increase of liver GSH up to 100 nmol/mg protein sustained for several days. 20 21 This BHA- or BHT-induced increase occurred also in starved animals. 22 Pretreatment with these compounds protected animals against various structurally dissimilar carcinogens23 24 as well as against xenobiotic-induced hepatotoxicity25,26 which could not be ascribed to the antioxidants themselves. 27 28 It has to be mentioned that BHT or BHA treatment leads to a concomitant induction of a variety of phase II enzyme activities of drug metabolism. 24 Recently, the hypothesis was presented that the enhancement of liver GSH to supraphysiological levels induced by phenolic antioxidants may be due to a shift from a primary hepatorenal circulation of GSH towards an enterohepatic circulation caused by a largely increased biliary glutathione efflux under the condition of chronic antioxidant treatment.29 A promising alternative to enhance liver GSH consists in the administration of various nontoxic, low molecular weight thiols which might be metabolically converted to a form where the sulfhydryl group becomes available for GSH biosynthesis. In such a comparative study in starved mice, equimolar doses (0.5 mmol/kg body weight) of different compounds were intravenously given and the liver GSH content was measured 2 h later. The following thiols failed to increase total soluble liver GSH: 10 cysteamine, 2,3-dimercaptopropanol, 2mercaptoethanesulfonic acid, A^-2-mercaptopropionylglycine, D-penicillamine, and dihydrolipoate. N-Acetylcysteine, L-methionine, GSH, or glutathione disulfide (GSSG) administra­ tion resulted in a significant increase in liver GSH but did not protect against drug-induced liver injury. Recently, we noticed an exception among these compounds: administration of


Glutathione: Metabolism and Physiological Functions

FIGURE 1. Enhancement of total soluble liver glutathione in starved male NMRI mice following intraperitoneal administration of 250 mg/kg thiazolidine carboxylate arginine salt.

TABLE 1 Effect of Pretreatment of Male, Starved NMRI Mice with 200 mg/kg Thiazolidine Carboxylate, Arginine Salt on Acetaminophen- or Allyl Alcohol-Induced Liver Injury (Assessed by Serum ALT) and Lipid Peroxidation (Assessed by Ethane Exhalation) and Residual Hepatic Total Glutathione Content Treatment None Allyl alcohol (1.1 mol/kg) Acetaminophen (400 mg/kg) Thiazolidine carboxylate + allyl alcohol Thiazolidine carboxylate + acetaminophen a b c d

Ethane exhalation* 2 .0

464 117 9 96

± ± ± ± ±


78 11 2C 14

Serum ALTb 47 1440 4950 95 3470

± ± ± ± ±

3 600 910 45° 1000

Liver glutathione*1 28.2 ± 0 . 6 0.4 ± 0.1 2.4 ± 0.4 8 . 0 ± 1 .0 e 3.4 ± 0.4

nmol/kg • h. Serum alanine aminotransferase activity, units per liter. p < 0.05 compared to disease control; n > 12; values: mean ± SEM. Nanomoles per milligram protein 4 h after acetaminophen or 1 h after allyl alcohol.

250 mg/kg of thiazolidine carboxylate arginine salt to starved mice led to a threefold increase of liver GSH within 2 h (Figure 1). When thiazolidinecarboxylate-pretreated mice were intoxicated 1 h after treatment with either acetaminophen14 or allyl alcohol, 30 they were significantly protected against allyl alcohol but not against acetaminophen (Table 1). Acetaminophen-induced liver injury is known to be caused by microsomal metabolism of the drug and is histologically observed in the centrolobular part of the organ. Conversely, allyl alcohol is metabolized by cytosolic alcohol dehydrogenase and the lesion is set in the periportal zone of the liver. Therefore, it seems likely that a preferential zonal utilization of a GSH precursor derived from thiazolidine carboxylate and/or intracellular compartmentation may be responsible for the differential pharmacological effect observed. As already mentioned, the total liver GSH is only a rough measure of the protective potential of GSH and detailed knowledge on the GSH content of different liver cell types is needed. An interesting advance in this direction has been recently reported on the different GSH contents of perivenous and periportal rat liver cells and their in vitro susceptibility to allyl alcohol cytotoxicity. 31

25 TABLE 2 Effect of Hepatic Glutathione Depletion on Lipid Peroxidation, Liver Cell Damage and Potentiation of Allyl Alcohol or Acetaminophen Toxicity

Treatment Controls Phorone (400 mg/kg) Diethylamaleate (3 x 300 mg/kg) Allyl alcohol (0.75 mmol/kg) Phorone -1- allyl alcohol (0.75 mmol/kg) Acetaminophen (200 mg/kg) Phorone + acetaminophen

Ethane MDA exhalation (nmol/kg * h) (pmol/mg protein)

Liver GSH (nmol/mg protein)

Serum ALT (U/l)

1 ± 0.5 11 ± 2.6 2 ± 0.5

55 ± 7 85 ± 12 54 ± 3

20 ± 3 1 ± 0.1a 4 ± 1

84 ± 21 69 ± 4b 75 ± 13b

85 ± 19a 450 ± 125a

190 ± 43a 150 ± 40

2.3 ± 0.5a 0.2 ± 0.1a

170 ± 46ac 520 ± 150ac

15 ± 5 145 ± 48a

84 ± 12 234 ± 63a

22.7 ± 1 0.5 ± 0.1a

89 ± 27 5400 ± 890ad

Note: Data ± SEM; n > 5; MDA, malondialdehyde, i.e., thiobarbituric acid-reactive material. a b c d

p < 0.05 compared to control. After 5 h. After 1 h. After 4 h.

B. DEPLETION OF LIVER GLUTATHIONE 1. Potentiation of Hepatotoxicity Following GSH Depletion Several ß-unsaturated chemicals, which are good substrates of GSH transferases, became established compounds for depleting GSH in various organs or tissue preparations. Since the liver contains many GSH transferase isoenzymes with very high activity, this organ is the major in vivo target of GSH depletion by compounds such as diethyl maleate, phorone (di-isopropylidene acetone), or cyclohexenone, to list the most popular ones. There is a continuing debate as to whether the absence of GSH as such is sufficient to induce reactions leading to cellular injury via lipid peroxidation. A very simple experiment conducted in vitro demonstrated that in liver homogenate diethyl fumarate (an extremely poor GSH transferase substrate) did not deplete GSH but led to a similar malondialdehyde and ethane production as the very efficient depletor, diethyl maleate. 32 When the parameters (in vivo, ethane exhalation or in vitro, malondialdehyde accu­ mulation) are taken for lipid peroxidation and serum transaminases release for the assessment of liver injury, there is no indication that almost total depletion of hepatic glutathione per se leads either to lipid peroxidation or acute liver injury (Table 2). However, when these animals were additionally challenged by allyl alcohol or acetaminophen, the hepatoxicity as well as lipid peroxidation induced by these compounds was greatly enhanced (Table 2) . 30,35 There are numerous further examples for the potentiation of liver injury induced by reactive metabolites following GSH depletion. 33 In some cases, a drug with a high first-pass effect in the liver may act as a GSH depletor after metabolic activation and, additionally, it may impose an oxidative stress because of side reactions of this activation. Examples of this type are acetaminophen and allyl alcohol. If given in subtoxic doses, depletion of liver GSH made the organ more susceptible to the xenobiotic. 2. Protection against Hepatotoxicity Following Glutathione Depletion Mice treated simultaneously with two per se subtoxic doses of D-galactosamine and endotoxin develop a fulminant inflammatory hepatitis within 9 h. This liver injury is not associated with measurable signs of lipid peroxidation or GSH depletion. Recent studies


Glutathione: Metabolism and Physiological Functions TABLE 3 Protection by Glutathione Depletion against Galactosamine/ Endotoxin (GalN/E)- or Galactosamine/Tumor Necrosis Factor-a-(GalN/TNF-a) Induced Hepatitis in Starved Mice Liver GSH* (nmol/mg)

Treatment Control Disease control Phorone -I- GalN/Ec DEM + GalN/Ee BSO + GalN/Ef

18 ± 1 18 ± 1 1 ± 0.1d 1.5 ± 0.1d 11.4 ± 1.5d

Note: Data ± SEM; n >


Serum ALTb (GalN/E) 40 4650 62 78 3170

± ± ± ± ±

4 1500 3d 13d 960

Serum ALTb (GalN/TNF-a) 40 4080 205 128

± ± ± ±

4 455 41d 35d


a b c d

At the time when GalN was given. After 9 h. Phorone: 250 mg/kg 90 min prior to GalN/E. p < 0.05 compared to GalN/E (700 mg/kg galactosamine + 33 M-g/kg endotoxin i.p.). e DEM (Diethylmaleate): 3 x 400 mg/kg i.p. 1 h prior to GalN/E. f BSO (Buthionine sulfoximine): 900 mg/kg i.p. 8 h prior to GalN/E. Modified from Tiegs, G. and Wendel, A Biochem. Pharmacol., 37, 2569, 1988. With permission.

show that the pathogenic mediator sequence involves lipoxygenase metabolites with special emphasis on leukotriene D4. 36 This cysteinyl leukotriene requires GSH for its biosynthesis. Hence depletion of GSH should result in an interruption of the amplification mechanism suggested for galactosamine/endotoxin hepatitis. 37 Data in Table 3 (left-hand panel) show that this holds true. Diethyl maleate as well as phorone pretreatment, i.e., drastic GSH depletion, protected mice in this model while mere inhibition of GSH synthesis by buthionine sulfoximine failed to do so. It has to be added that this study does not allow the conclusion that it is only the hepatic GSH depletion which is involved in the protective effect. Further studies suggested that galactosamine/endotoxin-induced liver injury includes a transient ischemia evoked by the vasoconstrictor leukotriene D4. After degradation of the leukotriene, a reperfusion injury is proposed for this model which can be experimentally supported by the protective effects of superoxide dismutase or allopurinol.38 The terminal cytotoxic mediator, however, is likely to be represented by the tumor necrosis factor-a (TNF), against which eicosanoid synthesis inhibitors, antagonists, reactive oxygen scav­ engers, and the xanthine oxidase inhibitor offered no protection. 39 Extraordinarily, GSH depletion also protected mice in vivo against galactosamine/TNF-induced fulminant hepatitis (Table 3, right-hand panel). The preventative protection by phorone, as well as by diethyl maleate pretreatment, was dose-dependent. Although it is difficult to find any rationale at present for this finding, the prospect of analyzing the time and site of action of this hepatoprotection against a highly effective endogenous inflammatory mediator is exciting.

IV. SUMMARY In vivo evidence in mice and rats suggests that enhancement of intrahepatic glutathione by administration of liposomally entrapped GSH or GSH methyl esters is a desirable means to protect the organ against various noxious chemicals or drugs. If challenged with this group of xenobiotics, the naturally (starved) or artificially (GSH transferase substrates)

27 depleted liver is much more susceptible. Without imposing an additional stress, no signs of acute liver injury are apparent following GSH depletion alone. On the contrary, artificial GSH depletion of mice protects the animals against fulminant inflammatory hepatitis induced either by galactosamine/endotoxin or by galactosamine/TNF administration. These findings require a revised and more sophisticated view of the effect of GSH depletion on liver injury.

REFERENCES 1. Flohé, L ., The glutathione peroxidase reaction: molecular basis of the antioxidant function of selenium in mammals, Curr. Top. Cell. Regul., 27, 473, 1985. 2. Yoshimura, S., Komatsu, N ., and Watanabe, K ., Purification and immunohistochemical localization of rat liver glutathione peroxidase, Biochim. Biophys. Acta, 621, 130, 1980. 3. Christophersen, B. O ., Formation of monohydroxy polyenic fatty acid hydroperoxides by a glutathione peroxidase, Biochim. Biophys. Acta, 164, 35, 1968. 4. Grossmann, A. and Wendel, A., Non-reactivity of the selenoenzyme glutathione peroxidase with enzy­ matically hydroperoxidized phospholipids, Eur. J. Biochem., 135, 549, 1983. 5. Ursini, F ., Maiorino, M ., and Gregolin, C., The selenoenzyme phospholipid hydroperoxide glutathione peroxidase, Biochim. Biophys. Acta, 839, 62, 1985. 6. Ursini, F. and Bindoli, A ., The role of selenium peroxidases in the protection against oxidative damage of membranes, Chem. Phys. Lipids, 44, 255, 1987. 7. Weitzel, F ., Ursini, F ., and Wendel, A ., Dependence of mouse liver phospholipid glutathione peroxidase on dietary selenium, in Selenium in Biology and Medicine, Wendel, A., Ed., Springer Verlag, Berlin, 1989, 29. 8 . Lawrence, R. A. and Burk, R. F ., Glutathione peroxidase activity in selenium-deficient rat liver, Biochim. Biophys. Res. Commun., 71, 952, 1976. 9. Cikryt, P ., Feuerstein, S ., and Wendel, A ., Selenium- and nonselenium-dependent glutathione peroxidases in mouse liver, Biochem. Pharmacol., 31, 2873, 1982. 10. Jaeschke, H. and Wendel, A ., Diurnal fluctuation and pharmacological alteration of mouse organ glu­ tathione content, Biochem. Pharmacol., 34, 1029, 1985. 11. Wendel, A. and Jaeschke, H ., Influences of selenium deficiency and glutathione status on liver metabolism, in Cellular Antioxidant Defense Mechanisms, Chow, C. K., Ed., CRC Press, Boca Raton, FL, 1988. 12. Tateishi, N. and Sakamoto, Y ., Nutritional significance of glutathione in rat liver, in Glutathione: Storage, Transport and Turnover in Mammals, Sakamoto, Y., Higashi, T., and Tateishi, N., Eds., Japan Scientific Society Press, Tokyo, 1983, 13. 13. Wendel, A. and Cikryt, P ., The level and half-life of glutathione in human plasma, FEBS Lett., 120, 209, 1980. 14. Wendel, A. and Feuerstein, S., Drug-induced lipid peroxidation. I. Modulation by monooxygenase activity, glutathione and selenium status, Biochem. Pharmacol., 30, 2513, 1981. 15. Hahn, R ., Wendel, A ., and Flohé, L ., The fate of extracellular glutathione in the rat, Biochim. Biophys. Acta, 539, 324, 1978. 16. Wendel, A., Jaeschke, H ., and Gloger, M ., Drug-induced lipid peroxidation. II. Protection against paracetamol-induced liver necrosis by intravenous liposomally entrapped glutathione, Biochem. Pharmacol., 31, 3601, 1982. 17. Wendel, A. and Jaeschke, H ., Drug-induced lipid peroxidation. III. Glutathione content of liver, kidney and spleen after intravenous administration of free and liposomally entrapped glutathione, Biochem. Phar­ macol., 31, 3607, 1982. 18. Smith, M. T ., Loveridge, N ., Wills, E. D ., and Cayen, J., The distribution of glutathione in the rat liver lobule, Biochem. J., 182, 103, 1979. 19. Anderson, M. E ., Powrie, F ., Puri, R. N ., and Meister, A ., Glutathione methyl ester: preparation, uptake by tissues, and conversion to glutathione, Arch. Biochem. Biophys., 239, 538, 1985. 20. Batzinger, R. P., Ou, S.-Y. L., and Bueding, E ., Antimutagenic effects of 2(3) tert.-butyl-4-hydroxyanisole and of antimicrobial agents, Cancer Res., 38, 4478, 1987. 21. Jaeschke, H. and Wendel, A ., Manipulation of mouse organ glutathione contents. II. Time and dose dependent induction of the glutathione conjugating system by phenolic antioxidants, Toxicology, 39, 59, 1986. 22. Jaeschke, H. and Wendel, A., Manipulation of mouse organ glutathione content. I. Enhancement by oral administration of butylated hydroxyanisole and butylated hydroxytoluene, Toxicology, 36, 77, 1985.


Glutathione: Metabolism and Physiological Functions 23. Wattenberg, L. W ., Inhibitors of chemical carcinogens, J. Environ. Pathol. Toxicol., 3, 35, 1980. 24. Kahl, R ., Synthetic antioxidants: biochemical actions and interference with radiation, toxic compounds, chemical mutagens and chemical carcinogens, Toxicology, 33, 185, 1984. 25. Ansher, S. S., Dolan, P., and Bueding, E., Chemoprotective effects of two dithiolthiones and of butylhydroxyanisole against carbontetrachloride and acetaminophen toxicity, Hepatology, 3, 932, 1983. 26. Miranda, C. L ., Henderson, M. C ., Schmitz, J. A., and Bühler, D. R ., Protective role of dietary butylated hydroxyanisole against chemical-induced acute liver damage in mice, Toxicol. Appl. Pharmacol., 69, 73, 1983. 27. Hammer, C. T. and Wills, E. D ., The role of lipid components of the diet in the regulation of the fatty acid composition of the rat liver endoplasmic reticulum and lipid peroxidation, Biochem. J., 174, 585, 1978. 28. Draper, H. H ., Polensek, L ., Hadley, M ., and Hirage, K ., Urinary malondialdehyde as an indicator of lipid peroxidation in the diet and in the tissues, Lipids, 19, 836, 1984. 29. Jaeschke, H. and Wendel, A ., Choleresis and increased biliary efflux of glutathione induced by phenolic antioxidants in rats, Toxicology, 52, 225, 1988. 30. Jaeschke, H ., Kleinwaechter, C., and Wendel, A ., The role of acrolein in allyl alcohol-induced lipid peroxidation and liver cell damage in mice, Biochem. Pharmacol., 36, 51, 1987. 31. Penttilä, K ., Allyl alcohol cytotoxicity and glutathione depletion in isolated periportal and perivenous rat hepatocytes, Chem. Biol. Interact., 65, 107, 1988. 32. Reiter, R. and Wendel, A., Chemically induced glutathione depletion and lipid peroxidation, Chem. Biol. Interact., 40, 365, 1982. 33. Reed, D. J. and Fariss, M. W ., Glutathione depletion and susceptibility, Pharmacol. Rev., 36, 258, 1984. 34. Kaplowitz, N., Aw, T. Y ., and Ookhtens, M ., The regulation of hepatic glutathione, Annu. Rev. Phar­ macol. Toxicol., 25, 715, 1985. 35. Jaeschke, H ., Werner, C., and Wendel, A., Disposition and hepatoprotection by phosphatidyl choline liposomes in mouse liver, Chem. Biol. Interact., 64, 127, 1987. 36. Wendel, A. and Tiegs, G ., Leukotriene D 4 mediates galactosamine/endotoxin-induced hepatitis in mice, Biochem. Pharmacol., 36, 1867, 1987. 37. Tiegs, G. and Wendel, A., Leukotriene-mediated liver injury, Biochem. Pharmacol., 37, 2569, 1988. 38. Wendel, A ., Tiegs, G ., and Werner, C ., Evidence for the involvement of a reperfusion injury in galactosamine/endotoxin-induced hepatitis in mice, Biochem. Pharmacol., 36, 2637, 1987. 39. Tiegs, G ., Wolter, M ., and Wendel, A., Tumor necrosis factor is a terminal mediator in galactosamine/ endotoxin-induced hepatitis in mice, Biochem. Pharmacol., 38, 627, 1989.

29 Chapter 4



Introduction...............................................................................................................30 A. Cellular Glutathione..................................................................................... 30 B. Mitochondrial Glutathione............................................................................30


Separation of the Subcellular Pools........................................................................ 30 A. Isolated Mitochondria.................................................................................. 30 B. Nonaqueous Fractionation of Tissues.......................................................... 31 C. Digitonin Treatment..................................................................................... 31 1. Cell Suspensions...............................................................................31 2. Cell Cultures..................................................................................... 32


Selective Depletion of Intracellular GSH................................................................ 33


Selective Changes of the Subcellular GSH Pools During Chemical Intoxication...............................................................................................................33 A. Liver............................................................................................................. 33 B. H eart............................................................................................................. 33 C. Isolated Hepatocytes..................................................................................... 34 D. Cultured Cells...............................................................................................34 1. Beating Heart Cells in Culture........................................................ 34 2. Rat Adrenal Cell Cultures................................................................ 35


General Comments....................................................................................................35

Acknowledgments................................................................................................................ 3 5 References............................................................................................................................. 36


Glutathione: Metabolism and Physiological Functions

I. INTRODUCTION A. CELLULAR GLUTATHIONE The thiol tripeptide glutathione (GSH) functions as an intracellular reductant and, in this way, plays an important protective role in conditions of oxidative stress or cell injury caused by a variety of chemicals. 1,2 During the course of the redox reactions, GSH is continuously oxidized to glutathione disulfide (GSSG) and rereduced by the enzymes GSHperoxidase (EC and GSSG reductase (EC, respectively. GSSG reductase uses reduced ß-nicotinamide-adenine dinucleotide phosphate (NADPH) as a specific sub­ strate and, in this way, the redox status of GSH is connected to that of NADPH and the cellular NADPH-generating systems. GSH may also be consumed nonenzymatically or in GSH/S-transferase (EC reactions with a variety of electrophilic sub­ strates. 3 Moreover, a continuous release of GSH and/or GSSG have been demonstrated in liver4,5 and heart.6 An active synthesis of new GSH from its precursor amino acids is thus required by the cells in order to retain a constant intracellular concentration. This release coincides in magnitude approximately with the ^-glutamyl transpeptidase (EC ac­ tivity of the tissue. This involves the so-called 7 -glutamyl cycle, which is discussed in detail elsewhere in this book. B. MITOCHONDRIAL GLUTATHIONE The presence of two separate intracellular pools of GSH was first proposed by Edwards and Westerfeld7 and later confirmed as intramitochondrial by Jocelyn and Kamminga8 and Jocelyn. 9 An amount equivalent to about 10% of the total hepatic GSH was found to be localized in the mitochondria after isolation of these organelles (for a recent review on this subject see Reference 10). By using a nonaqueous fractionation procedure, Wahlländer et al. 11 found a somewhat higher (13%) mitochondrial content in the liver, which was also in the range of that found in the heart with this same technique. 10 The biphasic decline in the GSH levels of mice liver and kidney after administration of buthionine sulfoximine (a specific inhibitor of 7 -glutamyl-cysteine synthetase [EC 6 .3.2.2], i.e., GSH synthesis) is also likely to be related to sequestration of GSH in the mitochondria. 3 As mitochondria also contain their own sets of GSH transferases, GSH peroxidase, GSSG reductase, and NADPH-gen­ erating systems, (i.e., the NADPH-specific isocitrate dehydrogenase and the energy-dependent NADH-transhydrogenase), they are capable of metabolizing H20 2 and other lipid hydroperoxides. The origin of the mitochondrial GSH, however, remains obscure. Since neither inward nor outward GSH transport through the mitochondrial membrane has been demonstrated, it was speculated that the mitochondrial GSH content was maintained by in situ synthesis. 1112 Subsequent research, however, was not able to detect the mitochondrial enzyme activities required for GSH synthesis. 13 Moreover, the assumed GSSG efflux as well as the GSH exchange through the mito­ chondrial membrane13 were not able to be confirmed. 14 Although a slow GSH transport through the inner mitochondrial membrane is an obvious possibility, the above contradicting results leave the question of the origin of mitochondrial GSH open to future investigation.

II. SEPARATION OF THE SUBCELLULAR POOLS A. ISOLATED MITOCHONDRIA One approach to the study of the role of the mitochondrial pool of GSH is, obviously, to do it in isolated mitochondria. The experimental model that can be used is described in References 8 and 9. In this series of papers, Jocelyn described the capacity of liver mito­ chondria to retain the majority of their GSH during the isolation procedure. Trials to prove if externally added GSH may enter the mitochondrial membrane have not been done since

31 Riley and Lehninger showed its swelling effect on the subcellular particles. 15 Nevertheless, isolated mitochondria have been used in different types of experiments implicating GSH due to the retention mentioned above, 1617 which very recently has been proposed to account also for GSSG, even under conditions of oxidative stress. 14 B. NONAQUEOUS FRACTIONATION OF TISSUES This technique to study compartmentation is based on the assumption that soluble en­ zymes and metabolites in each compartment are not separated from their respective com­ partment during fractionation of the tissue in solvents of low polarity. 1819 The tissue is freeze-clamped in liquid nitrogen with aluminum tongues and lyophilized at - 40°C. Tissue powder is homogenized in heptane/CCl4 (d = 1.23 g/ml) and subsequently fractionated on a heptane/CCl4 density gradient (d = 1.29 to 1.38 g/ml). The fractions obtained after centrifugation (16000 x g for 4 h) are divided in two aliquot portions. The activities of the mitochondrial marker enzyme citrate synthase and of the extramitochondrial marker enzyme phosphoglycerate kinase, as well as the protein content, are determined in one aliquot. The other is used for the determination of the metabolite content which is normally done after acid quenching and subsequent neutralization. From the specific activities of phosphoglycerate kinase and citrate synthase and the metabolite content in each fraction, information is extrapolated to determine metabolite contents in mitochondrial and extramitochondrial space according to Elbers et al. 20 The data are normally expressed per milligram compartmental protein, and the subcellular concen­ trations are obtained assuming a constant water content per milligram protein. The validity of the nonaqueous fractionation procedure has been demonstrated for glu­ tathione in liver11 and more recently in heart. 10 Moreover, the distributions of adenine nucleotides and other energy phosphates in rat liver, 20 heart, 21,22 and skeletal muscle23 have been reported using this procedure. The comparison of the results obtained in this way for these metabolites in liver cells with other fractionation techniques demonstrates the validity of this procedure in fractionation studies. 24 C. DIGITONIN TREATMENT Based on the fact that cholesterol content of the plasma membrane is higher than that of the mitochondrial membrane25 and that digitonin (DGT) reacts specifically with choles­ terol, a method for rapid separation of mitochondrial and cytosolic constituents has been developed. 26 The same principle has also been successfully used to separate the inner and the outer mitochondrial membranes27,28 as well as to deplete liver mitochondrial preparations from lysosomes. 29 According to this principle, isolated cells in suspensions or in monolayer cultures were incubated briefly with DGT in order to disrupt the plasma membrane while leaving the mitochondrial inner membrane intact. A subsequent rapid separation of the cell components allowed the localization and measurement of a variety of enzymes and cell metabolites in the cell compartments, assuming that no redistribution of the particular components takes place during the procedure. The fact that the mitochondrial inner membrane is essentially impermeable to GSH demonstrates the feasibility of this technique in assessment of the GSH status in the separated cell compartments. Somewhat modified procedures have been applied in freshly isolated hepatocyte suspensions1 and monolayer cell cultures. 2 1. Cell Suspensions Freshly isolated hepatocytes have been used extensively in recent years in studies de­ signed to assess selective subcellular changes of GSH during chemical intoxication. 12,30 33 This was accomplished by exposing samples of treated cells in suspension to increasing concentrations of DGT. At about 160 to 250 \xM DGT, the cell membrane was severely

Glutathione: Metabolism and Physiological Functions


TABLE 1 Subcellular Distribution of GSH in Different Tissues and Cell Types Glutathione concentration Total Heart tissue Liver Hepatocytes (suspension) Heart cells (culture) Adrenal cells (culture) a b c

1 .2 a 5.7a 37.4C 1 2 .2 C 23— 35c

Mitochondrial 7.9b 8 .8 b 3 .Ie 3.3e 3— 5C





2 0 .2 b

1 2 .8

31.0b 34.3C 8.9e 20—30°

92.8 87.2 91.7 73.0 85

8.3 27.0 15

Ref. 10 1 0 , 11

12, 33 35 36

jxmol GSH/g wet weight of tissue. nmol/mg compartmental protein. nmol/1 0 6 cells.

damaged and all the cytosolic constituents released while concentrations of DGT above 350 IxM were required in order to disrupt the mitochondrial membrane. 10 20 Rapid separation of the mitochondria by sedimentation through an inert oil (dibutyl phthalate, silicon) allowed the estimation of the mitochondrial pool of GSH. In agreement with previous methods, about 90% of the cellular GSH (—40 nmol/106 cells) was released concomitantly with the cytosolic marker enzymes while the rest ( 1 0 %) was associated with the mitochondrial com­ partment (Table 1). Electron microscopic examination of DGT-treated cells revealed a heavily damaged plasma membrane with the other cell constituents embedded in the endoplasmic reticulum. 26 2. Cell Cultures In accordance with cell suspensions, DGT treatment disrupted the plasma membrane and triggered the release of the cytosolic components in primary cultures of heart and adrenal cells from neonatal and adult rats respectively. 34' 37 DGT (200 \xM) was administered directly on the growth medium. After 30 s, the medium was withdrawn and the cells washed twice. Although the plasma membrane was severely damaged, allowing 95% of lactate dehydro­ genase (LDH) activity to leak out, the cells continued to be attached at the bottom of the petri dishes with no citrate synthase (the mitochondrial matrix marker enzyme activity) released. 35 It was concluded that the mitochondria remained in the attached “ leaky” cells. By measuring the amounts of “ releasable” and “ nonreleasable” GSH, an estimation of the cytosolic and mitochondrial pools was possible. The bulk of the cellular GSH, 85 and 73% for adrenal and heart cells, respectively, was released concomitantly with the cytosolic marker enzymes and was considered as cytosolic GSH while the rest (15 and 27%) was the mito­ chondrial fraction (Table 1). No mitochondrial marker enzyme activities were released when higher DGT concentrations and prolonged incubation were used in heart cell cultures in­ dicating a relative resistance of these particular mitochondria to DGT treatment. 35 The higher percentage of mitochondrial GSH in heart, as compared to adrenal and liver cells, correlates well with the higher content of mitochondria in the heart tissue. The discrepancy between the results obtained with DGT-treated myocytes35 and the nonaqueous fractionation technique applied to heart tissue10 is to be explained by the different experimental procedure. The latter procedure uses the amount of water per milligram compartmental protein to calculate the concentration of metabolite, and this magnitude certainly changes when heart cells are cultured. The total cellular GSH, however, was lower in heart (12.2 nmol/106 cells) as compared to liver and adrenal cells (37.4 and 23 to 35 nmol/106 cells, respectively) (Table 1).


III. SELECTIVE DEPLETION OF INTRACELLULAR GSH The chemically induced depletion of GSH in different mammalian organs and cell types was reviewed in 1981.38 Different substances may be used to irreversibly conjugate gluta­ thione leading to GSH depletion; this term should be distinguished from the oxidation of GSH to its disulfide GSSG caused by the administration of oxidants, which may either interact directly with GSH (e.g., diamide) 1,39 or act via GSH peroxidase (e.g., the reductive metabolism of organic hydroperoxides) . 40 The intraperitoneal treatment with, e.g., phorone (2,6-dimethyl-2,5-hepatidne-4-one), a substrate of the GSH S-transferases, leads to a de­ crease in cellular glutathione content in rat liver41 and heart. 10 However, no change is observed in GSH content in the brain 2 h after treatment. 10 This must be taken into account, since other compounds, such as xenobiotics or even natural products, have been reported to affect brain GSH when administered this way42'44 and their effect varied if oral treatment was used.45 The selective depletion of glutathione in the cell is somewhat more complicated to get than originally thought, since the substances used for it are not always devoid of direct or indirect peroxidative effects which may disturb the interpretation of the results obtained when they are used. On the other hand, the existence of membrane-surrounded organelles deserves the confirmation of whether the substance used penetrates them or not (for a recent review on this subject see Reference 10).

IV. SELECTIVE CHANGES OF THE SUBCELLULAR GSH POOLS DURING CHEMICAL INTOXICATION A. LIVER The hepatic mitochondrial pool of GSH has been shown to be sensitive to the abovementioned substrates when injected into animals. 141617 This can be demonstrated by meas­ uring GSH in the mitochondria isolated thereafter. The only precaution to be observed in such cases in to not attribute the induced GSH depletion the possible peroxidative damage observed, since it most probably is due to the substrate used . 10 B. HEART The heart is the first organ exposed to the xenobiotic that exceeds the capacity of the hepatic detoxication reactions, namely, GSH/GSH S-transferase (GST). It has been very recently shown that the mitochondrial pool of GSH is also sensitive to these GST substrates when injected intraperitoneally, only to an extent (50%) compatible with the life of the animal. 10 It is known that under normal conditions mitochondria can generate 0 2~ and H20 246 and indirect evidence has been brought forward that ischemic injury might generate free radicals that could be involved in damage to plasmatic and mitochondrial membrane.47 The latter has been reported to be more susceptible to free radical damage than the former.48 Moreover, once mitochondria are initially damaged, they may contribute to an increased rate of autoxidation reactions leading to an enhanced 0 2 formation. An intramitochondrial GSH depletion promoted by the so-called 0 2_ univalent leak46 under ischemic conditions or chemically induced depletion might trigger free-radical chain reactions in view of the small size of the mitochondrial pool in cardiac tissue. 10 A possible explanation of the “ stunned heart” reported in rats after induction of GSH depletion10 49 could be that mitochondrial GSH is being depleted over 50% of control values. If this is the case, the theoretical, but not demonstrated, protective role of mitochondrial GSH proposed in the liver12 might be applied to the heart as an explanation of the effects of the chemically induced GSH depletion10 49 or ischemia.50 The involvement of thiol groups


Glutathione: Metabolism and Physiological Functions

in the transfer of energy from mitochondria to cytosol has been suggested51' 53 and may be the explanatory mechanism for the changes in the mechanical function of the heart, i.e., this mitochondrial damage would impair adenosine triphosphate (ATP) production. C. ISOLATED HEPATOCYTES The mechanism of toxicity of a variety of chemicals has been studied by using isolated hepatocytes as a model system. In most of the cases of chemical intoxication, GSH was involved as a reducing agent, important for the protection of the cell, especially in cases of oxidative stress. The subcellular pools of GSH respond to such chemical treatments appar­ ently independently from each other. The fact that the onset of cell injury in isolated rat hepatocytes by ethacrynic acid and adriamycin plus l,3-bis(2-chloroethyl)-l-nitrosourea (BCNU) correlated well with the depletion of mitochondrial GSH led to the proposal that the maintenance of the mitochondrial pool is critical in protecting against cellular toxicity. 1231 Moreover, it was proposed that the reason why diethylmaleate (DEM) is not cytotoxic even if it depletes cytosolic GSH substantially is because it does not affect the mitochondrial pool. 12 Ku and Billings, however, have shown that considerable cell lysis was induced by formaldehyde in DEM-pretreated cells without significantly affecting the mitochondrial GSH pool. 54 It was concluded from these results that oxidation of the protein SH groups rather than mitochondrial GSH oxidation was the real cause of cell toxicity.54 A complete depletion of hepatic mitochondrial GSH with a combined treatment of phorone and L-buthionine sulfoximine (BSO) without affecting cell integrity also supports this statement. 32 The exact mechanism of cell damage induced by depletion of protein SH groups remains unanswered. Inactivation of the Ca2+-dependent plasma membrane ATPase and the subsequent loss of Ca2+ homeostasis have been implicated with oxidation of critical protein SH groups,55 thus leading to the proposal of the thiol/calcium hypothesis to explain the cytotoxicity of different xenobiotics. 56 Other SH proteins may also play a critical role in maintaining cellular integrity. The identification of these proteins requires further investigation. D. CULTURED CELLS Primary cultures of isolated single cells from both neonatal and adult animals provide a useful tool for studying biochemical and morphological alterations induced by a variety of chemicals. A particularly important advantage over freshly isolated cells is the possibility of mild toxin treatments for longer periods of time. This procedure allows a more distinct separation of the consequent events induced by the treatment. Specific subcellular GSH changes were apparent in an early phase during the treatment of heart1 and adrenal cells2 by the antitumor agent daunorubicin (DRB) and the carcinogen 7-hydroxymethyl-12-methylbenzanthracene (7-OHM), respectively. 34-37 1. Beating Heart Cells in Culture The effects of DRB treatment on biochemical and functional parameters of this model system were carefully recorded in order to elucidate the mechanism underlying the DRBinduced cardiotoxicity. 34,35 57-59 It is generally accepted that DRB, an anthracycline antibiotic, exerts its cytotoxicity by generating 0 2~ through redox cycling.60 61 The quinone moiety of anthracyclines has been shown to be capable of one electron reduction to a semiquinone free radical by both cytoplasmic and mitochondrial flavin dehydrogenases.62 63 The semi­ quinone radical rapidly reacts with oxygen to form 0 2~ and subsequently H20 2.62-64 Such a mechanism is conceivable to affect the status of cellular GSH, since GSH peroxidase is the main H20 2-metabolizing enzyme in the heart. Indeed, a decrease of the GSH content was observed among the early events following DRB administration to heart cell cultures. 34,35 The cells responded to the initial decrease of total GSH by activating the pathway of new GSH synthesis as indicated by recorded values higher than that of the control. However, as

35 the concentration of the added DRB increased, the cells were unable to compensate for the GSH decrease and toxic effects in the form of LDH release were observed. Interestingly, only the cytosolic pool was affected by DRB treatment while the mitochondrial GSH was essentially unchanged. It was observed that a decrease of about 30% of the level of cytosolic GSH was sufficient to drastically alter the permeability of the plasma membrane. 34,35 The apparent contradiction observed between cultured heart cells and hepatocytes re­ garding the effects of anthracycline treatment31,34,35 may be partially explained by the different treatments applied. While heart cells were treated with low concentrations and the changes recorded for a prolonged period of time (24 h), hepatocytes were treated with higher drug concentrations in combination with BCNU and severe toxicity was observed in short periods of time. Under such conditions, the consecutive events may overlap each other. For example, we were unable to detect any products of lipid peroxidation in DRB-treated myocytes, although their plasma membrane was severely damaged, indicating that lipid peroxidation observed in other systems may be a relatively late event. 2. Rat Adrenal Cell Cultures Exposure of cultured rat adrenal cells to 7-OHM caused a selective depletion of the mitochondrial GSH only, whereas the effect on the cytosolic GSH pool was negligible. 36 Although oxidation of mitochondrial GSH was observed, the total decrease could not be accounted for by oxidation to GSSG and formation of mixed disulfides with mitochondrial protein thiols has to be considered. That other poly aromatic hydrocarbons like dimethylbenz[a]anthracene or benzo[a]pyrene increase rather than decrease the cellular GSH may be explained by the activation of GSH synthesis as in the case of heart cells. A probable explanation for the selective decrease of mitochondrial GSH during 7-OHM treatment may be offered by the fact that the mitochondrial inner membrane contains high quantities of cytochrome P450. It is well known that cytochrome P450 enzymes in the presence of the so-called pseudosubstrates may generate 0 2~ and/or H20 2.65,66 It is possible that 7-OHM or some of its metabolites may represent such a pseudosubstrate inducing the generation of reactive oxygen species into the mitochondrial matrix and specifically activating the intramitochondrial defense mechanisms discussed above (Section I.B).

V. GENERAL COMMENTS The mitochondrial GSH pool is separate from that in cytoplasm with regard to sensitivity to chemical depletion. It appears that GSH depletion is not toxic per se but leaves the cells unprotected to further challenge. However, since most of the GSH-depleting agents are also oxidizing agents, they may further represent an oxidative challenge to the cells. The selective effects of the various chemicals on the mitochondrial or cytosolic GSH appears to be related to the distribution of the particular agent and/or the subcellular site of its metabolism. Decrease and/or oxidation of GSH in the cytosol or mitochondria may represent an increased susceptibility of critical SH protein groups in the respective compartments to oxidation, disturbing in this way the cell function and leading ultimately to cell death. Although both cytosol and mitochondria contain GSH-peroxidase and GSH-transferases as well as GSSG reductase and NADPH-generating systems, the latter lacks the enzymes required for GSH synthesis and the origin of their GSH remains obscure.

ACKNOWLEDGMENTS The authors wish to thank Barbara J. Knipe for typing this manuscript. Part of this work was supported by a grant No. PB87-0986 of the Direction General de Investigacion Cientifica y Técnica, Spain.


Glutathione: Metabolism and Physiological Functions

REFERENCES 1. Kosower, N. S. and Kosower, E. M ., Glutathione and cell membrane thiol status, in Functions of Glutathione: Biochemical, Physiological, Toxicological and Clinical Aspects, Larsson, A. L., Orrenius, S., Holmgren, A., and Mannervik, B., Eds., Raven Press, New York, 1983, 123. 2. Ceconi, C., Curello, S., Cargnoni, A., Ferrari, R., Albertini, A., and Visioli, O ., The role of glutathione status in protection against ischemic and reperfusion damage: effect of N-acetyl cysteine, J. Mol. Cell. Cardiol., 20, 5, 1988. 3. Meister, A. and Anderson, M. E ., Glutathione, Annu. Rev. Biochem., 52, 711, 1983. 4. Bartoli, G. M. and Sies, H ., Reduced and oxidized glutathione efflux from liver, FEBS Lett., 8 6 , 89, 1978. 5. Akerboom, T. P. M ., Bilzer, M ., and Sies, H ., The relationship of biliary glutathione disulfide (GSSG) efflux and intracellular GSSG content in perfused rat liver, J. Biol. Chem., 237, 4248, 1982. 6. Ishikawa, T. and Sies, H ., Cardiac transport of glutathione disulfide and S-conjugate. Studies with isolated perfused rat heart during hydroperoxide metabolism, J. Biol. Chem., 259, 3838, 1984. 7. Edwards, S. and Westerfeld, W. W ., Blood and liver glutathione during protein deprivation, Proc. Soc. Biol. Exp. Med., 79, 57, 1952. 8. Jocelyn, P. and Kamminga, A., Nonprotein thiol of rat liver mitochondria, Biochim. Biophys. Acta, 343, 356, 1974. 9. Jocelyn, P ., Some properties of mitochondrial glutathione, Biochim. Biophys. Acta, 396, 427, 1975. 10. Romero, F. J. and Romá, J., Careful consideration of the effects induced by glutathione depletion in rat liver and heart. The involvement of cytosolic and mitochondrial glutathione pools, Chem. Biol. Interact., 70, 29, 1989. 11. Wahlländer, A., Soboll, S., and Sies, H ., Hepatic mitochondrial and cytosolic glutathione content and the subcellular distribution of GSH-S-transferases, FEBS Lett., 97, 138, 1979. 12. Meredith, M. J. and Reed, D. J., Status of the mitochondrial pool of glutathione in the isolated hepatocyte, J. Biol. Chem., 257, 3747, 1982. 13. Griffith, O. W. and Meister, A., Origin and turnover of mitochondrial glutathione, Proc. Natl. Acad. Sei. U.S.A., 82, 4668, 1985. 14. Olafsdottir, K. and Reed, D. J., Retention of oxidized glutathione by isolated rat liver mitochondria during hydroperoxide treatment, Biochim. Biophys. Acta, 964, 377, 1988. 15. Riley, M. W. and Lehninger, A. L., Changes in sulfhydryl groups of rat liver mitochondria during swelling and contraction, J. Biol. Chem., 239, 2083, 1964. 16. Orrenius, S., Jewell, S. A., Bellomo, G ., Thor, H ., Jones, D. P ., and Smith, M. T ., Retention of calcium compartmentation in the hepatocyte — a critical role of glutathione, in Functions o f Glutathione: Biochemical, Physiological, Toxicological and Clinical Aspects, Larsson, A. L., Orrenius, S., Holmgren, A., and Mannervik, B., Eds., Raven Press, New York, 1983, 261. 17. Crane, D ., Häussinger, D ., Graf, P ., and Sies, H ., Decreased flux through pyruvate dehydrogenase by thiol oxidation during f-butyl hydroperoxide metabolism in perfused rat liver, Hoppe-Seyler’s Z. Physiol. Chem., 364, 977, 1983. 18. Soboll, S., Scholz, R., Freisl, M ., Elbers, R., and Heidt, H. W ., Distribution of metabolites between mitochondria and cytosol of perfused liver, in Use of Isolated Liver Cells and Kidney Tubules in Metabolic Studies, Tager, J. M., Söling, H.-D., and Williamson, J. R., Eds., North-Holland, Amsterdam, 1976, 29. 19. Akerboom, T. P. M ., van der Meer, R., and Tager, J. M ., Techniques for the investigation of intracellular compartmentation, Tech. Metab. Res., B205, 1, 1979. 20. Elbers, R ., Heidt, H. W ., Schmucker, P ., Soboll, S., and Wiese, H ., Measurement of the ATP/ADP ratio in mitochondria and in the extramitochondrial compartment by fractionation of freeze-stopped liver tissue in non-aqueous media, Hoppe-Seyler’s Z. Physiol. Chem., 355, 378, 1974. 21. Soboll, S. and Biinger, R ., Compartmentation of adenine nucleotides in the isolated working guinea pig heart stimulated by noradrenaline, Hoppe-Seyler’s Z. Physiol. Chem., 362, 125, 1981. 22. Kauppinen, R. A ., Hiltunen, J. K ., and Hassinen, I. E ., Subcellular distribution of phosphagens in isolated perfused rat heart, FEBS Lett., 112, 273, 1980. 23. Hebisch, S., Soboll, S., Schwenen, M ., and Sies, H ., Compartmentation of high-energy phosphates in resting and working rat skeletal muscle, Biochim. Biophys. Acta, 764, 117, 1984. 24. Soboll, S., Akerboom, T. P. M ., Schwenke, W .-D ., Haase, R., and Sies, H ., Mitochondrial and cytosolic ATP/ADP ratios in isolated hepatocytes. A comparison of the digitonin method and the non-aqueous fractionation procedure, Biochem. J., 192, 951, 1980. 25. Colbeau, A., Nachbaur, J., and Vignais, P. M ., Enzymic characterization and lipid composition of rat liver subcellular membranes, Biochim. Biophys. Acta, 249, 462, 1971. 26. Zuurendonk, P. F. and Tager, J. M ., Rapid separation of particulate components and soluble cytoplasm of isolated rat-liver cells, Biochim. Biophys. Acta, 333, 393, 1974.

37 27. Levy, M ., Toury, R ., and Andre, J., Separation des membranes mitochondriales. Purification et char­ acterisation enzymatique de la membrane externe, Biochim. Biophys. Acta, 135, 599, 1967. 28. Schnaitman, C ., Erwin, V. G ., and Greenawalt, J. W ., The submitochondrial localization of monoamine oxidase. An enzymatic marker for the outer membrane of rat liver mitochondria, J. Cell Biol., 32, 719, 1967. 29. Loewenstein, J., Schölte, H. R., and Wit-Peeters, E. M ., A rapid and simple procedure to deplete ratliver mitochondria of lysosomal activity, Biochim. Biophys. Acta, 223, 432, 1970. 30. Olafsdottir, K ., Pascoe, G. A ., and Reed, D. J., Mitochondrial glutathione status during Ca2+ ionophoreinduced injury to isolated hepatocytes, Arch. Biochem. Biophys., 263, 226, 1988. 31. Meredith, M. J. and Reed, D. J., Depletion in vitro of mitochondrial glutathione in rat hepatocytes and enhancement of lipid peroxidation by adriamycin and l,3-bis(2-chloroethyl)-l-nitrosourea (BCNU), Biochem. Pharmacol., 32, 1383, 1983. 32. Romero, F. J. and Sies, H ., Subcellular glutathione contents in isolated hepatocytes treated with l buthionine sulfoximine, Biochem. Biophys. Res. Commun., 123, 1116, 1984. 33. Romero, F. J., Sobol I, S., and Sies, H ., Mitochondrial and cytosolic glutathione after depletion by phorone in isolated hepatocytes, Experientia, 40, 365, 1984. 34. Rydström, J. and Galaris, D ., The mechanism of toxicity of anthracyclines in neonatal rat heart cells, Chem. Scr., 21 A, 157, 1987. 35. Galaris, D., Toft, E., and Rydström, J., Effect of daunorubicin on subcellular pools of glutathione in cultured heart cells from neonatal rats, Free Rad. Res. Comms., in press. 36. Hallberg, E. and Rydström, J., Selective oxidation of mitochondrial glutathione in cultured rat adrenal cells and its relation to PAH-induced cytotoxicity, submitted. 37. Hallberg, E. and Rydström, J., The mechanism of toxicity of polycyclic aromatic hydrocarbons in rat adrenal, Chem. Scr., 27A, 163, 1987. 38. Plummer, J. L ., Smith, B. R., Sies, H ., and Bend, J. R., Chemical depletion of glutathione, in vivo, Methods Enzymol., 77, 50, 1981. 39. Kosower, N. S. and Kosower, E. M ., The glutathione status of cells, Int. Rev. Cytol., 54, 109, 1978. 40. Lawrence, R. A. and Burk, R. F ., Species, tissue and subcellular distribution of non Se-dependent glutathione peroxidase activity, J. Nutr., 108, 211, 1978. 41. Van Doorn, R., Leijdekkers, C. M ., and Henderson, P. T ., Synergistic effects of phorone on the hepatotoxicity of bromobenzene and paracetamol in mice, Toxicology, 11, 225, 1978. 42. Vina, J., Romero, F. J., Sáez, G ., and Pallardó, F. V ., Effect of N-acetyl cysteine on GSH content in rat brain, Experientia, 39, 164, 1982. 43. Alverez de Laviada, T., Romero, F. J., Séz, G. T ., Antón, V ., Antón, V ., Jr., Romá, J., and Vina, J., A simple microassay for the determination of hydrazine in biological samples. Effect of hydrazine and isoniazid on liver and brain glutathione, J. Anal. Toxicol., 11, 260, 1987. 44. Romá, J., Alvarez de Laviada, T ., Antón, V ., Antón, V., Jr., Sáez, G. T ., Vina, J., and Romero, F. J., Hydraxine and glutathione concentration in liver, brain, and blood serum of the rat after hydrazine and isoniazid treatment, Naunyn-Schmiedeberg’s Arch. Pharmacol., 335, R21, 1987. 45. Estrela, J. M ., Sáez, G. T ., Such, L ., and Vina, J ., The effect of cysteine and TV-acetyl-cysteine on rat liver glutathione (GSH), Biochem. Pharmacol., 32, 3483, 1983. 46. Boveris, A ., Mitochondrial production of superoxide radical and hydrogen peroxide, Adv. Exp. Biol. Med., 78, 67, 1977. 47. Arroyo, C. M ., Kramer, J. H ., Dickens, B. F ., and Weiglicki, W. B ., Identification of free radicals in myocardial ischemia/reperfusion by spin trapping with nitrone DMPO, FEBS Lett., 211, 101, 1987. 48. Burton, K. P ., McCord, J. M ., and Ghai, G ., Myocardial alterations due to free radical generation, Am. J. Physiol, 246, H776, 1984. 49. Barssachi, R., Pelosi, G ., Camici, P ., Bonaldo, L ., Maiorino, M ., and Ursini, F ., Glutathione depletion increases chemiluminescence emission and lipid peroxidation in the heart, Biochim. Biophys. Acta, 804, 356, 1984. 50. Romero, F. J., Montoro, A ., Sáez, G. T ., Alberola, A ., Gil, F ., Vina, J ., and Such, L ., Myocardial glutathione alterations in acute coronary occlusion in the dog, Free Rad. Res. Comms., 4, 27, 1987. 51. Vignais, P. M. and Vignais, P. V ., Fuscin, an inhibitor of mitochondrial SH-dependent transport-linked functions, Biochim. Biophys. Acta, 325, 357, 1973. 52. Yagi, T. and Hatefi, Y ., Thiols in oxidative phosphorylation: inhibition and energy-potentiated uncoupling by monothiol and dithiol modifiers, Biochemistry, 23, 2449, 1984. 53. Hüther, F.-J. and Kadenbach, B ., Reactivity of the -SH groups of the mitochondrial phosphate carrier under native, solubilized and reconstituted conditions, Eur. J. Biochem., 143, 79, 1984. 54. Ku, R. H. and Billings, R. E ., The role of mitochondrial glutathione and cellular protein sulfhydryls in formaldehyde toxicity in glutathione-depleted rat hepatocytes, Arch. Biochem. Biophys., 247, 183, 1986.


Glutathione: Metabolism and Physiological Functions 55. Nicotera, P ., Moore, M ., Mirabelli, F., Bellomo, G ., and Orrenius, S., Inhibition of hepatocyte plasma membrane Ca2+-ATPase activity by menadione metabolism and its restoration by thiols, FEBS Lett., 181, 149, 1985. 56. Orrenius, S., McConkey, D. J., and Nicotera, P ., Mechanisms of cell toxicity — the thiol/calcium hypothesis, in Calcium-Dependent Processes in the Liver, Falk Symposium 48, Hellmann, C., Ed., MTP Press, Lancaster, England, 1988, 181. 57. Galaris, D. and Rydström, J., Enzyme induction by daunorubicin in neonatal heart cells in culture, Biochem. Biophys. Res. Commun., 110, 364, 1983. 58. Galaris, D ., Georgellis, A., and Rydström, J., Toxic effects of daunorubicin on isolated and cultured heart cells from neonatal rats, Biochem. Pharmacol., 34, 989, 1985. 59. Galaris, D ., Isberg, B ., Ahlberg, N. E ., and Rydström, J., Accumulation of Tc-99m-gluconate in daunorubicin-treated neonatal heart cells in culture, Acta Radiol. Oncol., 24, 177, 1985. 60. Goodman, J. and Höchstem, P ., Generation of free radicals and lipid peroxidation by redox cycling of adriamycin and daunomycin, Biochem. Biophys. Res. Commun., 77, 797, 1977. 61. Bachur, N. R ., Gordon, S. L ., and Gee, M. V ., Anthracycline antibiotic augmentation of microsomal electron transport and free radical formation, Mol. Pharmacol., 13, 901, 1977. 62. Davies, K. J. A. and Doroshow, J. H ., Redox cycling of anthracyclines by cardiac mitochondria. I. Anthracycline radical formation by NADH dehydrogenase, J. Biol. Chem., 261, 3060, 1986. 63. Doroshow, J. H ., Effect of anthracycline antibiotics on oxygen radical formation, Cancer Res., 43, 460, 1983. 64. Doroshow, J. H. and Davies, K. J. A., Redox cycling of anthracyclines by cardiac mitochondria. II. Formation of superoxide anion, hydrogen peroxide and hydroxyl radical, J. Biol. Chem., 261, 3068, 1986. 65. Premeruer, N., van den Branden, C., and Roels, F., Cytochrome P-450-dependent H20 2 production demonstrated in vivo. Influence of phénobarbital and allylisopropylacetamide, FEBS Lett., 199, 19, 1986. 66. Hornsby, P. J. and Crivello, J. F ., The role of lipid peroxidation and biological antioxidants in the function of the adrenal cortex. II, Mol. Cell. Endocrinol., 30, 123, 1983.

39 Chapter 5







Discussion................................................................................................................ 42


Conclusion................................................................................................................ 43

Acknowledgments................................................................................................................ 43 References............................................................................................................................. 44


Glutathione: Metabolism and Physiological Functions

I. INTRODUCTION Glutathione (GSH) is the most concentrated intracellular nonprotein thiol. It is an es­ sential element of the oxidoreduction status of many cells. 1 Due to its binding to proteins and by thiol/disulfide exchange, it plays a role in the regulation of enzyme activities. 2 It participates in the cellular defense mechanisms by terminating free-radical mediated chain reactions or by conjugating reactive intermediates of xenobiotics. Since glutathione plays such a fundamental role in normal cellular physiology, various attempts have been made to identify and to analyze the possible mechanisms of its hormonal regulation mainly in the liver. Fasting has been shown to reduce GSH level to 60% of its normal values. 3,4 That reduction has been attributed to a lower intake of cysteine, the limiting precursor of GSH synthesis. Furthermore, during starvation, the 7 -glutamyl transferase activity is enhanced and therefore induces an increase in GSH breakdown. 3' 5 Since the secretion of glucagon is stimulated during fasting, several authors have studied the influence of cyclic AMP and they have demonstrated that dibutyryl cyclic AMP decreased GSH levels.5,6 Previous reports have suggested that nonprotein thiol content in various organs (liver, kidney, heart) is replaced as a response to stress.7,8 Because epinephrine and glucocorticosteroids levels are increased in conditions of stress, the effects of those hormones on liver GSH content have been investigated. Levels of hepatic GSH in mice were shown to be significantly depressed by epinephrine, glucagon, and glucocorticosteroids. 9 The hepatic release of GSH across the sinusoidal plasma membrane was stimulated upon the addition of vasopressin, phenylephrine, and adrenaline. 10 The present work was carried out to evaluate possible hormonal regulation of liver GSH content by using suspensions of isolated hepa­ tocytes as an experimental model.

II. RESULTS When hepatocytes were incubated in the presence of glucagon, their intracellular GSH content was reduced as compared to untreated cells. When 10“ 6 M glucagon was added to the culture medium, the GSH level was kept at the initial control value (28.5 ± 2.7 nmol/ mg protein) whereas it continuously increased in unexposed hepatocytes (Figure 1). The effect of glucagon is mimicked by 10“ 3 M dibutyryl cyclic AMP (Figure 1). Figure 2 illustrates the concentration dependence for the glucagon effect on the cell GSH content, measured 3 h after the addition of the hormone. The maximum effect was observed at 10- 7 M glucagon. The half-maximum effect of glucagon was obtained at 5 • 10~ 9 M. Glucagon caused a parallel decrease of GSH content and total GSH equivalent thiol content (Figure 2). The effect of a variety of agents on GSH content is illustrated in Figure 3. The GSH loss in the presence of angiotensin was smaller than that induced by the other hormones. Addition of 10“ 5 M epinephrine to the culture medium prevented the accumulation of GSH within hepatocytes. The effect of 10” 7 M vasopressin was quicker than that of glucagon, since 1 h after its addition GSH loss was already significant. However, in contrast to the effect of glucagon, intracellular GSH content increased at a rate equivalent to that of control untreated hepatocytes ( ± 1 0 nmol/h). Figure 4 shows that, when maximal concentrations of glucagon and vasopressin were simultaneously added to the cells, the GSH loss was not larger than that observed in the presence of one of the hormones alone. After 1 h of incubation, glucagon had no effect on the intracellular GSH content, whereas glucagon plus vasopressin decreased the GSH level to the same extent as vasopressin alone. After 3 h of incubation, the GSH loss was slightly larger than that observed in the presence of vasopressin alone and was the same as that


FIGURE 1. Effect of glucagon and dibutyryl cyclic AMP on GSH content in isolated hepatocytes. Hepatocytes were incubated in Waymouth medium in the absence (•) or in the presence of 10 ~ 6 M glucagon (o) or 10 “ 3 M dibutyryl cyclic AMP (A). Values shown are means ± SEM for at least three separate experiments.

FIGURE 2. Dose-response curve of the effect of glucagon to decrease the GSH content (o) and the total equivalent GSH thiol content (□) in isoalted hepatocytes. Hepatocytes were incubated for 3 h in the presence of various concentrations of glucagon. Values shown are means ± SEM of at least three separate experiments; ★ or ★ ★ indicates a significant difference from control (t test for paired data: p ^ 0.05 or p ^ 0.01, respectively).


Glutathione: Metabolism and Physiological Functions

FIGURE 3. Effect of various hormones on GSH content in isolated he­ patocytes. Hepatocytes were incubated in the absence (•), or in the presence of 10”7 M angiotensin II (A), 10"7 M vasopressin (□), 10"5 M epinephrine (V), and 10-7 M glucagon (o). Values shown are means ± SEM of at least three separate experiments; ★ or ★ ★ indicates a significant difference from control (t test for paired data: p ^ 0.05 or p ^ 0.01, respectively).

FIGURE 4. Combined effect of glucagon and vasopressin on GSH con­ tent in isolated hepatocytes. Hepatocytes were incubated in the presence of 10~1 M glucagon (o), 10 ~ 7 M vasopressin (□), or in the presence of 10 7 M glucagon + 10 7 M vasopressin (A). Results are expressed as the percentage of control value at the same incubation time. Values are means ± SEM of three separate experiments.

observed in the presence of glucagon alone. Therefore, the effects of glucagon and vaso­ pressin were not additive, indicating that those hormones influenced GSH metabolism by the same mechanism even though vasopressin might act quicker than glucagon.

III. DISCUSSION In isolated hepatocytes surviving in suspension in experimental conditions which allow GSH synthesis, glucagon prevents the accumulation of intracellular GSH (Figure 1). This

43 effect of glucagon is probably mediated by cyclic AMP because dibutyryl cyclic AMP was shown to induce a similar effect (Figure 1). These data are in agreement with various in vivo observations showing that fasted rats have a decreased liver GSH content. 5 6 A lack of precursor amino acids (mainly cysteine and methionine) has been suggested as an explanation for the in vivo observations. 3 In our experimental conditions, however, such a lack of precursors does not exist since both control and glucagon-treated hepatocytes are incubated in the presence of the same concentrations of cysteine and methionine. As described in Figure 2, glucagon induced a similar decrease in GSH content and total GSH equivalent thiol content. Therefore, glucagon mainly affects the GSH level by itself instead of inducing a shift to oxidized forms of glutathione. Some additional experiments confirmed that glucagon modified neither the GSSG content nor the protein-bound glutathione level in hepatocytes (data not shown). Hence, we hypothesized that the effect of glucagon on the intracellular GSH content might result either from an inhibition of its synthesis or from an increase of its efflux. However, the thiol release from perfused rat liver is increased by phenylephrine and vasopressin, but not significantly by glucagon, 10 suggesting that glu­ cagon might interfere with GSH synthesis. A variety of other hormones also modifies the intracellular GSH content (Figure 3). It is now well established that epinephrine (via a r adrenergic receptors), vasopressin, and angiotensin act on hepatic metabolism by using inositol 1,4,5-triphosphate (IP3) as a second intracellular messenger; 12 these hormones raise the cytosolic free Ca2+ from the endoplasmic reticulum. The principal action of glucagon in liver has generally been attributed to the activation of physiological responses mediated by a rise in intracellular cyclic AMP. However, a number of recent reports suggests that glucagon can also elicit changes in cellular Ca2+ that lead to an increase in cytosolic free Ca2+ concentration. 13' 15 Glucagon mobilizes Ca2+ from the same pool as vasopressin, angiotensin II, and a r adrenergic agonists by a similar mech­ anism involving IP3. 16,17 This indicates that the modification in GSH content induced by the various hormones in isolated hepatocytes could be caused by an increase in cytosolic Ca2+

IV. CONCLUSION The data of the present paper provide evidence for hormonal regulation of GSH content in hepatocytes. That regulation could be mediated via changes in cytosolic free Ca2+ con­ centration. The specific physiological situation in which these modulations might occur in vivo remains to be defined, but it could be of significance in situations when glucagon secretion and parasympathetic activity are enhanced, e.g., exercise, hypoglycemia, and other forms of stress. Severe depletion in liver glutathione indeed occurs during physical exercise. 18 From our experiments and previous reports, 91019 it is clear that significant alterations in hepatic GSH may be created through endogenous physiological mechanisms. Since GSH is one of the most important antioxidant defense mechanisms in the liver, its depletion could enhance the vulnerability of that organ to injury induced by chemicals which are detoxified by GSH. Moreover, by releasing Ca2+ from the endoplasmic reticulum, those hormones could also potentiate a toxic effect which may result from a rise in free cytosolic Ca2+ concentration (e.g., oxidative stress). Finally, since the liver plays a major role in supplying glutathione and/or cysteine to other peripheral organs1 a hormonal modulation of hepatic GSH could also influence the thiol status of those tissues.

ACKNOWLEDGMENTS The authors wish to thank L. Hue for helpful discussions and V. Allaeys for her excellent technical assistance.


Glutathione: Metabolism and Physiological Functions

REFERENCES 1. Kaplowitz, N ., Aw, T. Y., and Ookhtens, M ., The regulation of hepatic glutathione, Annu. Rev. Pharmacol. Toxicol., 25, 715, 1985. 2. Gilbert, H. F ., Redox control of enzyme activities by thiol/disulphide exchange, Methods Enzymol., 107, 330, 1984. 3. Tateishi, N ., Higashi, T., Shinya, S., Naruse, A., and Sakamoto, Y., Studies on the regulation of glutathione level in rat liver, J. Biochem., 75, 93, 1974. 4. Harisch, G. and Mahmoud, M. F., The glutathione status in the liver and cardiac muscle of rats after starvation, Hoppe-Seyler’s Z. Physiol. Chem., 361, 1859, 1980. 5. Lauterburg, B. H. and Mitchell, J. R., Regulation of hepatic glutathione turnover in rats in vivo and evidence for kinetic homogeneity of the hepatic glutathione pool, J. Clin. Invest., 67, 1415, 1981. 6. Isaacs, J. and Binkley, F., Cyclic AMP-dependent control of the rat hepatic glutathione disulfide-sulfhydryl ratio, Biochim. Biophys. Acta, 498, 29, 1977. 7. Beck, L. V. and Linkheimer, W ., Effects of shock and cold on mouse liver sulfhydryls, Proc. Soc. Exp. Biol. Med., 81, 291, 1952. 8 . Jeffries, C. D ., Liver non protein sulfhydryl of endotoxin-treated mice, J. Bacteriol., 8 6 , 1358, 1963. 9. James, R. C ., Goodman, D. R ., and Harbison, R. D ., The perturbation of hepatic glutathione by alpha 2-adrenergic agonists, Fund. Appl. Toxicol., 3, 303, 1983. 10. Sies, H. and Graf, P ., Hepatic thiol and glutathione efflux under the influence of vasopressin, phenylephrine and adrenaline, Biochem. J., 226, 545, 1985. 11. Krack, G ., Gravier, O ., Roberfroid, M ., and Mercier, M ., Subcellular fractionation of isolated rat hepatocytes. A comparison with liver homogenate, Biochim. Biophys. Acta, 632, 619, 1980. 12. Charest, R ., Prpic, V., Exton, J. H ., and Blackmore, P. F., Stimulation of inositol triphosphate formation by vasopressin, epinephrine and angiotensin II and its relationship to changes in cytosolic-free Ca2+, Biochem. J., 227, 79, 1985. 13. Charest, R ., Blackmore, P. F., Berthon, B., and Exton, J. H ., Changes in free cytosolic Ca2+ in hepatocytes following alpha 1-adrenergic stimulation. Studies on Quin-2-loaded hepatocytes, J. Biol. Chem., 258, 8769, 1983. 14. Mauger, J.-P. and Claret, M ., Mobilization of intracellular calcium by glucagon and cyclic AMP analogues in isolated rat hepatocytes, FEBS Lett., 195, 106, 1986. 15. Combettes, L., Berthon, B ., Binet, A., and Claret, M ., Glucagon and vasopressin interactions in Ca2+ movements in isolated hepatocytes, Biochem. J., 237, 675, 1986. 16. Blackmore, P. F. and Exton, J. H ., Studies on the hepatic calcium-metabolizing activity of aluminium fluoride and glucagon. Modulation by cAMP and phorbol myristate acetate, J. Biol. Chem., 261, 11056, 1986. 17. Whipps, D. E ., Armston, A. E., Pryor, H. J., and Haiestrap, A. P ., Effects of glucagon and Ca2+ on the metabolism of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in isolated rat hepatocytes and plasma membranes, Biochem. J., 241, 835, 1987. 18. Pyke, S., Lew, H ., and Quintanilha, A., Severe depletion in liver glutathione during physical exercise, Biochem. Biophys. Res. Commun., 139, 926, 1986. 19. Roberts, S. M ., Skoulis, N. P ., and James, R. C., A centrally-mediated effect of morphine to diminish hepatocellular glutathione, Biochem. Pharmacol., 36, 3001, 1987.

45 Chapter






Glutathione Transport in Different Organs............................................................. 46 A. L iver............................................................................................................. 46 B. H eart............................................................................................................. 48 C. Kidney.......................................................................................................... 48 D. Erythrocytes..................................................................................................49 E. Plasm a.......................................................................................................... 49


Oxidative Stress........................................................................................................ 49 A. GSSG Release as an Indicator of Oxidative Stress................................... 49 B. Interactions between GSSG and GS-Conjugate Transport........................ 50 C. Interference of GSSG with Other Transport Systems................................ 50 D. Protection against Tissue Oxidative Stress by Plasma Glutathione....................................................................................................51 E. Hormonal Control of GSH Transport..........................................................51

Acknowledgments................................................................................................................ 52 References............................................................................................................................. 52


Glutathione: Metabolism and Physiological Functions

I. INTRODUCTION Most cells are equipped with a variety of antioxidant systems to protect them against toxic oxidative metabolites, including reactive oxygen species which are continuously gen­ erated during aerobic metabolism. The diversity of the defense mechanisms, ranging from low-molecular weight compounds to complex enzyme networks, allows an efficient protec­ tion at the intercellular and subcellular level. 1' 3 The naturally existing prooxidant/antioxidant balance is challenged under certain phys­ iological and pathophysiological conditions, e.g., during inflammatory processes, by the use of oxidative drugs or during severe exercise. The antioxidant role of glutathione, present at high concentrations in the aqueous compartments of the cell, is widely appreciated.4-6 With the GSH peroxidases, glutathione disulfide (GSSG) reductase and the ancillary NADPH supplying reactions, it forms a key defense system against oxidative stress in the cell. In many cells it has been shown that the occurrence of oxidative stress is accompanied by increased cellular efflux of glutathione in the form of the disulfide.7 There is some evidence that glutathione may also act in the exterior of the cell. Low but significant amounts of glutathione have been detected in blood plasma, 8 present mainly in the form of GSH.9 It was found in the rat that the plasma concentrations of the thiol differ considerably in the different regions of the blood circulation, 810 participating in an active interorgan turnover, in which the liver is the main organ exporting glutathione, while the kidney is very effective in extracting glutathione from the circulation. 11-13 Plasma glutathione may function in the maintenance of the thiol redox state of plasma proteins and in the protection against extracellular oxygen free-radical damage at inflammatory sites. Therefore, in the overall function of glutathione in the organism, membrane transport constitutes an essential feature. This overview deals with the transport properties of glutathione in different organs (as summarized in Table 1) and some aspects related to oxidative stress.

II. GLUTATHIONE TRANSPORT IN DIFFERENT ORGANS A. LIVER In perfused rat liver, glutathione is released from the organ predominantly in the form of GSH. GSH efflux occurs mainly into the sinusoidal space at a rate of 15 nmol/min/g liver; 1415 biliary GSH excretion occurs at a rate of 1 to 3 nmol/min/g liver. 15 A low rate of GSSG efflux of 0.4 nmol/min/g liver is observed, which occurs via biliary excretion. 1516 Transport of glutathione across the sinusoidal membrane of the hepatocyte is unidirectional; neither GSH nor GSSG is taken up by the intact liver. 1718 GSH efflux from the intact hepatocyte shows saturation kinetics with a of 3.3 mM and a Vmax of 20 nmol/min/g liver. 19’20 In uptake studies with isolated basolateral plasma membrane vesicles, two transport systems have been identified, a low-affinity system with a Kmof 3.3 mM and a Vmax of 12 nmol/min/mg, and a high-affinity system with substantially less activity. 21,22 It is not known at present whether there are two different translocases. Plasma membrane vesicles isolated from the sinusoidal domain represent a mixed population with an orientation of about 70% right side-out and 30% inside-out.23 It is therefore possible that the low-affinity system, bearing kinetic properties similar to those of efflux from the intact cell, represents uptake by the inside-out vesicles, whereas the high-affinity system with a low activity represents transport by the right side-out vesicles. This would be consistent with the unidirectional properties of GSH transport in the intact liver cell. Some controversy seems to exist regarding the energetics of GSH transport across the sinusoidal membrane. In isolated plasma membrane vesicles, transport was found to be independent of cations like sodium or potassium.21 However, another report indicates that GSH transport involves movement of K+ ions, 22 a mechanism favoring efflux from the intact liver.

TABLE 1 Kinetic Properties of Glutathione Transport in Different Organs of the Rat GSH

Liver Sinusoidal Perfused liver Isolated hepatocytes Membrane vesicles Low affinity High affinity Canalicular In vivo Perfused liver Membrane vesicles Heart Perfused Kidney Basolateral Membrane vesicles Luminal Membrane vesicles Erythrocytes Membrane vesicles Low affinity High affinity

3.2 3.5 3.9 3.3 0.05 0.34


vf max

Km (mM)


25b 12c 12d

Km (mM)

Efflux Efflux

nd nd

vT max



19, 26

nd nd


K + dependent N o K + dependency

22 21

0.33c 4.2d

22 21


16 15 27, 28

Efflux, ATP dependent

30 31


No saturation


No saturation 0.4






















3.5e 0.33e



40 41

ATP dependent ATP dependent

42 42

Note: nd: not determined. nmol/min/g tissue; 37°C. nmol/min/g tissue; 37°C, assuming that 108 cells correspond to 1 g wet weight of tissue. nmol/min/mg protein 25°C. nmol/min/mg protein; 37°C. nmol/min/ml cells; 37°C.


a b c d e


Glutathione: Metabolism and Physiological Functions

No saturation kinetics was observed for transport of GSH16 and GSSG15 across the canalicular plasma membrane in the intact organ. That GSH excretion is carrier mediated and not representing a simple diffusion process may be inferred from the finding that compounds like sulfobromophthalein (BSP) or phenol-3,6 -dibromophthalein disulfonate (DBSP) are able to inhibit canalicular GSH release. 24 Increased biliary efflux of GSSG14 25,26 or of the BSP-glutathione conjugate16,24,25 has no influence on GSH excretion, which suggests that GSH is transported via a separate system. Carrier-mediated transport of GSH27 and GSSG28 has also been shown with isolated canalicular plasma membrane vesicles. In this system, however, transport of GSH is inhibited both by GSSG and the benzyl-glutathione conjugate, unlike the results obtained from perfused liver. For GSSG, a linear relationship between the intracellular concentration and biliary excretion was found. 15 In perfusions with oxidizing agents like hydroperoxides or diamide, a tenfold increase of intracellar GSSG led to excretion rates up to 70 nmol/min/g liver without saturation kinetics. 18 An active transport mechanism most probably exists since GSSG is excreted into bile against a concentration gradient. 15 Indeed, it was shown that excretion of GSSG into bile is dependent on intracellular energy sources. 18 In the perfused rat liver, canalicular GSSG transport is inhibited by the glutathione thioethers of l-chloro-2,4-dinitrobenzene (DNP-SG) 29 and of sulfobromophthalein (BSPSG) . 25 Conversely, DNP-SG excretion into bile is suppressed under conditions of increased GSSG release, 29 suggesting that these compounds share a common translocator. B. HEART Both GSH and GSSG are released from the isolated perfused heart. GSH release amounts to 0.37 nmol/min/g tissue and GSSG release to 0.11 nmol/min/g tissue. 30 The infusion of increasing amounts of t-butyl hydroperoxide into the perfusate led to an increased release of the disulfide, which showed a Vmax of 7.5 nmol/min/g heart and an apparent Km of 30 nmol GSSG per gram heart. Thus, compared to the liver the capacity to transport GSSG out of the cell is low, which may be an explanation for the comparatively high sensitivity of this organ to oxidative stress. 30 As in the liver, the transport of the disulfide is inhibited by DNP-SG and vice versa. The transport rate of GSSG in heart depends on the cytosolic free ATP/ADP ratio31 (halfmaximal at a free ATP/ADP of about 10) independent of the membrane potential. Depo­ larization by infusion with 20 mM KC1, which leads to complete cardiac arrest, has no influence on the GSSG efflux rate. C. KIDNEY The kidney is the major organ for glutathione removal from the blood circulation, responsible for at least 50 to 65% of the net plasma glutathione turnover. 8,32 About 80% of both renal arterial GSH and GSSG8,10 is extracted after a single pass through the kidney. As the glomerular filtration capacity amounts to only 20 to 30%, peritubular mechanisms must contribute to the plasma disappearance of glutathione. The kidney possesses a high 7 -glutamyl-transpeptidase activity, which is mainly located on the luminal side of the brush border membrane. 33,34 Its role in the plasma extraction of glutathione has been established by the use of inhibitors such as AT-12510,32,35,36 and serine borate, 37 which caused the appearance of glutathione in the urine. Thus, after glomerular filtration, glutathione is degraded to its constituent amino acids which are reabsorbed across the brush border membrane. Basolateral catabolism of plasma glutathione is involved in the peritubular extraction of the tripeptide as evidenced by studies with AT-12510,35,38,38a and chromatographic analysis of venous glutathione metabolites. 38,383 However, renal extraction in the presence of AT-

49 125 still exceeded the glomerular filtration capacity,35,38,38* suggesting that transport of intact glutathione across the basolateral membrane also contributes to the renal handling of plasma glutathione. Experiments employing the multiple-dilution indicator method support this con­ tention. 383 Indeed, a Na+-dependent GSH transport system has been identified in renal basolateral plasma membrane vesicles. 39 Transport of glutathione across these membranes is electrogenic, involving cotransport of at least two Na+ ions, thus functioning as an extraction mechanism of GSH from the renal circulation in vivo. Since the active site of the 7 -glutamyl-transpeptidase is facing the tubular lumen, its functioning on intracellular glutathione as part of the intracellular turnover requires transport across the luminal plasma membrane.32,39* This process was recently characterized with isolated brush border membrane vesicles.40 In this system, GSH transport showed a Km of 0.21 mM and a Vmax of 0.69 nmol/min/mg and was dependent on the presence of a membrane potential favoring transfer of a negative charge. Transport was inhibited by S-benzyl-glutathione but not by the constituent amino acids glycine or glutamate. D. ERYTHROCYTES Red blood cells export GSSG but not GSH.41 Incubation of human erythrocytes with hydroperoxides leads to an efflux of the disulfide which is unidirectional and dependent on the intracellular level of ATP.41 Transport has been further characterized with isolated insideout vesicles in which two transport systems were identified, one high-affinity system with a Km of 0.1 mM and a Vmax of 0.33 nmol/min/ml cells, and one low-affinity system with a Km of 7.3 mM and a Vmax of 3.5 nmol/min/ml cells.42 For both systems, transport into the vesicles is strictly dependent on the presence of ATP. Also an ATP-dependent uptake of the dinitrophenyl conjugate of glutathione has been found,43 which inhibits the low-affinity GSSG transport system. Conversely, however high concentrations of GSSG (up to 10 mM) did not inhibit the conjugate transport44 so that the transport may not occur via a common translocator. Recently, ATPase activities in the erythrocyte plasma membrane were described which could be stimulated by the addition of GSSG45 or DNP-SG.46 The kinetic properties of these enzymes are very similar to those observed for transport, suggesting that these ATPases function in the active transport of the glutathione species. E. PLASMA Glutathione in blood plasma has a high turnover. Pharmacokinetic studies indicate a half-life of only a few minutes both in the rat47 and in humans. 48 Although GSH is prone to oxidation within the blood circulation due to the presence of glutathione oxidases on the surface of the basolateral plasma membrane in some tissues, 37’49 50 about 85% of total glutathione is normally in the reduced form . 9’51*52 A concentration of about 15 [xM is present in arterial blood. 10 The major part of plasma glutathione originates from the liver, 51,53 and plasma GSH is a reflection of the liver GSH content.51 Plasma glutathione is removed by other organs, notably the kidney, which is responsible for the extraction of about 50 to 65%.8,10 Thus, the plasma concentration of total glutathione is different in the various parts of the circulation. The highest concentrations are found in the venous blood coming from the liver (about 30 \xM), and low levels are found in renal venous blood (3 |xAf) . 10

III. OXIDATIVE STRESS A. GSSG RELEASE AS AN INDICATOR OF OXIDATIVE STRESS During oxidative stress, several organs, including liver, lung, heart, and erythrocytes, exhibit increased release of GSSG.7 In perfused liver, about 3% of GSSG generated by the intracellular GSH peroxidases is excreted into bile upon infusion of different amounts of t-


Glutathione: Metabolism and Physiological Functions

butyl hydroperoxide, and biliary GSSG release was proposed to be useful as an indicator of oxidative stress.54 Indeed, compounds which generate intracellular hydrogen peroxide within the liver, e.g., diquat, paraquat, or nitrofurans, have been shown to lead to increased biliary excretion of GSSG both in vitro15,55 and in vivo.51,56 In the absence of oxygen, biliary excretion of GSSG is lowered. 57,58 With carbon tetrachloride, a hepatotoxin causing lipid peroxidation, such increases were not observed. Likewise, chloroform and acetaminophen, known to cause lipid peroxidation as indicated by ethane and pentane production, 59 failed to elicit increased biliary GSSG release, and it was suggested that membrane-bound lipid hydroperoxides are poor substrates for GSH peroxidases.25 Recently, however, a membrane-bound Se-GSH peroxidase active towards lipid hydroperoxides has been identified.60 The lack of GSSG release may be due to a relatively low rate of lipid hydroperoxide generation or to a lowered glutathione content in the liver. In fact, acetaminophen even decreased biliary GSSG because intrahepatic glutathione is depleted by the formation and excretion of a glutathione conjugate. 59 This is in conjunction with the finding that acetaminophen treatment of the animal diminishes plasma GSH which is indicative of lowered liver values. 51 When rats are administered t-butyl hydroperoxide, paraquat, or diquat, increased plasma levels of GSSG are observed.51 Interestingly, GSSG increments were much more prominent in bile than in plasma with diquat, whereas with paraquat the increase was mainly in plasma. Measurement of plasma GSSG in the carotid artery and the vena cava at the level of the right atrium showed that with paraquat the lungs contribute to the increase. The liver appears to be the major target organ of diquat, releasing GSSG preferentially into the biliary com­ partment. B. INTERACTIONS BETWEEN GSSG AND GS-CONJUGATE TRANSPORT Inhibition of GSSG transport by glutathione-5 conjugates has been observed in tissues like erythrocytes,43,44 liver, 29 and heart. 30 Conversely, in liver and heart, but not in eryth­ rocytes,44 glutathione-5 conjugate release is suppressed under conditions of increased GSSG transport. This led to the assumption that in these organs a common translocator is involved in the transport of both GSSG and GS-conjugates. 29,30 In liver, the observation was made that the amount of GSSG, which accumulated within the cell during the inhibition by a glutathione-5 conjugate, exceeded the amount less ex­ ported. 61 Thus, additional effects by the conjugate must be responsible for the high accu­ mulation of the disulfide. Kinetic studies on purified glutathione reductase showed that this enzyme is inhibited by glutathione conjugates.61 Thus, the intracellular accumulation of GSSG under conditions of oxidative stress is amplified by simultaneous formation of glu­ tathione conjugates (Figure 1). This may become critical upon administration of drugs like menadione (2 -methyl-1,4-naphthoquinone), a redox cycling agent which elicits both oxi­ dative stress and high amounts of glutathione conjugate. 58 Efficient elimination of such conjugates from the cell is therefore of vital importance, especially in view of the fact that the glutathione conjugate of menadione is also able to undergo redox cycling.62 Increases in GSSG may provoke severe disturbances in the intracellular thiol homeostasis, influencing a variety of enzyme activities63 including transport systems (Figure 1, see below). C. INTERFERENCE OF GSSG WITH OTHER TRANSPORT SYSTEMS During increased hydroperoxide metabolism in the liver, induced either by the addition of hydroperoxides26 or by the addition of menadione,58 the biliary excretion of the bile acid taurocholate is inhibited. Evidence was presented that the increased formation of GSSG is responsible for the inhibition of bile acid excretion. The exact mechanism of inhibition by GSSG remains to be elucidated. Two possibilities are (1) competition for transport and (2) blockage of essential protein thiols on the translocator, potentially by the formation of mixed


FIGURE 1. Interactions between the formation and biliary excretion of glutathione conjugate (GSR) and glutathione disulfide (GSSG) during oxidative stress and possible interference with biliary taurocholate excretion.

disulfides. In recent studies using canalicular plasma membrane vesicles it was confirmed that GSSG can inhibit taurocholate efflux. 64 Transport could be blocked with the thiolalkylating agent NEM, indicating that the translocator indeed possesses sulfhydryl group(s) essential for its activity.64 D. PROTECTION AGAINST TISSUE OXIDATIVE STRESS BY PLASMA GLUTATHIONE Plasma glutathione can protect cells from oxidative damage. This has been shown for lung65 and intestinal cells.66 Significant protection against injury of the lung cells by paraquat and the intestinal epithelial cells by t-butyl hydroperoxide or menadione was achieved by the addition of extracellular glutathione. The protective effect was mediated via uptake of the thiol by these cells and was thus effected within the cell. In both cell types a sodiumdependent uptake of glutathione was identified.65,66 Significant protection was obtained at extracellular concentrations as low as 20 (jlAÍ, which is near-physiological. Uptake of GSH has also been observed in the perfused lung.67 E. HORMONAL CONTROL OF GSH TRANSPORT Transport of glutathione is an essential step in the interorgan turnover of this compound. There is increasing understanding that this process has functional significance under a variety of conditions. Hormones like vasopressin or angiotensin II and also adrenaline and phen­ ylephrine induce increased GSH efflux from liver.68 The increase in efflux is confined to the sinusoidal space. The changes are paralleled by changes in calcium movements across the plasma membrane. Omission of calcium from the perfusate or the addition of a calcium


Glutathione: Metabolism and Physiological Functions

chelator to the perfusate leads to a similar response of GSH efflux, indicating a link between the two phenomena.69 Also, electrical stimulation of the hepatic nerves around the portal vein causes increased GSH loss from the perfused liver.70 The increased rates of GSH release by the liver may function in the protection against oxidative stress at extrahepatic sites.71 For instance, it was shown in the rat that plasma levels of GSH initially increased during physical exercise, known to be accompanied by increased levels of plasma vasopressin, whereas the liver GSH content was decreased. 72 On this basis, it was thus suggested that there is increased supply of glutathione by the liver to fulfill increased demands in other tissues, for example, muscle which maintained a constant glutathione level under these conditions.73 During peripheral inflammation, the hepatic content of GSH was found to be decreased. 74 Thus, the generation of oxidative stress at inflammatory sites might be hormonally transmitted to the liver, which in turn responds by an increased GSH release to protect against oxidative tissue injury.71

ACKNOWLEDGMENTS This chapter was supported by the Deutsche Forschungsgemeinschaft, Grants Ak 8/11 and Si 255/8-1, and by the National Foundation for Cancer Research, Bethesda, MD.

REFERENCES 1. Sies, H ., Ed., Oxidative Stress, Academic Press, London, 1985. 2. Ishikawa, T ., Akerboom, T. P. M ., and Sies, H ., Role of key defense systems in target organ toxicity, in Target Organ Toxicity, Cohen, G. M., Ed., CRC Press, Boca Raton, FL, 1986, 129. 3. Sies, H ., Biochemistry of oxidative stress, Angew. Chem. Int. Ed. Engl., 25, 1058, 1986. 4. Kosower, N. S. and Kosower, E. M ., The glutathione status of cells, Int. Rev. Cytol., 54, 109, 1978. 5. Meister, A. and Anderson, M. E ., Glutathione, Annu. Rev. Biochem., 52, 711, 1983. 6 . Ishikawa, T. and Sies, H ., Glutathione as an antioxidant: toxicological aspects, in Glutathione: Chemical, Biochemical and Medical Aspects, Part B, Dolphin, D., Poulson, R., and Avramovic, O., Eds., John Wiley & Sons, New York, 1988, 8 6 . 7. Sies, H. and Akerboom, T. P. M ., Glutathione disulfide efflux from cells and tissues, Methods Enzymol., 105, 445, 1984. 8 . Häberle, D ., Wahlländer, A ., and Sies, H ., Assessment of the kidney function in maintenance of plasma glutathione concentration and redox state in anaesthetized rats, FEBS Lett., 108, 335, 1979. 9. Anderson, M. E. and Meister, A., Dynamic state of glutathione in blood plasma, J. Biol. Chem., 255, 9530, 1980. 10. Anderson, M. E ., Bridges, R. J., and Meister, A., Direct evidence for inter-organ transport of glutathione and that the non-filtration renal mechanism for glutathione utilization involves gamma-glutamyl transpep­ tidase, Biochem. Biophys. Res. Commun., 96, 848, 1980. 11. McIntyre, T. M. and Curthoys, N. P ., The interorgan metabolism of glutathione, Int. J. Biochem., 12, 545, 1980. 12. Inoue, M ., Interorgan metabolism and membrane transport of glutathione and related compounds, in Renal Biochemistry, Kinne, R. K. H., Ed., Elsevier, Amsterdam, 1985, 225. 13. Kaplowitz, N., Aw, T. Y., and Ookhtens, M ., The regulation of hepatic glutathione, Annu. Rev. Phar­ macol. Toxicol., 25, 715, 1985. 14. Bartoli, G. M. and Sies, H ., Reduced and oxidized glutathione efflux from liver, FEBS Lett., 8 6 , 89, 1978. 15. Akerboom, T. P. M ., Bilzer, M ., and Sies, H ., The relationship of biliary glutathione disulfide efflux and intracellular glutathione disulfide content in perfused rat liver, J. Biol. Chem., 257, 4248, 1982. 16. Kaplowitz, N ., Eberle, D. E ., Petrini, J., Touloukian, J., Corvasce, M. E ., and Kuhlenkamp, J., Factors influencing the efflux of hepatic glutathione into bile in rats, J. Pharmacol. Exp. Ther., 224, 141, 1983. 17. Hahn, R ., Wendel, A ., and Flohé, L ., The fate of extracellular glutathione in the rat, Biochim. Biophys. Acta, 539, 324, 1978.

53 18. Sies, H ., Reduced and oxidized glutathione efflux from liver, in Glutathione: Storage, Transport and Turnover in Mammals, Sakamoto, Y., Higashi, T., and Tateishi, N., Eds., Japan Scientific Society Press, Tokyo, 1983, 63. 19. Ookhtens, M ., Hobdy, K ., Corvasce, M. C., Aw, T. Y., and Kaplowitz, N ., Sinusoidal efflux of glutathione in the perfused rat liver, J. Clin. Invest., 75, 258, 1985. 20. Aw, T. Y ., Ookhtens, M ., Ren, C., and Kaplowitz, N ., Kinetics of glutathione efflux from isolated rat hepatocytes, Am. J. Physiol., 250, G236, 1986. 21. Inoue, M ., Kinne, R ., Tran, T ., and Arias, I. M ., Glutathione transport across hepatocyte plasma membranes. Analysis using isolated rat-liver sinusoidal-membrane vesicles, Eur. J. Biochem., 138, 491, 1984. 22. Aw, T. Y., Ookhtens, M ., Kuhlenkamp, J. F ., and Kaplowitz, N ., Trans-stimulation and driving forces for GSH transport in sinusoidal membrane vesicles from rat liver, Biochem. Biophys. Res. Commun., 143, 377, 1987. 23. Sips, H. J., Brown, D ., Oonk, R ., and Orci, L ., Orientation of rat-liver plasma membrane vesicles. A biochemical and ultrastructural study, Biochim. Biophys. Acta, 692, 447, 1982. 24. Ballatori, N. and Clarkson, T. W ., Sulfobromophthalein inhibition of glutathione and methylmercury secretion into bile, Am. J. Physiol., 248, G238, 1985. 25. Lauterburg, B. H ., Smith, C. Y ., Hughes, H ., and Mitchell, J. R ., Biliary excretion of glutathione and glutathione disulfide in the rat. Regulation and response to oxidative stress, J. Clin. Invest., 73, 124, 1984. 26. Akerboom, T. P. M ., Bilzer, M ., and Sies, H ., Relation between glutathione redox changes and biliary excretion of taurocholate in perfused rat liver, J. Biol. Chem., 259, 5838, 1984. 27. Inoue, M ., Kinne, R., Tran, T., and Arias, I. M ., The mechanism of biliary secretion of reduced glutathione. Analysis of transport process in isolated rat-liver canalicular membrane vesicles, Eur. J. Biochem., 134, 471, 1983. 28. Akerboom, T. P. M ., Inoue, M ., Sies, H ., Kinne, R ., and Arias, I. M ., Biliary transport of glutathione disulfide studied with isolated rat-liver canalicular-membrane vesicles, Eur. J. Biochem., 141, 211, 1984. 29. Akerboom, T. P. M ., Bilzer, M ., and Sies, H ., Competition between transport of glutathione disulfide (GSSG) and glutathione-S-conjugates from perfused rat liver into bile, FEBS Lett., 140, 73 1982. 30. Ishikawa, T. and Sies, H ., Cardiac transport of glutathione disulfide and S-conjugate. Studies with isolated perfused rat heart during hydroperoxide metabolism, J. Biol. Chem., 259, 3838, 1984. 31. Ishikawa, T ., Zimmer, M ., and Sies, H ., Energy-linked cardiac transport system for glutathione disulfide, FEBS Lett., 200, 128, 1986. 32. Griffith, O. W. and Meister, A ., Glutathione: interorgan translocation, turnover and metabolism, Proc. Natl. Acad. Sei. U.S.A., 76, 5606, 1979. 33. Horiuchi, S., Inoue, M ., and Morino, Y., Gamma-glutamyl transpeptidase: sidedness of its active site on renal brush-border membrane, Eur. J. Biochem., 87, 429, 1978. 34. Tsao, B. and Curthoys, N ., The absolute asymmetry of orientation of gamma-glutamyltranspeptidase and aminopeptidase on the external surface of the rat renal brush border membrane, J. Biol. Chem., 255, 7708, 1980. 35. Rankin, B. B. and Curthoys, N. P ., Evidence for the renal paratubular transport of glutathione, FEBS Lett., 147, 193, 1982. 36. Inoue, M ., Shinozuka, S., and Morino, Y ., Mechanism of renal peritubular extraction of plasma glu­ tathione. The catalytic activity of contraluminal gamma-glutamyltransferase is prerequisite to the apparent peritubular extraction of plasma glutathione, Eur. J. Biochem., 157, 605, 1986. 37. Ormstad, K ., Lastbom, T ., and Orrenius, S ., Evidence for different localization of glutathione oxidase and gamma-glutamyltransferase activities during extracellular glutathione metabolism in isolated perfused rat kidney, Biochim. Biophys. Acta, 700, 148, 1982. 38. Abbott, W. A., Bridges, R. J., and Meister, A ., Extracellular metabolism of glutathione accounts for its disappearance from the basolateral circulation of the kidney, J. Biol. Chem., 259, 15393, 1984. 38a. Rankin, B. B ., Wells, W ., and Curthoys, N. P ., Rat renal peritubular transport and metabolism of plasm (35S) glutathione, Am. J. Physiol., 249, F198, 1985. 39. Lash, L. H. and Jones, D. P ., Renal glutathione transport. Characteristics of the sodium-dependent system in the basal-lateral membrane, J. Biol. Chem., 259, 14508, 1984. 39a. Scott, R. D. and Curthoys, N. P ., Renal clearance of glutathione measured in rats pretreated with inhibitors of glutathione metabolism, Am. J. Physiol., 252, F877, 1987. 40. Inoue, M. and Morino, Y ., Direct evidence for the role of the membrane potential in glutathione transport by renal brush-border membranes, J. Biol. Chem., 260, 326, 1985. 41. Srivastava, S. K. and Beutler, E ., The transport of oxidized glutathione from human erythrocytes, J. Biol. Chem., 244, 9, 1969. 42. Kondo, T ., Dale, G. L ., and Beutler, E ., Studies on glutathione transport utilizing inside-out vesicles prepared from human erythrocytes, Biochim. Biophys. Acta, 645, 132, 1981.


Glutathione: Metabolism and Physiological Functions

43. Kondo, T ., Murao, M ., and Taniguchi, N., Glutathione S-conjugate transport using inside-out vesicles from human erythrocytes, Eur. J. Biochem., 125, 551, 1982. 44. LaBelle, E. F ., Singh, S. V., Srivastava, S. K ., and Awasthi, Y. C., Evidence for different transport systems for oxidized glutathione and S-dinitrophenyl glutathione in human erythrocytes, Biochem. Biophys. Res. Commun., 139, 538, 1986. 45. Kondo, T ., Kawakami, Y ., Taniguchi, N ., and Beutler, E., Glutathione disulfide-stimulated Mg2+ATPase of human erythrocyte membranes, Proc. Natl. Acad. Sei. U.S.A., 84, 7373, 1987. 46. LaBelle, E. F ., Singh, S. V ., Ahmad, H ., Wronski, L., Srivastava, S. K ., and Awasthi, Y. C ., A novel dinitrophenylglutathione-stimulated ATPase is present in human erythrocyte membranes, FEBS Lett., 228, 53, 1988. 47. Ammon, H. P. T., Melien, M. C. M ., and Verspohl, E. J., Pharmacokinetics of intravenously admin­ istered glutathione in the rat, J. Pharm. Pharmacol., 38, 721, 1986. 48. Wendel, A. and Cikryt, P ., The level and half-life of glutathione in human plasma, FEBS Lett., 120, 209, 1980. 49. Lash, L. H. and Jones, D. P ., Localization of the membrane-associated thiol oxidase of rat kidney to the basal-lateral plasma membrane, Biochem. J., 203, 371, 1982. 50. Lash, L. H. and Jones, D. P ., Characterization of the membrane-associated thiol oxidase activity of rat small-intestinal epithelium, Arch. Biochem. Biophys., 225, 344, 1983. 51. Adams, J. D ., Lauterburg, B. H ., and Mitchell, J. R ., Plasma glutathione and glutathione disulfide in the rat: regulation and response to oxidative stress, J. Pharmacol. Exp. Ther., 227, 749, 1983. 52. Lash, L. H. and Jones, D. P ., Distribution of oxidized and reduced forms of glutathione and cysteine in rat plasma, Arch. Biochem. Biophys., 240, 583, 1985. 53. Lauterburg, B. H ., Adams, J. D ., and Mitchell, J. R ., Hepatic glutathione homeostasis in the rat: efflux accounts for glutathione turnover, Hepatology, 4, 586, 1984. 54. Sies, H. and Summer, K .-H ., Hydroperoxide-metabolizing systems in rat liver, Eur. J. Biochem., 57, 503, 1975. 55. Brigelius, R. and Anwer, M. S., Increased biliary GSSG-secretion and loss of hepatic glutathione in isolated perfused rat liver after paraquat treatment, Res. Commun. Chem. Pathol. Pharmacol., 31, 493, 1981. 56. Dubin, M ., Moreno, S. N. J., Martino, E. E ., Docampo, R ., and Stoppani, A. O. M ., Increased biliary secretion and loss of hepatic glutathione in rat liver after nifurtimox treatment, Biochem. Pharmacol., 32, 483, 1983. 57. Cummings, S. W ., Hill, K. E ., Burk, R. E ., and Ziegler, D. M ., Effect of hypoxic perfusion on hepatic concentration and biliary release of glutathione disulfide, Biochem. Pharmacol., 37, 967, 1988. 58. Akerboom, T. P. M ., Bultmann, T ., and Sies, H ., Inhibition of biliary taurocholate excretion during menadione metabolism in perfused rat liver, Arch. Biochem. Biophys., 263, 10, 1988. 59. Smith, C. V ., Hughes, H ., Lauterburg, B. H ., and Mitchell, J. R ., Chemical nature of reactive metabolites determines their biological interactions with glutathione, in Functions of Glutathione: Bio­ chemical, Physiological, Toxicological, and Clinical Aspects, Larsson, A., Orrenius, S., Holmgren, A., and Mannervik, B., Eds., Raven Press, New York, 1983, 125. 60. Ursini, F ., Maiorino, M ., and Gregolin, C., The selenoenzyme phospholipid hydroperoxide glutathione peroxidase, Biochim. Biophys. Acta, 839, 62, 1985. 61. Bilzer, M ., Krauth-Siegel, R. L ., Schirmer, R. H ., Akerboom, T. P. M ., Sies, H ., and Schulz, G. E ., Interaction of a glutathione S-conjugate with glutathione reductase. Kinetic and X-ray crystallographic studies, Eur. J. Biochem., 138, 373, 1984. 62. Wefers, H. and Sies, H ., Hepatic low-level chemiluminescense during redox-cycling of menadione and the menadione-glutathione conjugate: relation to glutathione and NAD(P)H: quinone reductase (DT-diaphorase) activity, Arch. Biochem. Biophys., 224, 568, 1983. 63. Ziegler, D. M ., Role of reversible oxidation-reduction of enzyme thiols-disulfides in metabolic regulation, Annu. Rev. Biochem., 54, 305, 1986. 64. Griffiths, J. C., Sies, H ., Meier, P. J., and Akerboom, T. P. M ., Inhibition of taurocholate efflux from rat hepatic canalicular membrane vesicles by glutathione disulfide, FEBS Lett., 213, 34, 1987. 65. Hagen, T. M ., Brown, L. A ., and Jones, D. P ., Protection against paraquat-induced injury by exogenous GSH in pulmonary alveolar type II cells, Biochem. Pharmacol., 35, 4537, 1986. 6 6 . Lash, L. H ., Hagen, T. M ., and Jones, D. P ., Exogenous glutathione protects intestinal epithelial cells from oxidative injury, Proc. Natl. Acad. Sei. U.S.A., 83, 4641, 1986. 67. Dawson, J. R ., Vähäkangas, K ., Jernström, B ., and Moldeus, P ., Glutathione conjugation by isolated lung cells and the isolated perfused lung. Effect of extracellular glutathione, Eur. J. Biochem., 138, 439, 1984. 6 8 . Sies, H. and Graf, P ., Hepatic thiol and glutathione efflux under the influence of vasopressin, phenylephrine and adrenaline, Biochem. J., 226, 545, 1985.

55 69. Graf, P. and Sies, H ., Hepatic glutathione efflux and calcium, in Calcium-Dependent Processes in the Liver, Gerok, W ., Heilmann, C., Herrmann, M., and Keppler, D ., Eds., MTP Press, Lancaster, England, 1988, 191. 70. Häussinger, D ., Stehle, T ., Gerok, W ., and Sies, H ., Perivascular nerve stimulation and phenylephrine responses in rat liver. Metabolic effects, Ca2+ and K + fluxes, Eur. J. Biochem., 163, 197, 1987. 71. Sies, H. and Cadenas, E., Oxidative stress: damage to intact cells and organs, Philos. Trans. R. Soc. London, Ser. B, B311, 617, 1985. 72. Lew, H ., Pyke, S., and Quintanilha, A ., Changes in the glutathione status of plasma, liver and muscle following exhaustive exercise in rats, FEBS Lett., 185, 262, 1985. 73. Pyke, S., Lew, H ., and Quintanilha, A ., Severe depletion in liver glutathione during physical exercise, Biochem. Biophys. Res. Commun., 139, 926, 1986. 74. Bragt, P. C. and Bonta, I. L ., Oxidant stress during inflammation: anti-inflammatory effects of antiox­ idants, Agents Actions, 10, 536, 1980.

57 Chapter 7

GLUTATHIONE AND ALCOHOL Luis A. Videla and Consuelo Guerri




Experimental Liver GSH Depletion Induced by Acute Ethanol Ingestion....................................................................................................................58 A. Characteristics...............................................................................................58 B. Mechanisms.................................................................................................. 59 1. Reaction of Ethanol-Derived Acetaldehyde with GSH or Its Precursor L-Cysteine....................................................................59 2. GSH Utilization in Ethanol-Induced Oxidative Stress................... 59 3. Efflux of Hepatic Glutathione into Bile and Blood and Synthesis of Hepatic G S H ...............................................................60


Effect of Prolonged Ethanol Ingestion on Hepatic GSH in Experimental Animals and Man...............................................................................61


Concluding Remarks.................................................................................................62

Acknowledgments.................................................................................................................63 References............................................................................................................................. 63


Glutathione: Metabolism and Physiological Functions

I. INTRODUCTION Reduced glutathione (GSH) is known to play a critical role in detoxication processes of cells, including hydroperoxide catabolism (GSH-peroxidase-glutathione disulfide (GSSG) reductase system), conjugation with electrophiles (GSH transferases), and direct interception of free radicals or quenching of excited states. 1-3 From this viewpoint, the relationship between GSH and ethanol has been extensively explored in the past years in connection with the development of an oxidative stress condition in the liver as a possible lesioninducing mechanism leading to alcoholic hepatic necrosis. 2,4,5 Cellular oxidative stress is a disturbance in the prooxidant/antioxidant balance in favor of the former, 6 which can be achieved by either an enhancement of oxidative reactions with production of reactive oxygen species, a diminution of antioxidant defenses, or both. Thus, an alteration in the GSH content of the liver by ethanol could represent a major component of oxidative stress in the tissue2 and could also lead to an imbalance in the interorgan GSH homeostasis, as the liver appears to have a central role in supplying extrahepatic tissues with GSH or its component amino acids. 7 In this review, the current knowledge of the effect of ethanol on the glutathione status of the liver and other tissues is examined. Discussion is centered on the molecular mechanisms involved in the effects elicited by acute and chronic ethanol ingestion as well as their contributory factors.

II. EXPERIMENTAL LIVER GSH DEPLETION INDUCED BY ACUTE ETHANOL INGESTION A. CHARACTERISTICS The administration of single doses of ethanol (2.3 to 6.0 g/kg of body weight) to experimental animals (mouse, rat, baboon) reduces hepatic GSH by 21 to 64%.8-21 The effect has been predominantly studied in the fasted state9,10,12-20 and is produced regardless of the sex of the animals. 12 An ethanol dose-response relationship with the hepatic GSH has been established in the range of 1 to 6 g of ethanol per kilogram with significant GSH decreases starting with 3 g/kg and with a maximal effect at 5 g/kg. 1012«14»19 The study of the time course of ethanol-induced liver GSH depletion has indicated a lack of change early in intoxication, 10,12,14,17,18 20 significant GSH reduction after 3 h of treatment, and maximal effects following 6 h , 10,12,17,20 with levels returning to control values 8 to 1 2 h after ethanol administration (3 to 5 g/kg) . 12,14 The maintenance of low levels of hepatic GSH for periods longer than 1 2 h cannot be taken as a long-lasting action of ethanol, 10 but would rather reflect the GSH-decreasing effect of fasting itself, 12 which can be for as long as 40 h of food deprivation. 10 These considerations provide a possible explanation for negative results reported in trials with low doses of ethanol ( 2 g/kg) 22 or unsuitable periods of intoxication ( 1 , 2 , or 16 h) . 22-24 The diminution in hepatic GSH by ethanol given in vivo has been reproduced in isolated hepatocytes incubated with 10 to 50 mM ethanol25,26 and in isolated perfused rat liver after the infusion of 45 mM ethanol for 25 min. 27 The effect can be exacerbated by several conditions which act as additional noxious challenges to the organ such as previous chronic alcohol intake, 13 iron overload, 28,29 or hypoxia, 30 and it is diminished during aging31 or by pretreatment with the antioxidants cyanidanol, 32 silymarin, 33 and desferrioxamine. 16 Also, an enhanced availability of GSH in the liver tissue has been shown to completely reverse the decrease of the tripeptide by ethanol. This was found for methionine or S-adenosyl-Lmethionine in isolated hepatocytes25,34 and in in vivo conditions. 21 It is interesting to note that the studies on the inhibition of the enzymes involved in ethanol oxidation by the alcohol dehydrogenase pathway point to acetaldehyde as the mediator of alcohol-induced liver GSH

59 depletion. In fact, the alcohol dehydrogenase inhibitor pyrazole prevents the decrease in GSH levels produced by ethanol in vivo11 or added to isolated hepatocytes, 25 26 while in­ hibition of aldehyde dehydrogenase by disulfiram1125 26 or tolbutamide35 potentiates the effect. This view is in agreement with studies indicating a direct GSH-decreasing action exerted by acetaldehyde on the liver, either when given in vivo36 or added to isolated liver cells. 2526 B. MECHANISMS The study of the mechanisms responsible for the effect of acute ethanol intoxication on liver GSH has suggested several actions of the xenobiotic on different aspects of GSH metabolism. 1. Reaction of Ethanol-Derived Acetaldehyde with GSH or its Precursor L-Cysteine The in vitro incubation of GSH with acetaldehyde at physiological pH and temperature conditions has indicated a nonenzymatic condensation between them, with a drastic dimi­ nution (55 to 84%) in the concentration of the tripeptide. 25’38 This effect is not due only to GSH oxidation, as the total GSH equivalents (GSH + 2GSSG) in the system were decreased by 50%.38 In these studies, unreacted GSH was determined enzymatically, 25’38 which might explain the negative results found when sulfhydryl groups were detected with 5,5'-dithiobis(2-nitrobenzoate), (DTNB) , 39 a reagent that probably displaces acetaldehyde from the con­ densation product.40 The reaction is a spontaneous process with a standard free energy change of - 3.0 kcal/mol and an interaction constant of 134, assessed by polarographic techniques,40 being reversible upon removal of acetaldehyde by hydrazine. 25 The analysis of the conden­ sation products found in vitro revealed the presence of free primary amino groups (positive reaction with ninhydrin) and substituted sulfhydryl moieties (negative reaction with DTNB) , 38 probably indicating the formation of the corresponding thiohemiacetal and dithioacetal de­ rivatives. Although the nonenzymatic reaction of GSH with acetaldehyde assayed in a reconstituted alcohol dehydrogenase system at equilibrium was reported to account for less than 10% of the in vivo GSH depletion induced by ethanol, 17 the exact contribution of this mechanism in the hepatocyte has not been established. Under cellular conditions, however, thiohemiacetal formation could proceed undetected mainly due to instability of the adduct40 and to its biotransformation into cysteine conjugates, which can be further N-acetylated (mercapturic acid formation) 41 or subjected to ring closure (2-methylthiazolidine-4-carboxylic acid [2-MTCA] formation) . 42 Binding of acetaldehyde to the GSH-precursor cysteine has been shown to occur in vitro17’39’43 at a rate which is five times greater than that with GSH and about one third of the rate of GSH depletion by ethanol. 17 However, in vitro, 17 as well as in vivo,44 studies have failed to detect the formation of 2-MTCA as the result of the reaction of acetaldehyde with cysteine. Also, the in vivo administration of 2-MTCA to rats resulted in undetectable levels of the metabolite in 24-h urine samples. 44 Together, these observations support the contention that both formation and degradation of 2-MTCA may occur in vivo under con­ ditions of ethanol intoxication.43 44 Catabolism of 2-MTCA could be explained by the oxi­ dative pathway described in mitochondria,45 dissociation of 2-MTCA into its constituents as observed upon incubation at physiological conditions, 17 or both. 2. GSH Utilization in Ethanol-Induced Liver Oxidative Stress Acute ethanol intoxication has been shown to be associated with the development of an oxidative stress condition in the liver tissue. 2 This condition is characterized by a lipid peroxidative response2’4’5’9’12-14’20’26' 29’31’36 which can impose an enhanced GSH utilization for peroxide and free-radical catabolism. 17 Although hepatic lipid peroxidation evaluated by tissue accumulation of malondialdehyde (MDA) or conjugated dienes (CD) has produced


Glutathione: Metabolism and Physiological Functions

conflicting results after ethanol ingestion, 2,418 2648 the phenomenon has received strong support when assessed by techniques which are noninvasive for the organ. These include measurements of ( 1 ) hydrocarbon evolution in the isolated perfused rat liver,49 50 a response which is also observed in vivo5153 and partially mediated by inhibition of hydrocarbon metabolism by ethanol;54 (2) biliary release of MDA in the anesthetized rat as a reflection of hepatic MDA production;28 29 (3) spontaneous chemiluminescence of the in situ rat liver, 55 a technique related to the steady-state level of oxidative free radicals;6,50 and (4) oxygen uptake associated with the lipid peroxidative process (antioxidant-sensitive respiration as­ sessed in the perfused rat liver) . 27,56,57 Ethanol-induced liver lipid peroxidation seems to be mediated by its metabolite acetaldehyde2,26,36,49,50,57 and involves the generation of free radicals, as ethane release from perfused rat liver,49,50 chemiluminescence of the liver in situ,55 and CD33 or MDA32 accumulation in the liver tissue were completely abolished by the free-radical scavengers cyanidanol and silymarin. Furthermore, ethanol-induced release of glutamate-pyruvate transaminase and sorbitol dehydrogenase from perfused livers into the perfusate was suppressed by superoxide dismutase, catalase, and desferrioxamine, sug­ gesting a role for oxygen free radicals and iron in alcohol-induced hepatotoxicity. 58 In this respect, it has been shown that hydroxyl (H 0‘) or hydroxyl-like radicals can be produced in a Fenton-type reaction between Fe2+ and H20 2 generated by the microsomal electron transport system. 59,60 The oxidation of ethanol into acetaldehyde by microsomal enzymes would imply HO’ generation and interaction with the molecule of ethanol to form 1-hydroxyethyl radicals (CH3-CH-OH), as recently detected by spin-trapping with nitrone de­ rivatives and characterized by electron paramagnetic resonance spectroscopy, both in in vitro61 and in vivo62 conditions. The exact nature of the cellular stores providing active iron for free-radical generation required in both microsomal ethanol oxidation and lipid peroxi­ dation is not currently known.63 It has been suggested, however, that ferritin may function in vivo as a physiological iron donor as 0 2 produced by xanthine oxidase is able to mobilize iron from ferritin64 and promote lipid peroxidation. 65 Thus, oxygen- and ethanol-derived free radicals involved in microsomal ethanol oxidation and acetaldehyde oxidation by xan­ thine oxidase in the hepatocyte may directly induce lipid peroxidation. Alternatively, these radicals may enhance the process through iron release from hepatic ferritin as shown in a reconstituted system consisting of the reaction catalyzed by alcohol dehydrogenase, xanthine oxidase, ferritin, and microsomes as a membrane target for peroxidation.66 An enhanced hepatic GSH utilization imposed by the prooxidative pressure developed by ethanol would lead to an increase in the GSSG content in the tissue, as reported in in v/w?28,29,32,33,67 and in vitro26 studies, but not confirmed in one trial. 17 This interrelation is supported by the effect of the antioxidants cyanidanol32 and silymarin33 which, when given prior to ethanol, abolished both liver GSSG accumulation and lipid peroxidation increase by ethanol. However, the increase in the GSSG content of the liver following ethanol ingestion was found to account for only 19% of the GSH depletion observed, indicating the involvement of other mechanisms such as changes in the glutathione release into the bile and/or blood67 or in the synthesis of the tripeptide. 15,17 3. Efflux of Hepatic Glutathione into Bile and Blood and Synthesis of Hepatic GSH Glutathione is known to be released from the hepatocyte. 7,68 GSH efflux occurs mainly into the circulation to maintain an interorgan GSH homeostasis7 while GSSG (and glutathione-S-conjugates) is transported into the bile, 7,68 representing flux through the glutathione peroxidase reaction in the tissue. 68 The evaluation of the effect of acute ethanol ingestion on the biliary release of glutathione has consistently revealed a diminution of the process for both GSSG and GSH15,28,29,67 at experimental times in which ethanol was present in the blood of the animals. Although the exact nature of this effect of ethanol is not known, it has been related to the decreased intrahepatic content of glutathione15 and to the in vivo

61 formation of GSH-acetaldehyde conjugates.67 Alternatively, ethanol reduction of biliary GSSG efflux could represent a direct action on the biliary export system which has been reported to be carrier mediated.7,68 Efflux of hepatic GSH into the circulation has been reported to account for a major part of the turnover of the tripeptide in the liver.69 Acute ethanol intoxication in the rat was found to elicit a marked increase in the plasma level of total glutathione which was assessed in arterial blood samples. 67,70 The time course study of the changes in plasma glutathione revealed a maximal increase after 3 h of ethanol ingestion. 70 Since at this time the effect of ethanol coincided with a maximal decrease in red blood cell glutathione70 and negligible changes in the liver tissue, 12 the enhancement in plasma glutathione observed was suggested to be of erythrocyte origin. After 3 h of intoxication, however, erythrocyte glutathione returned to control values and the plasma concentration was stabilized at a level significantly higher than control values (time zero), for up to 6 h of treatment. 70 Since after 3 to 6 h of ethanol treatment the liver becomes maximally depleted of GSH, 12 the increased plasma glutathione found in this time interval was suggested to be mainly due to hepatic sinusoidal transport. 70 This view was confirmed by data showing elevated posthepatic plasma levels of total glutathione in rats given ethanol for 3.5 h , 17 while no changes were observed in prehepatic plasma levels. 17 These results indicate that acute ethanol ingestion induces a loss of GSH from the liver into the circulation that could constitute a major mechanism for the intrahepatic depletion of the tripeptide, 17,70 provided that the nonhepatic splanchnic contri­ bution to plasma GSH is taken into account. The effect of acute ethanol intoxication on the rates of synthesis and utilization of liver GSH has been studied after pulse-labeling of the tripeptide with its radioactive precursors 35S-cysteine15,17 or 35S-methionine. 15 The analysis of the specific activity-time curves has indicated a significant diminution in the rate of incorporation of GSH precursors into the hepatic glutathione pool. 15,17 Decreased liver GSH synthesis by ethanol was found to occur without changes in the rate of GSH utilization, as estimated by analysis of the two-com­ partment (precursor-product) model for GSH turnover. 15 In the experimental conditions used, rates of GSH synthesis and utilization can be roughly estimated as (1) tracer precursors that can be incorporated into proteins with slower turnover than GSH15 and (2) steady-state conditions that are needed for turnover measurements but are not attained in animals fasted for 18 to 24 h 15,17 because they exhibit a decline in hepatic GSH content. 7 Although the inhibition of the hepatic synthesis of GSH by ethanol might be due to a decreased availability of cysteine, its level has been found to be unchanged15 or only slightly diminished17 in the liver of ethanol-treated rats. In the latter case, it was calculated that the decrease in cysteine could account for only 17% of the total loss of liver GSH after 4 and 5 h of ethanol intoxication. 17 Thus, the mechanisms by which acute ethanol ingestion leads to an inhibition of liver GSH synthesis remain to be studied. Possible actions of ethanol and/or acetaldehyde could include (1) inhibition of the enzymes of GSH synthesis, namely, 7 -glutamyIcysteine and GSH synthetases and (2) reduction in the content of the intermediate 7 -glutamylcysteine either by conjugation with acetaldehyde or by enhanced utilization by 7 -glutamylcyclotransferase.71

III. EFFECT OF PROLONGED ETHANOL INGESTION ON HEPATIC GSH IN EXPERIMENTAL ANIMALS AND MAN The study of the effect of chronic ethanol administration to experimental animals (rat, baboon) on the hepatic content of GSH has produced conflicting results, with decreases, 11,13,72 increases, 74 80 or no changes81 being reported. The discrepancies observed could be ascribed to differences in the experimental designs used. Chronic alcohol treatment was carried out for 2 to 23 weeks (in rat studies) using either Leiber-de Carli liquid diets or 20% ethanol


Glutathione: Metabolism and Physiological Functions

in the drinking fluid, and determinations were made using different methods (e.g., in in­ toxicated animals or after ethanol withdrawal, under fed or overnight fasting condi­ tions) . 11,13,72 81 Concomitantly, with the assessment of the effect of chronic ethanol consumption on hepatic GSH, an enhancement has been reported in the activities of the enzymes involved both in the synthesis (7 -glutamylcysteine and GSH synthetases) 17,19 and degradation (7 -glutamyl transferase [7 -GT] ) 11,79 of the tripeptide. The increase in liver 7 -GT activity with chronic ethanol ingestion has been shown to be the result of enzyme induction at micro­ somal82"85 and plasma membrane80 levels. These results are in agreement with the increased glutathione turnover79 and sinusoidal efflux of GSH73,81 after chronic alcohol ingestion, changes that were similar in magnitude and not accounted for by the enhancement in 7 -GT activity. 81 It has been established that the rate of GSH efflux from the liver into plasma approximates (90 to 95%) the turnover rate of hepatic GSH and, therefore, that of GSH synthesis, while 5 to 10% of the rate of GSH utilization is due to conjugation reactions, degradation via the 7 -glutamyl cycle and oxidation to GSSG. 7 Thus, if chronic ethanol ingestion increases both liver GSH synthesis as suggested, 11,79,86 and sinusoidal GSH efflux73,81 to a similar extent, a lower steady-state level of hepatocytic GSH would be established, as reported by some authors. 11,13,72,73 Moreover, the increases in the activity of hepatic glutathione-5-transferases , 75,77,78 glutathione peroxidase , 77,87 7 -GT , 11,79,80,182-85 and in lipid peroxidation13,62,76,77,87'92 reported after chronic ethanol intake would also contribute to a diminished GSH level in the liver tissue. Although the increased hepatic GSH content observed following chronic alcohol treatment74' 80 was suggested to reflect an adaptive change against ethanol-induced lipid peroxidation,77 the mechanisms responsible for this effect of ethanol remain uncertain. Studies of liver biopsy samples from alcoholic patients have indicated a decreased content of hepatic GSH93 96 when compared to values obtained in nonalcoholics93,95,96 or reported references values.97 This change was found to be unrelated to the nutritional status of the patients93 and influenced by the length of abstinence and the presence of liver necrosis. 94 In fact, after prolonged alcohol withdrawal, the hepatic GSH content was higher in patients without liver necrosis than in those with necrosis. Since fibrosis did not influence the positive linear correlation between liver GSH levels and the period of abstinence, it was suggested that the alcohol diminution of liver GSH is related more to the acute hepatocellular damage than to its late consequences.94 Liver GSH depletion observed in alcoholic patients occurs concomitantly with an enhanced free-radical prooxidative activity, with elevated indexes of lipid peroxidation being reported in the liver tissue,93,98 serum,99 103 and breath. 104 Reduction of hepatic GSH and enhancement of cellular free-radical activity are evidence for the de­ velopment of an oxidative stress condition in the liver of alcoholic subjects, as found in experimental animals,2,4,26 which may constitute an important mechanism in the pathogenesis of alcoholic liver cell necrosis and in the progression and persistence of alcoholic liver disease. The oxidative stress phenomenon could be exacerbated by contributory factors related to prolonged alcohol consumption such as ( 1 ) induction of liver microsomal enzymes associated with free-radical and H20 2 generation, 105 (2) induction of the hepatic GSH degradating enzyme 7 -GT, 106 (3) hepatic accumulation of iron, 107 a powerful lipid peroxidation inducer, and (4) nutritional depletion. The latter factor could involve a decreased ingestion and/or absorption of essential nutrients, which would limit the availability of the cellular antioxidant vitamins E 102,108 and A , 109 the precursors for GSH biosynthesis, trace elements such as selenium102,108,110' 112 (constituent of glutathione peroxidase) and zinc108 (constituent of superoxide dismutase), and possibly other molecules needed for repair of cellular com­ ponents altered by oxidative damage.6

IV. CONCLUDING REMARKS The experimental data presented in this work indicate that ethanol ingestion elicits a

63 significant diminution in hepatic GSH. The mechanisms involved in this action of ethanol include, in acute models, the conjugation of acetaldehyde with GSH or its precursor cysteine, GSH oxidation due to ethanol-induced oxidative stress, efflux of GSH into blood, and depressed GSH synthesis. In experimental animals, chronic alcohol consumption has pro­ duced conflicting results. However, an enhanced glutathione turnover in the liver tissue might lead to a lower steady-state content of GSH, as found in liver biopsies from alcoholic subjects. The steady-state content of GSH and the rates of GSH synthesis and turnover seem to determine the availability of the tripeptide in the liver tissue.7 81 A low availability of GSH would be crucial in determining the extent of hepatic damage produced by prolonged alcohol intake, known to have a lipid peroxidative response, 2,4,26 or by drugs subjected to activation to reactive electrophilic intermediates such as acetaminophen113 and other hepatotoxins. 114,115 Although the effect of ethanol in decreasing GSH is quantitatively more important in the liver, it has also been observed in the kidney, 11,76 heart, 11 brain, 11,116 and erythrocytes70 to a minor extent. However, the mechanisms involved and the relevance of these findings remain to be determined.

ACKNOWLEDGMENTS Studies of Luis A. Videla presented in this work were supported by grants B-1860 from Departamento Técnico de Investigación, Universidad de Chile, and by a grant for sabbatical visits from Dirección General de Investigación Científica y Técnica of Spain.

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69 Chapter


GLUTATHIONE IN PROKARYOTES Gerald L. Newton and Robert C. Fahey


Introduction............................................................................................................... 70


Thiols in Prokaryotes............................................................................................... 70


Evolution of Glutathione Biosynthesis.................................................................... 73


Why Glutathione?..................................................................................................... 75


Conclusions............................................................................................................... 76

Acknowledgments................................................................................................................ 76 References............................................................................................................................. 76


Glutathione: Metabolism and Physiological Functions

I. INTRODUCTION There is now a very substantial body of evidence showing that glutathione (GSH) plays a key role in protecting cells against oxidative challenge and toxic substances. 12 Until recently it was widely considered that glutathione is ubiquitous among living organisms and essential for life. However, most of the data on glutathione metabolism has come from animal studies and much less is known about glutathione in plants, fungi, and microorganisms. In 1978 we reported the results of an initial survey of GSH in bacteria which indicated that many species of bacteria, including both aerobes and anaerobes, do not make glutathione. 3 The bacteria did contain other nonprotein thiols but these could not be identified at the time. Since there must have been extensive diversification among prokaryotes prior to the evolution of the cyanobacteria and the subsequent accumulation of oxygen in the atmosphere, a study of the thiols produced by widely divergent bacterial species promised to provide insights into the factors which led to GSH playing such a dominant role in eukaryotes. In the past decade we have developed methods based upon fluorescent labeling of thiols followed by separation using high-performance liquid chromatography (HPLC) which permits the specific determination of a wide range of thiols of biological interest, including GSH.4 These have been used to survey the thiols produced by a broad selection of prokaryotes. 5,6 We summarize here the results of these studies, including results not previously published, and attempt to assess their implications for the evolution and function of glutathione.

II. THIOLS IN PROKARYOTES In assessing whether a given species of bacteria produces glutathione or not, care must be taken to evaluate the conditions under which it is cultured. If the growth medium itself contains glutathione, then a false positive identification of glutathione associated with the organism can result. This has proven to be the case with some bacilli which can accumulate significant levels of GSH from the medium and even appear to produce a glutathione reductase. 7 However, this glutathione reductase activity represents only a nonspecific action of a more general disulfide reductase and no GSH is produced when the organism is cultured on glutathione-free medium. 3,8 The first step in conducting a survey of prokaryotes is to establish a basis for choosing the species to be studied. Traditionally, bacteria have been classified according to the Gram test as Gram-positive or Gram-negative. Our current information on the low molecular weight thiol composition of Gram-positive bacteria is summarized in Table 1 and that for Gramnegative bacteria in Table 2. Ribosomal RNA sequence data provide an objective basis for the more definitive classification of bacteria and allow the construction of a tree describing the evolutionary descent of contemporary bacterial species.9,10 Two major kingdoms of bacteria, the archaebacteria and the eubacteria have been defined by such studies, and a third kingdom, the eukaryotic cytoplasms, has been defined through such studies. We will utilize here the elaboration of this scheme by Stackebrandt11 in which the arachaebacteria are subdivided into two main lines of descent or groups, and the eubacteria are subdivided into 11 distinct groups. In Tables 1 and 2 we have indicated the group to which the bacteria are assigned based upon RNA sequence information. First we will consider the eubacteria, starting with group 1, the Gram-positive bacteria. Although GSH and enzymes of glutathione metabolism have been the subject of occasional reports for Gram-positive bacteria, there is no decisive evidence that any of these bacteria synthesize GSH. We have examined nine members of the group 1 eubacteria, including representative of each of the two subgroups, the “ Clostridium” and “ Actinomyces” subgroups, using HPLC methods to specifically identify GSH and taking care to grow the bacteria on glutathione-free media (Table 1). GSH was absent in all cases. In our earliest

71 TABLE 1 Major Thiols in Gram-Positive Eubacteria pmol/g residual dry weight*

o2 Group Dep Micrococcus roseus Streptomyces griseus Streptococcus mutans Arthrobacter globiformis Bacillus cereus B. subtilis Staphylococcus aureus Clostridium pasteurianum C. kluyveri Deinococcus radiodurans

1A 1A 1A 1A 1C 1C 1C 1C 1C 2

Ae Ae Ae Ae F F F An An Ae


y GC


SS 0 3

< 0.1 < 0.1 0.08

< 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1

< 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1

< 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1

0 .1 0

0.14 0.07 0.16 < 0.1 < 0.1 < 0.1

CoA 1.4 2

H2S 0.56

U25-6 U25-6 0 .6 — Erg-4 1.0 0.18 P-0.12 0.3 HC-0.11 0.18 U19-0.4 < 0.1 U60-3.5 U60-1.7 1.8 0.44 1.0

1.4 0.8

0.18 ND 2



Ref. 5 5 __b __c 5 __d __e 3 3 5

Note: Ae, aerobic; F, facultative anaerobe; Mi, microaerophilic; An, anaerobic; Cys, cysteine; 7 -GC, 7 -glutamyl cysteine; GSH, glutathione; S S 03, thiosulfate; CoA, coenzyme A; P, pantetheine; U, unknown; Erg, ergothioneine; HC, homocysteine; and ND, not determined. a b c d e

Approximately 80% of the total dry weight. See Reference 6 for extraction procedure. Strain GS-5 was obtained from E. Thomas (St. Jude Children’s Hospital, Memphis, TN) and was cultured at 37°C on Todd-Hewitt media (Difco) that was specifically depleted of GSH . 30 ATCC Strain 8010 was cultured on Nutrient Broth (Difco) at 26°C. Strain 36.1 was obtained from W. Loomis (University of California, San Diego). ATCC strain 25923 was cultured on Trypticase Soy Broth (BBL) at 37°C.

studies using an enzymatic assay for determining GSH we did see substantial levels of GSH in Streptococcus agalactiae and S. lactis, but these results require verification using HPLC methods before they can be considered a valid exception. Generally, the Gram-positive bacteria accumulate Coenzyme A (CoA) at millimolar levels, and a disulfide reductase capable of reducing CoA disulfide and related disulfides is produced by bacilli. 12 The group 1 bacteria also produce several unidentified thiols, thiols with retention times on HPLC that do not correspond to known standard thiols. These unknown thiols are tabulated with the identification “ U” followed by the retention time observed using HPLC “ method 1” as described by Fahey and Newton.4 The major thiol in the two species of Clostridia analyzed by HPLC is the unknown U-60, and the unknown thiol U-25 is produced in large amounts by Micrococcus roseus and Streptomyces griseus. An unusual thiol, ergothioneine (the betaine of 2-mercaptohistidine) was found to be the major thiol in Arthrobacter globiformis (Table 1). This thiol has also been reported to be present in many species of the Gram-positive Mycoplasma13 and the fungus Neurospora crassa.14 The group 2 eubacteria are a small group consisting of the genera Deinococcus. This group is represented by D. radiodurans (formerly Micrococcus radiodurans) which produces no GSH but, like the group 1 eubacteria, accumulates large amounts of CoA (Table 1). The cyanobacteria constitute group 3 of the eubacteria (Table 2). They are the only prokaryotes capable of oxygenic photosynthesis and are considered to be the precursors of the chloroplasts of plants. 1516 All nine species studied, which include species of the genera Anacystis, Anabaena, Plectonema, Synechococcus, Microcoleus, and Oscillatoria produce significant levels of GSH (Table 1). In addition glutathione reductase has been purified to homogeneity from Anabaena sp. strain 711917 and from Spirulina maxima.1* Group 4 is comprised mainly of the taxa Bacteroides, Cytophage, and Flavobacterium and constitutes a major line of the eubacteria including both aerobic and anaerobic species. The only representative examined thus far for thiol content is B. fragilis, an anaerobe which was found not to produce GSH (Table 2).


TABLE 2 Major Thiols in Gram-Negative Eubacteria and Archaebacteria

Group Dep Eubacteria Cyanobacteria (9 species) Bacteriodes fragilis Spirochaeta halophila Chloroflexus aurantiacus Chlorobium thiosulfatophilum Rhizobium leguminosarum R. phaseoli R. trifolii Photosynthetic purple (3 species) Escherichia coli B Psuedomonas fluorescens Chromatium vinosum D Archaebacteria Halobacteria (6 species) Sulfolobus acidocaldarius Note:

a b c d e f 8

3 4 5 9 10 11a 11a 11a lla ,ß II 7 II7 II 7 1



Ae An F F An Ae Ae Ae F F Ae Mi

c6 » C3, whereas the order C3 > > C6 > C5is proposed for acceptor-substitutedp-benzoquinones.7The addition of GSH to a /7-benzoquinone proceeds far more rapidly (2 x 106 m _1 s-1)9 than that to 1,4-naphthoquinones, which may be expected to be about 104-fold slower. The rate of GSH addition to /?-benzoquinones is inversely related to the number of -CH3 substituents, being nondetectable for duroquinone.9


B. 1,4-NAPHTHOQUINONES For the case of 1,4-naphthoquinones, the addition is only possible at C2 and/or C3 in the quinoid ring, but the position and rate of formation of the thioether is dependent on whether the substituent is present in either the benzenoid or the quinoid ring. 1. Quinoid Ring-Substituted Naphthoquinones Quinoid ring-substituted 1,4-naphthoquinones reacts with GSH at substantially lower rates; for the case of menadione the rate was calculated as 28 M -1s -1 (at pH 9.3)10 and, in general, substituents in the quinoid ring, regardless of their electron-donating or electronwithdrawing properties seem to decrease the rate of addition.10 An electron-donating sub­ stituent at C2, such as an -OH group in the case of 2-hydroxy-1,4-naphthoquinone or lawsone, prevents nucleophilic addition.11


Glutathione: Metabolism and Physiological Functions


2. Benzenoid Ring-Substituted Naphthoquinones Benzenoid-ring, donor-substituted 1,4-naphthoquinones at C5 (Reaction 4) or C6 (Re­ action 5) determine C2 or C3 as the most preferable sites for nucleophilic attack, respectively, with acceptor-substituted compounds at these positions having the opposite effect.7 At var­ iance with an -OH substituent in the quinoid ring, an -OH group in the benzenoid ring, as in the case of 5-hydroxy-1,4-naphthoquinone or juglone, does not prevent nucleophilic addition. This substitution pattern was proposed, on rather ambiguous grounds, to determine C3 as the site for sulfur addition; this, of course, would be an exception in the nucleophilic additions to juglone, which otherwise are expected to take place at C2 (Reaction 4).7



C. QUINONE EPOXIDES Quinone epoxides, formed during the HOO“ addition to the -C 2=C3- double bond1213 undergo nucleophilic addition at slower rates than the parent quinones; these rates are further diminished upon -CH3 substitution of quinone epoxides. The reductive addition to quinone epoxides involves formal rearomatization of the quinoid ring followed by epoxide ring opening.14 For the case of p-benzoquinone and 1,4-naphthoquinone epoxides, the primary product is a hydroxy-glutathionyl-hydroquinone;1415 the substituents are located at opposite sides in p-benzoquinones (Reaction 6), whereas the -OH substituent is vicinal to the glu­ tathionyl substituent in 1,4-naphthoquinones (Reaction 7). The mechanism for the reductive addition to quinone epoxides is sequentially similar to their reduction via two electrons mediated by DT-diaphorase,16 except that in the latter case the product is a hydroxy-hydroquinone. The subsequent chemistry of hydroxy- and hydroxy-glutathionyl-hydroquinones is described below.



283 D. EFFECT OF THE GLUTATHIONYL SUBSTITUENT ON THE REDUCTION POTENTIAL OF THE QUINONE The glutathionyl substituent is expected to exert only minor changes in the one-electron reduction potentials [E(Q /Q“)] values of quinones, in agreement with the weak electronwithdrawing properties of the thioether substituents,17 which have Hammet constants op « 0 and am ~ 0.1 to 0.2. Accordingly, the glutathionyl substituent raises slightly the oneelectron reduction potential values relative to that of menadione by about 11 mV.17 Although no one-electron reduction potential values for glutathionyl-p-benzoquinones are available, similar minor changes as observed with 1,4-naphthoquinones17 are expected. However, the half-wave reduction potential (E1/2) values of glutathionyl quinones are more negative than the parent compounds and the glutathionyl substitution of menadione lowers the E1/2 value by 45 mV.18 The aryl-thio substituent decreases the E1/2 values of unsubstituted- and methyl-substituted p-benzoquinone, but it raises the reduction potential value of 2,6-dimethyl-p-benzoquinone, probably by preventing the overall polar effect of the substituents to be exerted.19

III. SUBSEQUENT CHEMICAL REACTIVITY OF GLUTATHIONYL-HYDROQUINONE CONJUGATES The primary product resulting from the thiol addition to quinoid compounds is in every case a thioether derivative, customarily named for the case of GSH, glutathionyl-hydroquinone conjugate (Reactions 1 to 7). The subsequent chemical reactivity of these thioether derivatives is by a large extent determined by the changes in the reduction potentials of the molecule, the presence of substituents, and environmental factors, such as medium polarity, pH, solvent cage, and solvation energy. The possible reactions to which glutathionyl-hydroquinones are subjected include cross oxidation, autoxidation, disproportionation, oxidative elimination, diconju­ gation, free radical interactions, and enzymic reduction. A. CROSS OXIDATION, AUTOXIDATION, AND DISPROPORTIONATION The overall oxidation of the conjugate, RS-QH2 —* RS-Q, occurs through one-electron transfer reactions by means of different sequences comprised in cross oxidation, autoxidation, and disproportionation. The variables controlling these redox transitions are complex and a single mechanism for the oxidation of the glutathionyl-hydroquinone conjugate can be ruled out. Evidence for one-electron transfer reactions is obtained by the observation with the ESR technique of a glutathionyl-semiquinone adduct of p-benzo- and 1,4-naphthoquinones; the occurrence of the transient semiquinone species was accounted for in terms of cross­ oxidation reactions.20 The prevalence of either reaction is determined by the relative concentrations and the reduction potential of, on the one hand, the thioether derivative and, on the other, the parent quinone and 0 2/0 2*~ couple. The former reduction potential is an expression of the overall effect exerted by the substituents including those involved in the thioether linkage. Scheme I gives an overall view on these possible redox interactions, which provide the general trend for the sequences involved in the oxidation of the thioether: RS-QH2—> RS-Q; these sequences are mainly determined by the type of quinone involved.


Glutathione: Metabolism and Physiological Functions


One-electron transfer reactions of glutathionyl-hydroquinone conjugates.

1. Reductive Addition —> Cross Oxidation -» Disproportionation p-Benzo- and 1,4-naphthoquinone undergo sulfur nucleophilic additions at relatively high rates and this process does not represent a limiting step for the following redox tran­ sitions. The conversion R S -Q H 2 —» R S -Q is likely to be accomplished by the sequence cross oxidation —» disproportionation on the following accounts: 1.


The thiol substituent exerts only minor changes in the reduction potential of unsubstituted- or methyl-substituted quinones17 therefore, it can be expected that the redox chemistry of the former will not be substantially different from that of the latter. However, the E1/2 values of glutathionyl-substituted p - benzo-19 and 1,4naphthoquinones11’13’14’18 are up to 40 mV more negative than their parent compounds, thus favoring electron transfer from the former to the latter, i.e., cross oxidation, as observed for the case of thiol-substituted p-benzo-19 and 1,4-naphthoquinones.11 The subsequent RS-Q*- —» RS-Q transition is plausibly accomplished through dis­ proportionation rather than autoxidation on the following grounds. First, the secondorder rate constant for the disproportionation reaction is about 108 M “ 1 s~1for several /7-benzo- and 1,4-naphthosemiquinones.21 Second, this rate is either several orders of magnitude higher than the rate of electron transfer to 0 2, for the case of /7-benzosemiquinone, or at least equal to the rate of electron transfer to 0 2 for the case of 1,4naphthosemiquinone. Third, the equilibrium of the autoxidation reaction (Q*~ + 0 2 Q + Q2‘ ) is substantially displaced either towards the left for p-benzosemiquinone or the forward and backward reaction have similar second-order rate constants as for 1,4-naphthosemiquinones. Lastly, but not least, the spontaneous disproportionation of 0 2~, which might have been the driving force favoring the autoxidation reaction, proceeds at a rate slower than either rate comprised in the autoxidation process. Thus, significant autoxidation of /7-benzosemiquinones is observed only in the presence of superoxide dismutase,22 which displaces the equilibrium of the above autoxidation reaction towards the right. At an experimental level superoxide dismutase was found to enhance the overall autoxidation of glutathionyl-/?-benzohydroquinones,15 a process probably supported by a similar mechanism.

2. Reductive Addition —» Autoxidation —» Autoxidation The reduction potential of hydroxyquinones with an -OH substituent in the benzenoid ring is slightly more positive than that of the parent compound,23'25 whereas that of hy­ droxyquinones with an -OH substituent in the quinoid ring is 200 to 300 mV more negative than the parent compound.26 The presence of a glutathionyl substituent in hydroxyquinones

285 is likely not to affect significantly these values and the subsequent chemistry. The -OH substituent in the benzenoid ring of naphthoquinones stabilizes the semiquinone species by displacing the equilibrium of the disproportionation reaction towards the left [2 Q*~ Q* + QH2];27 it could be expected, therefore, that disproportionation would contribute negligibly to the RS-Q*- —» RS-Q transition. Hydroxysemiquinones with the -OH substituent in the quinoid ring have a very negative reduction potential and autoxidize rapidly. At an exper­ imental level, the DT-diaphorase-catalyzed two-electron reduction of 2-hydroxy- and 5hydroxy-1,4-naphthoquinone was accompanied by a 6- to 12-fold higher autoxidation than that of the parent compound lacking the -OH substituent.18 Thus, autoxidation of hydroxyhydroquinones is a favored mechanism regardless of the position of the -OH substituent in the compound. 3. Reductive Addition —» Cross Oxidation —> Autoxidation The reductive addition of GSH to quinone epoxides, either of the p-benzo- or 1,4naphthoquinone series, is slower than that to the parent quinones.14 This facilitates cross­ oxidation reactions of the resulting hydroxy-glutathionyl-hydroquinone primary product with unconjugated quinone. The sequence RS-Q*- —» RS-Q, however, is mediated by autoxi­ dation rather than disproportionation for the reasons outlined above, that is, once the hydroxysemiquinone is formed by overcoming the limiting steps, its autoxidation prevails substan­ tially over its disproportionation. B. OXIDATIVE ELIMINATION Oxidative elimination reactions are limited to those hydroquinones bearing a good leaving group, e.g., -OH, -OCOCH3, -Cl, and -Br, and are influenced by the electron density of the hydroquinone nucleus, i.e., its reduction potential. Oxidative elimination is involved in the chemistry subsequent to the GSH reductive addition to naphthoquinone epoxides,14 in the molecular mechanism underlying the vitamin K epoxide reductase activity,28 and in the enzymic reduction of or reductive addition to bioreductive alkylating agents.10 29 The primary product following the reductive addition of GSH to 1,4-naphthoquinone epoxide (Reaction 7) undergoes oxidative elimination (Reaction 8) with formation of glu­ tathionyl-1,4-naphthoquinone. This accounts for about 30% of the molecular product dis­ tribution observed, whereas oxidative elimination is the only reaction following the reductive addition to 2,3-epoxy-2-methy 1-1,4-naphthoquinone or menadione epoxide (Reaction 9). Thus, 2-methyl-3-glutathionyl- 1,4-naphthoquinone is the common end-stable molecular product following the GSH reductive addition to menadione (Reaction 3) and menadione epoxide (Reaction 9).



The thiol addition to dimethyl-substituted 1,4-naphthoquinone epoxide, as a model for vitamin K, also involves thiol addition to open the epoxide ring, yielding a 2-thio-3-hydroxy adduct. Reductive cleavage of this adduct by a second thiolate anion and elimination of H20 yields the corresponding quinone.28 The term bioreductive alkylation is used to define a mechanism involving three sequential


Glutathione: Metabolism and Physiological Functions

steps: (1) two-electron reduction of a quinone bearing a leaving group, (2) oxidative elim­ ination of the leaving group with formation of a quinone methide, and (3) electrophilic addition to a cellular target.29 The requirement of a two-electron reduction of quinones with leaving groups for the subsequent oxidative elimination is encountered, for instance, by the two-electron transfer flavoprotein DT-diaphorase.30 An alternative mechanism might involve the two-electron reductive addition with thiols,10 although the process is slowed down considerably if the leaving group is in the quinoid ring of 1,4-naphthoquinones. The GSH addition to a quinone with a leaving group results in the formation of a thioether derivative of the quinone. Following the oxidative elimination process, the sulfur-substituted quinone methide can hypothetically undergo a second nu­ cleophilic addition at the exocyclic methide moiety to yield a diconjugate. The overall process may be regarded as involving sequential intra- and extra-ring conjugation, as illustrated in Reaction 10.

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C. DICONJUGATION The oxidized thiol-quinone conjugate, formed by any of the mechanisms for RS-QH2 —» RS-Q outlined in Sections A and B, could theoretically undergo a second conjugation reaction, provided that it is not hindered by either the overall polar or the steric effect of the substituents. For example, un- and mono-methyl-substituted p-benzoquinone and un­ substituted 1,4-naphthoquinone are prone to undergo a second conjugation, though it is expected that the second reaction would proceed slower than the first. Glutathionyl-methyl1,4-naphthoquinone (product in Reaction 9) or glutathionyl-hydroxy-/?-benzoquinone (prod­ uct in Reaction 6) do not undergo second additions, in the latter case being unfavored by the electron-donating properties of the -OH substituent — located on one side of the ring — and by the steric hindrance created by the glutathionyl moiety located on the opposite side of the ring. D. FREE RADICAL INTERACTIONS On thermodynamical grounds, semiquinones with a reduction potential between +460 and —330 mV can reduce H20 2 to a hydroxyl radical.31 Experimental evidence for this reaction has been provided with ESR using the spin-trapping technique,32 33 although coun­ terevidence has also been brought forward.34 Given the similarity in the redox properties of quinones and their glutathionyl adducts, it may be expected that the latter will participate in similar reactions. The oxidation of certain semiquinones by HO* yields an excited quinone of triplet multiplicity as evidenced by spectral analysis of photoemission.12 The role of glutathionylhydroquinone derivatives in this regard seems to be related to the deactivation of the triplet state by either (1) electron transfer from the glutathionyl-hydroquinone to triplet quinone [RS-QH2 4 Q* —» RS-QH* 4 QH*] or (2) deactivation of the excited state by the S-C bond, involving energy transfer [RS-QH2 4 Q* —» RS-QH2 4 -






1 2 ,3 5

287 E. ENZYMIC REDUCTION OF GLUTATHIONE-QUINONE CONJUGATES Glutathione conjugates of 1,4-naphthoquinone, 2-methyl-1,4-naphthoquinone (mena­ dione), 5-hydroxy-1,4-naphthoquinone (juglone), and 2-methyl-5-hydroxy-1,4-naphthoqui­ none (plumbagin) can be reduced efficiently via two electrons by the flavoprotein DTdiaphorase (Reactions 11 and 12) . 18 The rate of reduction of the respective glutathionyl quinone derivatives by DT-diaphorase is about 30 to 40% lower than that of the parent compounds. However, autoxidation of the two-electron reduced glutathionyl derivatives is 1 2 - to 16-fold higher than the two-electron reduced quinones lacking a glutathionyl substi­ tuent.

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IV. ELECTROCHEMICAL EVALUATION OF QUINONEGLUTATHIONE CONJUGATES High-performance liquid chromatography (HPLC) with electrochemical detection allows the identification and quantification of minute amounts of quinones . 36 This technique is a useful tool for the evaluation of quinone metabolism at a cellular level. 37 Substances that have the property to participate in redox reactions, i.e., electroreducible and electrooxidizable compounds, are suitable for detection in column effluents at very low concentrations by selective electrochemical detection measurements. HPLC with electro­ chemical detection is an amperometric determination, which involves heterogenous electron transfer from one phase to another phase. The electrochemical detector is composed of a three-pole potentiostat system. On the basis of the potential of a reference electrode, constant potential electrolysis is given between the working and the auxiliary electrodes. This current is measured for finding the concen­ tration of the electrochemically active material. The electrode measures current at a fixed potential, which is subsequently converted to a voltage capable of driving a recorder. Electron transfer from the solute to the electrode surface is known as oxidative electro­ chemical detection, whereas electron transfer from the electrode surface to the solute is known as reductive electrochemical detection. The former case requires a positive applied potential, whereas the latter case requires a negative applied potential. At fixed-specific potentials, electrochemical detection makes it possible to detect some substances which are reduced or oxidized at that potential, while others, which have lower reducing or oxidizing potentials, remain undetected. Glutathionyl-quinone conjugates retain the redox properties of the parent quinone com­ pounds and, therefore, they can be analyzed by HPLC with electrochemical detection . 12,14 The analysis of glutathionyl-quinone conjugates does not require derivatization or prolonged incubations as otherwise necessary for the identification of glutathionyl conjugates of com­ pounds lacking redox properties. The thioether derivative is of a more polar character than the parent quinone and it is eluted with much shorter retention times when analyzed by reversed-phase HPLC. The separation of these conjugates requires the use of buffered mobile phases, containing substances capable of ion-pair formation with charged groups on the tripeptide.


Glutathione: Metabolism and Physiological Functions

FIGURE 1. Hydrodynamic voltamogram of 2-methyl-1,4-naphthoquinone and 2-methyl-3-glutathionyl- 1,4naphthoquinone. Data from References 13 and 14.

As with quinones, the redox state of quinone-glutathionyl conjugates can be evaluated by the use of either reductive- or oxidative-electrochemical detection . 1214 The E 1/2 values of quinone-glutathionyl conjugates can be obtained by means of hydrodynamic voltamograms, i.e., the midpoint value obtained through current against applied potential plots (Figure 1). These values are useful to predict possible redox interactions that quinoneglutathionyl conjugates can undergo. The glutathionyl substituent lowers slightly the E 1/2 of quinones by about 40 to 45 mV. For example the E 1/2 values, analyzed with reductive electrochemical detection, for 1,4-naphthoquinone, 2-methyl-1,4-naphthoquinone, and 5hydroxy-1,4-naphthoquinone are - 1 8 0 , -2 2 5 , and - 1 4 0 mV, respectively, and their corresponding glutathionyl derivatives possess E 1/2 values of -2 2 5 , -2 6 5 , and —195 mV, respectively . 18 Glutathionyl-p-benzohydroquinone, analyzed with oxidative electrochemical detection, shows a E 1/2 value of + 220 mV, 20 mV more positive than the parent compound (E 1/2 = + 200 mV ) . 12 The changes in E 1/2 values exerted by the glutathionyl substituent are exemplified for menadione and menadione-glutathione conjugate in the hydrodynamic voltamograms in Figure 1.

V. BIOLOGICAL IMPLICATIONS OF ARYLATION REACTIONS The biological implications of the bimolecular reaction of electrophilic quinones with cellular sulfur nucleophiles is of a critical character, and leads to perturbations of biochemical pathways and, eventually, to the expression of cytotoxicity. The following considerations need to be taken into account when analyzing the influence of this critical bimolecular reaction on cellular metabolism.

A. THIOL DEPLETION AS THE BASIS FOR CYTOTOXICITY Given the cellular distribution of GSH, the reductive addition reaction should take place in the hydrophilic milieu, whereas the electrophilic counterpart — the quinoid compound — depending on its partition coefficient, tends to be largely distributed in the hydrophobic milieu. The partition coefficient of quinoids has been shown to correlate well with their cytotoxicity . 38 GSH may serve as a buffer system, which protects against and precedes the electrophilic

289 attack of quinones to more vital cellular targets such as DNA . 38 Thus, at an experimental level, formation of glutathione-quinone conjugates in isolated hepatocytes has been reported to precede the onset of toxicity . 10’39,40 On the one hand, the cellular concentration of GSH is usually high and depletion of the thiol upon reductive addition with electrophilic quinones seems an unlikely single mechanism leading to the expression of cytotoxicity. On the other hand, the cell is a heterogenous system and depletion of GSH within defined microenvironments can easily lead to overall changes in metabolism without altering significantly the total GSH concentration.

B. CELLULAR EXPRESSION OF THE REDOX CHEMISTRY OF GLUTATHIONYL-QUINONE CONJUGATES The 1,4-reductive addition of GSH to quinones is formally a two-electron transfer process. The glutathionyl-hydroquinone conjugates are endowed with a particular chemistry, which has been briefly outlined in Section III. These chemical reactions could be considered as entailing a proxidant character subsequent to the bimolecular reaction between quinones and GSH. Hydroquinones, whether conjugated with GSH or not, are furnished with a less elec­ trophilic character than the parent quinones. However, hydroquinones are not redox-inert compounds and their apparent stability is due to their protonated state at physiological pH; it is known that the protonated compounds are less prone to undergo electron transfer reactions than their anionic counterparts.41 For example, hydroquinones substituted with electrondonating groups are not more stable than semiquinones, in spite of the radical character of the latter, and participate readily in redox interactions. Therefore, the effect of substituents on the hydroquinone redox chemistry is of crucial importance. The glutathionyl substituent, though slightly altering the redox properties of the quinone, exerts changes that involve an at least eightfold higher rate of autoxidation than the parent hydroquinones . 1518 This reflects only an apparent discrepancy with the sequence reductive addition —» cross oxidation —> disproportionation described in Section III.A. 1. The latter sequence is supposed to be involved in the initial steps following the addition of GSH to the quinone and, as the reaction progresses, the uneven distribution of organic reactants and relatively high concentration of 0 2 shift the sequence reductive addition —> autoxidation —> disproportionation towards autoxidation. If autoxidation occurs, a second conjugation with GSH is likely to contribute to cellular thiol depletion, provided that the conditions given in Section III.D are encountered.

C. ROLE OF SUPEROXIDE DISMUTASE IN QUINONE METABOLISM It has been proposed that glutathione transferases display activities that are comple­ mentary to the activity of DT-diaphorase in the biotransformation and detoxication of elec­ trophilic compounds that may arise as products of oxidative metabolism .42 Similarly, the sequential activities of DT-diaphorase and superoxide dismutase may serve as a main de­ toxication pathway for electrophilic quinones. These complimentary activities require, on the one hand, the occurrence of organic compounds which break down with formation of semiquinones and 0 2*~, this process being brought about by DT-diaphorase and, on the other, the reduction of the semiquinones by 0 2,_ , this process being mediated by superoxide dismutase. The role of superoxide dismutase in connection with DT-diaphorase consists, therefore, of inhibition of semiquinone autoxidation by facilitating its reduction at the expense of 0 2*~. This activity has been termed superoxide:semiquinone oxidoreductase,43 its overall mechanism formulated as Q-“ + 0 2*_ + 2H + QH 2 + 0 2, and interpreted in terms of current quantum chemistry and X-crystallography informations (Reactions 13 and 14). SOD-Cu++ + 0 2-“ — * SOD-Cu+ + 0 2



Glutathione: Metabolism and Physiological Functions

( 14)

It is important that substrates for superoxide dismutase are those molecules which can break down with the formation of semiquinone radicals and 0 2*~; thus, it is expected that superoxide dismutase will be inefficient in its superoxide:semiquinone oxidoreductase func­ tion to reduce semiquinones generated during the NADPH-cytochrome P450 reductase activ­ ity. The two-electron reduction of hydroxy quinones, such as hydroxy-/?-benzoquinones, hy­ droxy-1,4-naphthoquinones bearing an -O H substituent in the benzenoid ring, and quinone epoxides, regardless of the mechanism involved in their reduction, is followed by an intense autoxidation, which is prevented by superoxide dismutase. The autoxidation of the gluta­ thionyl derivatives of these hydroxyquinones is likewise efficiently prevented by superoxide dismutase (Reaction 14). The sequence DT-diaphorase —» superoxide dismutase or sulfur reductive addition —> superoxide dismutase provides, therefore, a new means for protection against quinone tox­ icity .43 Of note, not all semiquinones can be reduced at the expense of 0 2*~ within a reaction involving superoxide dismutase; the autoxidation of unsubstituted and methyl-substituted pbenzoquinones is enhanced by superoxide dismutase, because the enzyme displaces the equilibrium of the autoxidation reaction [Q,_ + 0 2 ^ Q + 0 2,_] towards the right. In summary, there is an apparent discrepancy on the overall effect of superoxide dis­ mutase on reduced quinone autoxidation: ( 1 ) the enzyme enhances autoxidation of those quinones that have little tendency to autoxidize, be it because of their high reduction potential or the weak polar effect of substituents; in general, these quinones have one-electron reduction potential values for the intermediate step [E(Q*- /Q2 -)] that span between +473 and -1-426 mV and (2) superoxide dismutase inhibits the autoxidation of those reduced quinones, which otherwise tend to participate in electron-transfer reactions at high rates; in general, these are quinones with a substantially more negative reduction potential, [E(Q*~/Q2 -)], contributed by the strong inductive effects of their electron-donating substituents, whose values span between +193 and —77 mV. Another consideration regarding the effect of superoxide dismutase on the autoxidation of this second group of reduced quinones is the discrepancy between the reduction potential of the redox couples involved, which, on thermodynamical grounds, will make this reaction unfavorable. Thus, the reduction potential of the (Cu++/ Cu +) couple in superoxide dismutase is about +280 mV, whereas the [E(Q*- /Q2 -)] values for several quinones, as, for example, 1,4-naphthoquinone, are +270 mV or below. D. SULFUR REDUCTIVE ADDITION AS THE BASIS FOR QUINONE CELLULAR DISPOSAL The two-electron reduction of quinones can be accomplished by either DT-diaphorase or the sulfur nucleophilic addition. At variance with what was outlined in Section III, the two-electron reduction implied in the 1,4-reductive addition of GSH to quinones could bear an antioxidant character, for it fulfills the chemical requirements, that is, the occurrence of -O H groups in the terminal positions, to facilitate the conjugation with UDP-glucuronate by means of UDP-glucuronosy 1-transferase. Although the reaction is believed to operate in connection with the two-electron transfer flavoprotein DT-diaphorase, the sulfur nucleophilic addition similarly involves a two-electron transfer process (Scheme II).



Two-electron reduction processes and glucuronyl conjugation of hydroquinones.

DT-diaphorase may contribute to the UDP-glucuronosyl-transferase-mediated conjuga­ tion of glutathionyl-quinones by reducing the latter via two electrons [RS-Q —» RS-QH2], as recently observed 18 (Section III.E and Scheme II). It is expected that the GSH-conjugation of the quinone, which yields a compound with hydrophilic character located in the cytosol, facilitates its further conjugation with UDP-glucurononate. It remains to be determined whether the glutathionyl moiety may exert some type of steric hindrance against the subsequent formation of an ether linkage between the activated glucuronate and the -O H groups in the glutathionyl-hydroquinone molecule. The cellular disposal of glutathionyl-glucuronate-quinone conjugates seems, however, a likely means of detoxication of electrophilic quinones.

VI. GENERAL COMMENTS The addition of sulfur nucleophiles, such as GSH, to the quinoid double bond is facilitated by activation of the latter by the electron-withdrawing character of the conjugated exocyclic oxygen atoms. The nature and position of substituents present in the quinoid compound determine its electronic character and influence largely the rate of GSH addition as well as the position of the glutathionyl adduct. The effect of the glutathionyl substituent on the reduction potential of the quinoid compound is ambivalent. On the one hand, the oneelectron reduction potential [E(Q/Q- )] values are slightly raised over that of the unsubstituted


Glutathione: Metabolism and Physiological Functions

quinone , 17 44 whereas the E 1/2 values are lowered by about 40 to 50 mV relative to the parent compound. The hydroquinone-thioether derivative is liable to participate in redox reactions that comprise cross oxidation, autoxidation, disproportionation, oxidative elimination, free-rad­ ical interactions, and enzymic reduction. The prevalence of either pathway is determined by ( 1 ) the inherent physicochemical properties of the quinone, mainly its reduction potential, (2 ) the concentration of and reduction potentials of oxidants such as 0 2 (e.g., the 0 2/ 0 2-~ couple) or unreacted quinone, and (3) solvent-related factors, such as the polarity of the medium. The materialization of the bimolecular collision between an electrophilic quinone and a sulfur nucleophile will be primarily determined by the distribution of both reactants in the cellular environment. The partition coefficient of most quinones suggests that the electrophile is held in apolar matrices. Conversely, the bulk of the low molecular weight thiol, GSH, is distributed in the aqueous cytosolic milieu. This uneven distribution of reactants will apparently be the major limiting factor for the bimolecular collision to occur. However, quinone-glutathionyl conjugates are formed during the metabolism of, for example, mena­ dione in hepatocytes, as evidenced by a depletion, rather than oxidation, of GSH . 39’40 The product of this bimolecular reaction, the glutathionyl-hydroquinone conjugate, is of a rather hydrophilic character and, thus, it will display its chemical reactivity in the aqueous environment represented by the cytosol. The expression of this chemical reactivity is assumed to be influenced by the balance in the biological milieu between potential activating and detoxifying activities, including ( 1 ) the activity of cellular reductases, such as DT-diaphorase, (2 ) conjugating enzymes, such as UDP-glucuronosyl-transferase, and (3 ) the intracellular 0 2 concentration. Neither of the latter is necessarily evenly distributed in the cell and this may give rise to local alteration of this balance, which, eventually, may be expressed as an impairment of vital cell functions. A hypothetical antioxidant role of thiol addition to quinones may arise from the possibility that the glutathionyl-hydroquinone conjugates are endowed with the chemical requirements for formation of an ether linkage with activated glucuronate via UDP-glucuronosyl-transferase (Scheme II). This mechanism, which can be considered as coupled to detoxification, or disposal, of the quinones, remains to be evaluated. The likelihood of this reaction may be limited by the high rate of autoxidation of glutathionyl-hydroquinone conjugates . 15,18 The predominance in a certain environment of either autoxidation or glucuronosyl-conjugation determines whether the conjugation of quinones with GSH will entail a proxidant or an­ tioxidant character.

ACKNOWLEDGMENTS Supported by grants 7679 and 4481 from the Swedish Medical Research Council, grant 2703-B89-01XA from the Swedish Cancer Foundation, and a grant from Linköping Regionsjukhusets Forskningsfonder to Anders Brunmark.

REFERENCES 1. Miller, E. C. and Miller, J. A., Searches for ultimate chemical carcinogens and the reactions with cellular macromolecules, Cancer, 47, 2327, 1981. 2. Hanzlik, R. P., Effect of substituents on reactivity and toxicity of chemically reactive intermediates, Drug Metab. Rev., 13, 207, 1982. 3. Jocelyn, E. C., Biochemistry of the SH Group. The Occurrence, Chemical Properties, Metabolism and Biological Function of Thiols and Disulfides, Academic Press, London, 1972, 47.

293 4. Gleicher, G. J., Theoretical and general aspects, in The Chemistry of Quinonoid Compounds, Patai, S., Ed., John Wiley & Sons, London, 1974, 1. 5. Thomson, R. H., Naturally Occurring Quinones. III. Recent Advances, Chapman & Hall, London, 1987. 6. Finley, K. T., The addition and substitution chemistry of quinones, in The Chemistry of Quinonoid Compounds, Patai, S., Ed., John Wiley & Sons, London, 1974, 877. 7. Rozeboom, M. D., Tegmo-Larsson, I. M., and Houk, K. N., Frontier molecular orbital theory of substituent effects on regioselectivities of nucleophilic additions and cyclo-additions to benzoquinones and naphthoquinones, J. Org. Chem., 46, 2338, 1981. 8. Flaig, W., Beutelspacher, H., Riemer, H., and Kalke, E., Einfluss von Substituenten auf das Redox­ potential substituirter Benzochinone-(l,4), Liebigs Ann. Chem., 719, 96, 1968. 9. Rossi, L., Moore, G. A., Orrenius, S., and O’Brien, P. J., Quinone toxicity in hepatocytes without oxidative stress, Arch. Biochem. Biophys., 251, 25, 1986. 10. Wilson, I., Wardman, P., Tai-Shun, L., and Sartorelli, A. C., Reactivity of thiols towards derivatives of 2- and 6-methyl-1,4-naphthoquinone bioreductive alkylating agents, Chem. Biol. Interact., 61, 229, 1987. 11. Nickerson, W. J., Falcone, G., and Strauss, G., Studies on quinone-thioethers. I. Mechanism of formation and properties of thiodione, Biochemistry, 2, 537, 1963. 12. Brunmark, A. and Cadenas, E., Electronically-excited state generation during the reaction ofp-benzoquinone with hydrogen peroxide. Relation to product formation: 2-hydroxy- and 2,3-epoxy-p-benzoquinone. The effect of glutathione, Free Radical Biol. Med., 3, 169, 1987. 13. Brunmark, A., Cadenas, E., Lind, C., Segura-Aguilar, J., and Ernster, L., DT-diaphorase-catalyzed reduction of various p-benzoquinone- and 1,4-naphthoquinone epoxides, Free Radical Biol. Med., 5, 133, 1988. 14. Brunmark, A. and Cadenas, E., 1,4-Reductive addition of glutathione to quinone epoxides. Mechanistic studies with h.p.l.c. with electrochemical detection under aerobic and anaerobic conditions and evaluation of chemical reactivity in terms of autoxidation reactions, Free Radical Biol. Med., 6, 149, 1989. 15. Brunmark, A. and Cadenas, E., Reductive addition of glutathione to p-benzoquinone, 2-hydroxy-pbenzoquinone, and p-benzoquinone epoxides. Effect of hydroxy- and glutathionyl substituents on p-benzohydroquinone autoxidation, Chem. Biol. Interact., 68, 273, 1988. 16. Brunmark, A., Cadenas, E., Segura-Aguilar, J., Lind, C., and Ernster, L., DT-diaphorase-catalyzed reduction of quinone epoxides, Free Radical Biol. Med., 3, 181, 1987. 17. Wilson, I., Wardman, P., Tai-Shun, L., and Sartorelli, A. C., One-electron reduction potential of 2and 6-methyl-1,4-naphthoquinone bioreductive alkylating agents, J. Med. Chem., 29, 1381, 1986. 18. Buffinton, G. D., Öllinger, K., Brunmark, A., and Cadenas, E., DT-Diaphorase-catalyzed reduction of 1,4-naphthoquinone derivatives and glutathionyl-quinone conjugates. Effect of substituents on autoxi­ dation rates, Biochem. J., 257, 561, 1989. 19. Brown, E. R., Finley, K. T., and Reeves, R. L., Steric effects of vicinal substituents on redox equilibria in quinonoid compounds, J. Org. Chem., 36, 2849, 1971. 20. Gant, T. W., d’Arcy Doherty, M., Odowole, D., Sales, K. D., and Cohen, G. M., Semiquinone anion radicals formed by the reaction of quinones with glutathione or amino acids, FEBS Lett., 201, 296, 1986. 21. Meisel, D., Free energy correlation of rate constants for electron transfer between organic systems in aqueous solution, Chem. Phys. Lett., 34, 263, 1975. 22. Yamazaki, I., Free radicals in enzyme-substrate reactions, in Free Radicals in Biology, Vol. 3, Pryor, W. A., Ed., Academic Press, New York, 1977, 183. 23. Land, E. J., Mukherjee, T., Swallow, A. J., and Bruce, J. M., Reduction of the naphthazarin molecule as studied by pulse radiolysis. I. Addition of a single electron, J. Chem. Soc. Faraday Trans. 1, 79, 391, 1983. 24. Mukherjee, T., One-electron reduction of juglone (5-hydroxy-1,4-naphthoquinone): a pulse radiolysis study, Radiat. Phys. Chem., 29, 455, 1987. 25. Ashnagar, A., Bruce, J. M., Dutton, P. L., and Prince, R. C., One- and two-electron reduction of hydroxy-1,4-naphthoquinones and hydroxy-9,10-anthraquinones. The role of internal hydrogen bonding and its bearing on the redox chemistry of the anthracycline antitumour quinones, Biochem. Biophys. Acta, 801, 351, 1984. 26. d’Arcy Doherty, M., Rodgers, A., and Cohen, M. G., Mechanisms of toxicity of 2- and 5-hydroxy1,4-naphthoquinone; absence of a role for redox cycling in the toxicity of 2-hydroxy-1,4-naphthoquinone to isolated hepatocytes, J. Appl. Toxicol., 7, 123, 1987. 27. Dodd, N. J. F. and Mukherjee, T., Free radical formation from anthracycline anti-tumour. Agents and model systems. I. Model naphthoquinones and anthraquinones, Biochem. Pharmacol., 33, 379, 1984. 28. Silverman, R. B., Chemical model studies for the mechanism of vitamin K epoxide reductase, J. Am. Chem. Soc., 103, 5939, 1981. 29. Lin, J., Cosby, L. A., Shansky, C. W., and Sartorelli, A. C., Potential bioreductive alkylating agents. I. Benzoquinone derivatives, J. Med. Chem., 15, 1247, 1972.


Glutathione: Metabolism and Physiological Functions

30. Talcott, R. E., Rosenblum, M., and Levin, V. A., Possible role of DT-diaphorase in the bioactivation of antitumour quinones, Biochem. Biophys. Res. Commun., I l l , 346, 1983. 31. Koppenol, W. H. and Butler, J., Energetics of interconversion reactions of oxygen radicals, Adv. Free Radical Biol. Med., 1, 91, 1985. 32. Kalyanaraman, B., Sealy, R. C., and Sinha, B. K., An electron spin resonance study of the reduction of peroxides by anthracycline semiquinones, Biochem. Biophys. Acta, 799, 270, 1984. 33. Nohl, H. and Jordan, W., The involvement of biological quinones in the formation of hydroxyl radicals via the Haber-Weiss reaction, Bioorg. Chem., 15, 374, 1987. 34. Sushkov, D. G., Gritsan, N. P., and Weiner, L. M., Generation of hydroxyl radical during the enzymic reduction of 9 ,10-anthraquinone-2-sulfonate. Can semiquinone decompose hydrogen peroxide?, FEBS Lett., 225, 139, 1987. 35. Encinas, M. V., Lissi, E. A., and Olea, A. F., Quenching of triplet benzophenone by vitamins E and C and by sulfur-containing amino acids and peptides, Photochem. Photobiol., 42, 347, 1985. 36. Rappaport, S. M., Jin, Z. L., and Xu, X. B., High-performance liquid chromatography with reductive electrochemical detection of mutagenic nitro-substituted aromatic hydrocarbons in diesel exhausts, J. Chromatogr., 240, 145, 1982. 37. Fluck, D. S., Rappaport, S. M., Eastmond, D. A., and Smith, M. T., Conversion of 1-naphthol to naphthoquinone metabolites by rat liver microsomes: demonstration by high-performance liquid chroma­ tography with reductive electrochemical detection, Arch. Biochem. Biophys., 235, 351, 1984. 38. Powis, G., Hodnett, E. M., Santone, K. S., See, K. L., and Melder, D. C., Role of metabolism and oxidation-reduction cycling in the cytotoxicity of antitumour quinone imines and quinone diimines, Cancer Res., 47, 2363, 1987. 39. Bellomo, G., Thor, H., Eklöw-Lastbom, C., Nicotera, P., and Orrenius, S., Oxidative stress — mechanisms of toxicity, Chem. Scripta, 27A, 117, 1987. 40. Ross, D., Thor, H., Orrenius, S., and Moldéus, P., Interaction of menadione (2-methyl-1,4-naphthoquinone) with glutathione, Chem. Biol. Interact., 55, 177, 1985. 41. Stenken, S., Oxidation of phenolates and phenylenediamines by two alkanoxyl radicals produced from 1,2-dihydroxy- and l-hydroxy-2-alkoxy-alkyl radicals, J. Phys. Chem., 83, 595, 1979. 42. Mannervik, B., The roles of different classes of glutathione transferase in the detoxication of reactive products of oxidative metabolism, Chem. Scripta, 21A, 121, 1987. 43. Cadenas, E., Mira, D., Brunmark, A., Lind, C., Segura-Aguilar, J., and Ernster, L., Effect of superoxide dismutase on the autoxidation of various hydroquinones — a possible role of superoxide dismutase as a superoxide:semiquinone oxidoreductase, Free Radical Biol. Med., 5, 71, 1988. 44. Brunmark, A. and Cadenas, E., One-electron reduction of glutathionyl-naphthoquinones and rate of electron transfer to oxygen. A pulse radiolysis study, manuscript in preparation.

295 Chapter 27



Introduction............................................ .......................................................................296


Conjugation of Xenobiotics with Glutathione.......................................................... 296 A. Glutathione 5-Transferases.............................................................................296 B. Effects of Glutathione D epletion...................................................................298 C. Modification of Glutathione 5-Transferase Activities.................................301 D. Binding Properties of Glutathione 5-Transferases........................................302


Canalicular Excretion of Xenobiotics........................................................................303 A. Biliary Transport of GSH and GSSG........................................................ 303 B. Biliary Transport of Glutathione Conjugates................................................304


Glutathione and M etals................................................................................................306


Other Effects Related to Hepatobiliary Transport................................................... 307


Concluding Remarks.....................................................................................................309



Glutathione: Metabolism and Physiological Functions

I. INTRODUCTION The tripeptide glutathione plays an important role in a large number of biological phe­ nomena. Its function is particularly important in hepatic detoxification processes due to reactions catalyzed by glutathione peroxidases and glutathione 5-transferases. The latter enzyme catalyzes the interaction between glutathione and many electrophilic metabolites of xenobiotics, increasing the hydrophilicity of ligands and facilitating the hepatic excretory function. Glutathione 5-transferases are also able to bind nonsubstrate hydrophilic compounds and a transport function related to this binding property has been suggested. The effects of glutathione 5 -transferases-mediated biotransformation on the excretory process of different xenobiotics can be studied in several ways including depletion of cellular glutathione or modifications in the enzyme activity. Glutathione is not only involved in the biotransfor­ mation process but also influences the canalicular transport of xenobiotics. A competition between canalicular glutathione and glutathione conjugates transport via the same carrier system has been reported. This review focuses on the different aspects of the relationship between glutathione and the hepatobiliary transport of xenobiotics.

II. CONJUGATION OF XENOBIOTICS WITH GLUTATHIONE A. GLUTATHIONE S-TRANSFERASES Glutathione is able to react with many compounds to form conjugates. Although at alkaline pH conjugation can be spontaneous, this enzymatic process is normally catalyzed by gluthathione 5-transferases. These enzymes catalyze the reaction between reduced glu­ tathione (GSH) and electrophilic atoms serving as points for nucleophilic attack. The inter­ action of glutathione and the enzyme might facilitate the ionization of glutathione by decreasing its pKa to form the glutathione thiolate ion (GS~) responsible for the reaction . 1 The conjugates are further metabolized to mercapturates mainly by the kidney. The brush border of the proximal tubular epithelium catalyzes the degradation by means of three different enzymes: ( 1 ) 7 -glutamyltransferase which removes the 7 -glutamyl moiety, (2 ) dipeptidase which removes the glycine moiety, and (3) Af-acyltransferase which N-acetylates the cysteine conjugate to yield the corresponding mercapturic acid .2 Glutathione 5-transferases (EC are a family of multifunctional enzymes present in the cytosol of most cells. They are mainly found in the liver, representing 10% of total cytoplasmic protein in the rat . 3 Glutathione 5-transferase activity has also been found in other tissues such as intestine, heart,4 lung ,5 gonads ,6 or placenta .7 The enzyme activity shows a broad phylogenetic distribution and is present in crustaceans, insects, worms, fish, birds, and mammals . 8,9 The glutathione 5-transferases of rat liver are dimeric proteins and comprise subunits with mol wt of 25,500 (Ya), 26,500 (Yn), 27,000 (Ybl,Yb2), and 28,500 (Y c) . 10 The Ya and Yc subunits are products of different mRNAs which are derived from two related yet different genes . 1112 The Ybl and Y b2 subunits seem to be derived from genes different from the YaYc family . 1314 With the use of 5-hexylglutathione-linked Sepharose® 6 B affinity chroma­ tography, at least 11 transferase activity peaks can be resolved including both homodimers and heterodimers of the above subunits. 15 The glutathione 5-transferases of rat liver show an overlapping pattern of substrate specificities that are essential to their multiple roles in drug biotransformation and detoxification. In the human liver at least 13 forms of glutathione 5-transferases have been isolated . 16 The basic enzymes seem to be composed of at least two immunochemically distinct poly­ peptides designated Bj and B 2 and exist as homodimers that are able to hybridize to form

297 a heterodimer. 17 Other authors have characterized at least four immunologically different subunits . 18 The closely related amino acid composition and immunological characterization of glutathione 5-transferases has led to the conclusion that deamination is an important process for generation of the isoenzymes . 19 Nevertheless it has recently been demonstrated that the multiple basic human glutathione 5-transferases are products of separate genes and most likely encoded by a number of two or more different classes of mRNAs . 20,21 Given the large number of glutathione 5-transferase isoenzymes found both in the rat and other species, different means of naming have been used in the past. The different isoenzymes of rat liver are now named on the basis of their constituent subunits, each of which is identified by an arabic numeral.22 Glutathione 5-transferases 1-1, 2-2, 3-3, 3-4, 4-4, and 5-5 correspond to B, AA, A, C, D, and E in the previous nomenclature . 22 Based in structural properties, substrate specificities, and immunoreactivity, three different classes of mammalian cytosolic glutathione 5-transferases have been identified. The classes have been named a , |x, and it .23 Each class is comprised of isoenzymes with similar structural and enzymatic properties. Rat liver isoenzymes containing subunits 1 and/or 2 belong to class a , while glutathione 5-transferases formed by combination of subunits 3 and/or 4 are members of class |x . 23 Glutathione conjugation catalyzed by glutathione 5-transferases can involve endogenous metabolites. Reports have been made on the formation of glutathione conjugates from prostaglandins . 24 Conjugation with glutathione also functions in the m etabolism of leukotrienes25 and evidence also exists for conjugation of D5-3-ketosteroids. 26 Nevertheless, the bulk of identified electrophilic metabolites which react with glutathione are drugs, carcinogens, and metabolites of poly cyclic aromatic hydrocarbons . 1,27 Conjugation of these xenobiotics led to the formation of metabolites with an increased polarity and molecular weight that are then excreted into bile. Glutathione 5-transferases normally function as an important detoxificating metabolic system and only occasionally has the formation of more reactive and toxic metabolites been described .28,29 Liver glutathione 5-transferases show an acinar distribution. With selectively isolated periportal and perivenous hepatocytes by digitonin collagenase perfusion, a higher cytosolic activity has been found perivenous cells . 30 On the other hand, the glutathione content in the liver of both normal and phenobarbital-treated rats seems to be higher in periportal hepa­ tocytes . 31 When the formation of conjugates is studied by surface reflectance spectropho­ tometry it appears that both l-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro nitrobenzene (DCNB) are mainly conjugated by periportal cells in phenobarbital-treated rats, while similar rates of DCNB conjugation for both types of cells are present in normal livers and following retrograde perfusion, CDNB is conjugated in both regions in glutathione-depleted livers . 32 The conjugation of sulfobromophthalein (BSP) both after normal and retrograde perfusion is apparently similar indicating that all acinar zones contribute to conjugation of the dye . 33 It therefore appears that the conjugate formation in the different zones of the liver acini is determined by a variety of factors. Glutathione 5-transferase in adult rat liver is subject to sex-related differences and appears to be regulated by hormonal factors including testosterone. 34' 36 Differences in the relative concentrations of individual glutathione 5-transferases are apparent. Thus, isoenzymes 3-3 and 3-4 are present at higher concentrations in male liver cytosol, whereas isoenzyme 1-3 is present at higher concentrations in female liver . 37 Age-associated alterations have also been found although marked differences have been detected as a function of substrates, with no change for CDNB, a maximum at 1.5 months for BSP or a maximum between 6 to 12 months for iraw,s-4-phenyl-3-butane-2-one. 38 These selective alterations are always higher in males and are apparently due to changes both in the relative and total quantity of isoen­ zymes . 38 Interestingly, the development of glutathione levels does not follow a similar course in rat liver. Intracellular free glutathione increases from the 3- to 21-d postnatal periods and values are decreased in adult tissues . 39


Glutathione: Metabolism and Physiological Functions

Hepatic glutathione 5-transferases activities are inducible by metabolic inducers. The effects of phénobarbital, 3-methylcholanthrene, or 3,4-benzo(a)pyrene are well docu­ mented .40'42 The response, which is similar to that of microsomal enzymes, suggest a close functional relationship between both detoxificating systems . 1 Certain other factors may also lead to an increased activity of glutathione 5-transferases. Thus, acute ethanol treatment,43 streptozotocin-induced diabetes ,44 45 or hyperthyroidism 45 have been shown to induce the enzyme. Chronic administration of propylthiouracyl46 or treatment with dithiolthiones from cruciferous vegetables47 have a similar effect. Not only total glutathione 5-transferase activity but also isoenzyme patterns may be changed. Phénobarbital, 3-methylcholanthrene, or hexachlorobenzene cause elevated levels of subunits 1 and 3, while benzylisothiocyanate causes an increase in subunit 2 .48 All inducers elevate a class subunits to a greater extent than |x class subunits.48 Thus, it appears that the synthesis of individual subunits is regulated independently, although the mechanisms of induction of hepatic glutathione 5-transferase is still unknown. Glutathione 5-transferases are not only present in the cytosol fraction but also in the microsomal fraction of rat liver.49 50 Although the specific activity of the enzyme is less than 1 0 % of that of the cytosolic enzymes for a large variety of substrates and does not apparently play any role in the conjugation of foreign compounds, it has been demonstrated that in some cases, such as the conjugation of hexachloro-l:3-butadiene, it can be higher.51 The activity of the microsomal enzymes show no induction with phénobarbital and is apparently regulated in vivo through the formation and/or cleavage of a mixed disulfide bound with oxidized glutathione (GSSG) . 52 This form probably plays a role in detoxification reactions near the membrane milieu. B. EFFECTS OF GLUTATHIONE DEPLETION Current methods of investigating the involvement of conjugation with glutathione in the amelioration of xenobiotic-induced toxicity and in the enhancement of their biliary excretion rely on depletion of cellular glutathione. Hepatic levels of the tripeptide can be lowered by means of compounds that react enzymatically with glutathione to form conjugates. The most widely used depleting agent within this group is diethyl maleate, an a,ß-unsaturated carbonyl compound . 53 Intraperitoneal (i.p.) administration of this compound rapidly reduces gluta­ thione levels in the liver of rats , 53,54 dogs ,54 and rabbits , 55 leading to an increased toxicity of different xenobiotics . 56 Other substrates for glutathione 5-transferases can also deplete glutathione by enzyme-catalyzed reactions, such as iodomethane, phorone, or aromatic halocompounds.57 Thiol oxidants such as diamide have also been used57 although they are not adequate because they lead to large increases in GSSG concentration and result in the oxidation of cell components .27 The influence of modifications of hepatic biotransformation on the biliary excretion of compounds that are conjugated with GSH has usually been tested with the dye BSP. This is a compound that has been extensively employed in the clinical diagnosis of impaired liver function and as a model substance for the study of the hepatobiliary transport of organic anions .58 Following i.v. administration of the dye, it is rapidly removed from the blood mainly by the liver. Within the hepatocytes much of the BSP is conjugated with GSH by means of glutathione 5-transferases with liberation of HBr. Conjugation forms the thioester BSP-GSH, although a number of other conjugates including BSP-cysteinyl-glycine and BSPcysteine also appear. Conjugated BSP is the main form excreted into bile of the rat, although to a lesser extent the parent compound is also excreted .59,60 Conjugation is not an obligatory step for the biliary excretion of the dye but plays an extremely important role in the overall hepatic transport of the anion and biliary BSP excretion is known to be greater after its administration in conjugated form than when free .61


FIGURE 1. Effect of diethyl maleate (4.3 mmol/kg mixed 1:1 with com oil and given i.p.) on hepatic content of GSH (upper panel) and bile flow (lower panel) in the rat. Arrow point indicated the point at which diethyl maleate was administered. Values at each point are means ± SD. * p T 3 > T 4 = rT 3.7 Although in general conjugation serves to facilitate the urinary and biliary excretion of various sub­ stances by increasing their water solubility, sulfation and glucuronidation of thyroid hormone have different purposes.



Sequential deiodination of T4.

TABLE 1 Three Types of Iodothyronine Deiodinase

Location Site of deiodination Substrate preference Enzyme kinetics PTU Hypothyroidism Hyperthyroidism




Liver, kidney, thy­ roid Inner and outer ring rT3 > sulfates > T4 —t 3 Ping-pong Inhibition Decrease Increase

Brain, BAT, pituitary Outer ring T4 > rT3

Brain, skin, placenta Inner ring t 3> t 4

Sequential No effect Increase Decrease

Sequential No effect Decrease Increase

Note: For reviews, see References 4 to 6.

In normal rats, T4 and T 3 undergo significant biliary clearance predominantly through excretion of their glucuronide conjugates. However, if ID-I activity is inhibited, for instance, with PTU or in the hypothyroid state, the biliary excretion of sulfate conjugates is strongly augmented . 812 In combination with studies using isolated rat hepatocytes , 1316 the view has emerged that T 4 is conjugated in liver largely with glucuronic acid, T 3 to similar extents with glucuronic acid and sulfate, and 3,3'-T 2 predominantly with sulfate. Normally, sulfation results in the irreversible elimination of iodothyronines since the conjugates are degraded rapidly by ID-I . 14"18 Iodothyronine glucuronides are not substrates for ID-I and are excreted intact in bile. These conjugates are hydrolyzed by intestinal bacteria 19 and at least part of the liberated iodothyronines is reabsorbed . 20 Therefore, glucuronidation does not lead to an irreversible loss of thyroid hormone, although the actual extent of the enterohepatic cycle initiated by this reaction remains to be determined in humans and rats.


Glutathione: Metabolism and Physiological Functions


Pathways of T4 metabolism.

Metabolic reactions other than deiodination and glucuronidation play a minor role in the elimination of thyroid hormone (Figure 2). Oxidative deamination of the alanine side chain accounts for only 2% of the metabolism of T4, although up to 20% of T 3 disposal may occur by conversion to its acetic acid derivative.2 Ether bond cleavage of T 4 with resultant formation of diiodotyrosine is negligible under physiological circumstances21 but is induced to an unknown extent in certain pathological conditions such as bacterial infection . 2 After administration of radioiodine-labeled T 4 to rats, roughly equal proportions of radioactivity are excreted as iodide in the urine or as free iodothyronines in the feces . 22 23 The latter are probably derived from intestinal hydrolysis of biliary conjugates that have escaped enterohepatic circulation. In humans, as much as 85% of injected radioactive T 4 ultimately appears as radioiodide in the urine , 24 underscoring the importance of deiodination in the metabolism of T 4 in man. This is also concluded by comparison of the sum of the T 3 and rT 3 production rates with the disposal rate of T4 (see above). However, a low fecal clearance may not be equated with insignificant conjugation of iodothyronines in man because of the high susceptibility of the sulfates for deiodination and the possible enterohepatic circulation of the glucuronides.

II. TYPE I IODOTHYRONINE DEIODINASE A. PROPERTIES AND DISTRIBUTION By definition, ID-I is the enzyme which catalyzes the monodeiodination of the inner or the outer ring of various iodothyronine analogues by a PTU-sensitive mechanism. Especially liver, kidney, and thyroid have high deiodinase activities, but low ID-I levels occur in many other tissues .4 6 The enzyme is present in the microsomal fraction of tissue homogenates and strong evidence has been put forward that in rat liver it is associated with the endoplasmic reticulum. ID-I is an integral membrane protein that shows significant deiodinase activity only in the presence of cytoplasmic cofactor(s). In vitro, requirement for the latter is obviated by addition of synthetic thiols such as dithiothreitol (DTT) . 4 6 Active ID-I has been solubilized from microsomal tissue fractions using a number of ionic and nonionic detergents at higher concentrations than those required for the release of luminal proteins. Recently, a procedure has been described leading to the 2 x 103-fold purification of ID-I from rat liver microsomes25’26 and some preliminary properties of the enzyme have been determined . 25' 28 The lowest molecular weight estimates for active detergent-dispersed ID-I are in the 50- to 60-kDa range, and the delipidated enzyme has a pi value of 9.3. Affinity labeling of ID-I with bromoacetylated substrates followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has identified a possible subunit with mol wt 27 kDa . 29,30

321 TABLE 2 Substrate Specificity of Rat Liver Type I Iodothyronine Deiodinase Substrate





t4 T4 sulfate t4 T4 sulfate rT3 rT3 sulfate t3 T3 sulfate

Outer ring

2.3 ND 1.9 0.3 0.06 0.06 6.2 4.6

30 ND 18 526 559 516 36 1050

13 ND 9 2020 8730 8600 6 230

Inner ring Outer ring Inner ring

Note: Kinetic parameters were determined using rat liver microsomes in 0.1 M phosphate (pH 7.2), 2 mM EDTA, and 3 to 5 mM DTT. Km is expressed in |xM and Vmax in pmol/ min/mg protein; ND, not detectable. Data are taken from References 17, 18, and 35.

In view of the low level of ID-I in rat liver microsomes, the recent proposal that it is identical with the abundant enzyme protein disulfide isomerase (PDI) is surprising . 31 Also, with respect to the acidic nature of PDI (pi 4.2), its identification as a single polypeptide (mol wt 56 kDa), its localization in the lumen of the endoplasmic reticulum, and its different tissue distribution , 32 this hypothesis must be rejected. Interestingly, ID-I activity is decreased in livers from selenium-deficient animals . 33 It is too premature to suggest that ID-I is a selenoenzyme, although its exquisite sensitivity to iodoacetate34 is compatible with the presence of a selenocysteine residue.

B. SUBSTRATES Table 2 gives the kinetic parameters of the deiodination of T4, T3, and rT 3 as well as their sulfate conjugates by ID-I of rat liver microsomes in the presence of 3 to 5 mM DTT as determined in our laboratory . 1718 35 Besides the Vmax and Km values themselves, Table 2 also provides the ratios of these parameters as a cofactor-independent measure of the effi­ ciency of the different catalyzed reactions. Although the Vmax values for these reactions are lower in human liver microsomes, the substrate preferences of human and rat ID-I are very similar. 36 Among the nonsulfated iodothyronines, rT 3 is clearly the preferred substrate for ID-I and is converted quantitatively by ORD to 3,3'-T2. The Vmax/Km ratio for this reaction is at least 500-fold higher than for the deiodinations of T4 and T3. The nonselective nature of ID-I is illustrated by the fact that T4 undergoes both ORD to T 3 and IRD to rT3, although normally little rT 3 accumulates because of its rapid further conversion to 3,3'-T 2. 35,36 As with T4, deiodination of T 3 is a relatively slow process and it is not known to occur other than by IRD to 3,3'-T2. Sulfation of the phenolic hydroxyl group has dramatic effects on the ID-I substrate behavior of different iodothyronines except rT3. Not only is the IRD of T 3 greatly accelerated by sulfation but also the product 3,3'-T 2 sulfate is deiodinated much faster than nonsulfated 3,3'-T 2. 14 Interestingly, the deiodination pattern of T4 is changed completely by sulfation . 18 The IRD of T 4 sulfate is facilitated, leading to the rapid successive formation of rT 3 sulfate and 3,3'-T 2 sulfate and finally to the complete deiodination of the molecule. In contrast to T4 itself, however, ORD of T 4 sulfate does not occur, excluding T 4 sulfate and T 3 sulfate


Glutathione: Metabolism and Physiological Functions

as possible intermediates in the generation of T 3 from T4. As noted above, therefore, sulfation induces the irreversible deiodinative clearance of T4 and T3. The reason for the high reactivity of sulfated substrates has not been established but may be due to a better interaction with basic residues of the enzyme in keeping with its high pi value. This is supported by the findings that other acidic analogues generated by the acétylation or removal of the aN H 2 group are improved substrates for ID-I. 37

C. INHIBITORS A large number of competitive inhibitors of the deiodination of iodothyronines by IDI have been recognized, especially aromatic substances with halogen substituents ortho to hydroxyl or amino groups. They comprise (1) iodothyronine analogues acting as competitive substrates,4 5 (2) simple iodinated phenols and anilines, including several X-ray-contrast agents, 38 (3 ) halogenated phenolphthalein and fluorescein derivatives such as bromophenol blue , 39 erythrosin ,40 and rose bengal,41 (4) coumarin compounds,42 and (5) the polyaromatic flavonoids.43 It has been suggested that the active site of ID-I contains an essential cysteine residue based on the inactivation of the enzyme by SH group-blocking reagents. Iodoacetate is particularly reactive in this respect, and short incubations with micromolar concentrations of this inhibitor suffice to produce substantial enzyme inactivation .34 The high potency of iodoacetate suggests that its target is a strong nucleophile, i.e., a reactive SH group of cysteine or, as speculated above, perhaps a SeH group of selenocysteine. However, it may also be explained in part by the interaction of the C 0 2” group of the reagent with basic residues in the enzyme-active site. This notion is supported by the finding that iodoacetamide is about tenfold less reactive. 34 In comparison with carboxymethylation, ID-I is much less susceptible to inactivation with iV-alkylmaleimides which are only inhibitory at > 0 .1 mM concentrations. 34 Enzyme inactivation with iodoacetate is prevented in the presence of ID-I substrates, especially rT3, suggesting that its target is located in the enzyme-active center. Leonard and Visser have described a method for the specific labeling of ID-I in rat kidney microsomes with [3H]iodoacetate. 34 The proportion of [3H]acetate incorporation into rT3-protectable sites was determined, and the kinetics of this process were found to be identical with the rate of enzyme inactivation. At increasing [3H]iodoacetate concentrations, labeling approached a maximum corresponding to 2.5 pmol/mg microsomal protein . 34 Af-Bromoacetyl (BrAc)-iodothyronine derivatives represent an interesting class of ID-I inhibitors, and recognition of their extreme reactivity towards the enzyme has led to their application as useful affinity labels .29,30 BrAcT3 is a highly potent inhibitor of ID-I, for instance, exhibiting a KAvalue towards the ORD of rT 3 of 0.1 nM compared with a value of 10 [xM for T3. SDS-PAGE of microsomes labeled with [125I]BrAcT3 has demonstrated incorporation into predominantly two protein bands with mol wt 27 and 56 kDa . 29 The smaller protein has been identified as a subunit of ID-I since the rate of its labeling is strongly correlated with the rate of enzyme inactivation which are both prevented by substrates and competitive inhibitors. With increasing [125I]BrAcT 3 concentrations, incorporation of label into rT3-protectable sites shows a maximum of 2.5 pmol/mg microsomal protein . 29 It may be speculated that BrAcT 3 reacts with the same enzyme cysteine residue as that modified by iodoacetate but histidine and lysine are potentially alternative targets. Observations of ID-I inactivation by diethylpyrocarbonate and rose bengal-induced photooxidation indeed suggest the presence of an essential histidine residue.41 Inhibition of ID-I by thiouracils is uncompetitive with iodothyronine substrate and com­ petitive with thiol cofactor.4' 6’36 Inactivation of ID-I by thiouracil requires the presence of substrate and has been correlated with the incorporation of radioactive thiouracil into enzyme protein .44 45 [125I]5-iodothiouracil appears a suitable affinity label in this respect but so far



Mechanism of type I iodothyronine deiodination.

only the covalent binding of [35S]thiouracil to ID-I has been properly quantified. Injection of rats with increasing doses of the radioactive inhibitor resulted in the maximal incorporation of 6 pmol 35S per milligram kidney microsomal protein .45 This probably represents an overestimate of the actual ID-I content of rat kidney microsomes since subsequent treatment in vitro with 10 mM DTT released 33% of protein-bound 35S but restored PTU-inhibited ID-I activity by 60 to 70%.45 These results, therefore, are in close agreement with the specific affinity labeling of ID-I in vitro using iodoacetate and BrAcT3, indicating that about 2.5 pmol of enzyme is present per milligram microsomal protein in both liver and kidney.

D. MECHANISM The reductive deiodination of iodothyronines by ID-I is supported by thiol cofactors, and enzyme kinetics have mostly been determined using DTT as the reductant. These studies have pointed to a ping-pong type reaction mechanism, indicating that iodothyronine substrate and thiol cofactor react with alternating forms of the enzyme.4-6 Especially the uncompetitive inhibition of ID-I by thiouracil suggests that an enzyme sulfhydryl group plays a central role in the catalytic mechanism. Apparently, in the native state this group is highly reactive towards iodoacetate while transformation by substrate is thought to make it susceptible to reaction with thiouracil. This is supported by the finding that iodoacetate modification of ID-I is prevented by prior enzyme-thiouracil complex formation . 34,45 Since thiourea deriv­ atives show a profound reactivity for sulfenyl iodide groups, the formation of such an E-SI intermediate in the deiodination process is implicated .4' 6 The current view of the catalytic mechanism of ID-I is illustrated in Figure 3. This is thought to concern the transfer of an iodinium (I+) ion from the substrate (in this case T4) to the enzyme-sulfhydryl (E-SH) group, resulting in the generation of the monodeiodinated product (in this case T3) and the enzyme-sulfenyl iodide (E-SI) intermediate. The latter represents an oxidized form of ID-I which is reduced back to the native state by cofactor (in this case DTT), resulting in the release of iodide and oxidation of the cofactor to the disulfide. In case of a monovalent thiol (RSH) as the cofactor, formation of a discrete ESSR mixed-disulfide intermediate will precede reaction with a second cofactor molecule to produce fully reduced enzyme and oxidized cofactor RSSR. Compatible with its competitive inhibition towards cofactor, thiouracil also reacts with E-SI under the formation of an enzymethiouracil mixed disulfide. This inactive enzyme complex is very stable and is only reduced in vitro by very high DTT concentrations at elevated pH . 34 Little is known about the molecular mechanism of catalysis by ID-I. It is different from that underlying the deiodination of iodotyrosines by a flavoenzyme located in the particulate fractions of thyroid and liver. This enzyme is driven by a NADPH-dependent reductase and is not inhibited by PTU .46 There is some analogy between the reaction catalyzed by ID-I and the dehalogenation of 5-bromo- or 5-iodo-2'-deoxyuridylate by thymidylate synthetase.47 The latter also appears to involve the participation of an enzyme SH group and is also strongly stimulated by DTT.


Glutathione: Metabolism and Physiological Functions

Electrophilic displacement of I by H has been suggested as the mechanism for the nonenzymatic deiodination of iodotyrosines with cysteine, where the latter is thought to act as acceptor for I + under formation of a sulfenyl iodide .48 Such a reaction is facilitated by the electron-donating effect of an ortho hydroxyl group, and for this reason it only applies to the deiodination of a phenolic ring, i.e., the outer ring of iodothyronines. However, IDI also catalyzes deiodination of substrate loci lacking a free hydroxyl group, i.e., the inner ring and also the outer ring of sulfated substrates. Therefore, the catalytic mechanism of ID-I remains to be fully explored.

E. TURNOVER NUMBERS As discussed above, quantitation of ID-I in rat liver and kidney microsomes with different affinity labels has provided surprisingly close estimates of the enzyme contents of these fractions, i.e., 2.5 pmol/mg protein. The turnover numbers of the liver enzyme in the deiodination of different substrates in the presence of 3 mAÍ DTT are obtained by dividing the Vmax values reported in Table 2 by the amount of enzyme, both expressed per milligram microsomal protein. This yields turnover numbers of 30/2.5 = 12 min - 1 for T4 and 556/ 2.5 = 220 min - 1 for rT3, which are low values compared with many other enzymatic reactions. That rat liver contains approximately 50 mg microsomal protein per gram wet weight49 allows for the estimation of turnover numbers of ID-I in isolated hepatocytes. In incubations of 2 x 106 hepatocytes in monolayer with a saturating concentration of rT 3 (1 jjlM) in the absence or presence of 1 mM DTT, Vmax values were obtained of 7.6 and 78 pmol I “ per minute, respectively (unpublished observations). Since 2 x 106 cells contain roughly 1 mg microsomal protein and, therefore, 2.5 pmol enzyme, turnover numbers amount to about 3 and 30 m in - 1 under these conditions. Considering the lower DTT concentration and the possibly restricted access to the enzyme in intact cells, the latter value is in reasonable agreement with the value of 220 min - 1 for microsomes. However, in the absence of added DTT the enzyme clearly operates at only a fraction of its maximal capacity in hepatocytes. Based on the disposal rate of thyroid hormone in normal rats and the total liver ID-I content, the turnover number of the enzyme under physiological conditions may also be approximated. The production of T4 in rats amounts to roughly 1 nmol per 100 g body weight per day . 50 For this calculation it is assumed that the complete deiodination of T 4 takes place in the liver, producing 4 nmol I “ per 100 g body weight per day. The relative liver weight in rats is about 4 g per 100 g body weight, containing 200 mg microsomal protein and, therefore, 0.5 nmol ID-I. Thus, the turnover number of the deiodinase in the intact animal amounts to approximately 8 d _1, an extremely low value indeed. Because of the assumptions made, this probably represents an overestimation of the true situation. A summary of these calculations is presented in Table 3.

III. ROLE OF GLUTATHIONE A. EFFECTS OF GLUTATHIONE AND OTHER THIOLS Several methods have been used to identify the physiological cofactor(s) for ID-I present in the soluble fraction of liver and other tissues. First, different natural cofactors have been tested for deiodinase-stimulating activity in comparison with the effects of synthetic thiols. Second, attempts have been made to associate the changes in the activity of rat liver cytosol to support microsomal deiodination with changes in the level of glutathione (GSH) under different pathophysiological conditions. Third, multiple factors have been isolated from rat liver cytosol that alone or in combination with other compounds show stimulation of ID-I activity. Initial studies of the subcellular distribution of ID-I in liver showed a complete loss of deiodinase activity following separation of cytosol from the particulate fractions .51 It was

325 TABLE 3 Effects of DTT and other Thiols on the Conversion of rT3 to 3,3'-T2 by Rat Liver Microsomes Production of 3,3'-T2 (pmol.min " protein ~l) Thiol

_ ME

1 mM 5 mM MES 1 mM 5 mM DMP 1 mM 5 mM DMPS 1 mM 5 mM GSH 1 mM 5 mM

0 mM DTT

1 mM DTT

5 mM DTT