Oxidative Stress: Eustress and Distress [1 ed.] 0128186062, 9780128186060

Table of contents :
Cover
Oxidative
Stress:
Eustress and Distress
Copyright
Contributors
Preface
Part 1: Conceptual
1
Oxidative eustress and oxidative distress: Introductory remarks
Introduction
What is new?
On the development of stress response concepts
Merits and pitfalls: Usefulness of oxidative stress concept
Outlook
Acknowledgments
References
2
Epistemological challenges of the oxidative stress theory of disease and the problem of biomarkers
Causation and the OSTD
Association versus causation: The problem of confounders and reverse causation
Using Koch’s postulates and Bradford Hill’s criteria in OSTD studies
Experimental approach to the study of the OSTD
The problem of multiple causes
Measuring OS and the problem of biomarkers
Biomarkers as signs
Is OS just a biochemical derangement? Physiological support versus pharmacological therapy
References
Further reading
3
Systems biology and network medicine: An integrated approach to redox biology and pathobiology
Introduction
Introduction to network medicine
Basic network concepts and network components
Protein-protein interaction network and the human interactome
Protein-protein interactions
Human interactome
Posttranslational modifications (PTM) of proteins and PPI networks
Human disease network
Systems biology approach to understand the redox system
Redox system
Mitochondrial molecular networks
Redox couples and redox environment network
Protein thiols and redox proteomics
Network analysis of antioxidant systems
Omics studies of redox biology and gene regulatory network inference
Redox transcriptomics and gene regulatory network
Redox proteomics
Redox metabolomics
Integration of multiomic networks
Dynamic networks and flux analysis
Network modeling of multicompartment redox system
Current challenges
Future directions
Acknowledgment
References
4
The reactive species interactome
Introduction
Chemical interactions among reactive species
Characteristics of the reactive species interactome
Role of the reactive species interactome in the response to stress and evolution of life
The redox interactome
The RSI in the context of stratified medicine
Summary and conclusions
References
Part 2: Oxidative eustress and distress: Processes and responses
5
Oxidative stress and the early coevolution of life and biospheric oxygen
Oxygen and the early history of our planet
Oxygen and life: First contact
An oxygenic throttle?
Resistance is not futile
Rise of the aerobes
An anaerobic mass extinction?
Phylogenetic evidence
Redox biology as an integral part in multicellular evolution
Conclusion
Acknowledgments
References
6
How imaging transforms our understanding of oxidative stress
Introduction
Lesson 1: H 2 O 2 is highly compartmentalized
Lesson 2: Thioredoxin system is powerful regulator of H 2 O 2 patterns
Lesson 3: Basal H 2 O 2 concentration in the cell is low nanomolar
Lesson 4: H 2 O 2 transport across membranes is facilitated by aquaporins
What is missing?
References
Further reading
7
In vivo applications of chemogenetics in redox (patho)biology
Introduction
Chemogenetics versus optogenetics
Chemogenetic approaches to study oxidative stress in vivo
Multiparametric single-cell imaging approaches using chemogenetic tools and genetically encoded biosensors
References
Further reading
8
Quantification of intracellular H2O2: Methods and significance
Introduction
Biophysical measurement of intracellular H2O2
Fluorescent reporters
Kinetic models
Comparing findings
Outlook
References
9
The Keap1-Nrf2 pathway: From mechanism to medical applications
Introduction
The discovery of Nrf2
The discovery of Keap1
Regulation of Nrf2 by Keap1
p62-dependent regulation of Nrf2 through a hinge and latch mechanism
Integration of the Keap1-Nrf2 pathway into the cellular primary metabolism circuitry
The Keap1-Nrf2 pathway and inflammation
Future perspectives
Conclusion
References
10
Ferroptosis: Physiological and pathophysiological aspects
A short introduction to the field of cell death
Ferroptosis: Regulated cell death or just a cellular catastrophe?
A physiological meaning to ferroptosis?
Ferroptosis in pathophysiology
Ferroptosis induction: A promising new anticancer therapeutic strategy
Concluding remarks
Acknowledgments
References
11
Aquaporins: Gatekeepers in the borders of oxidative stress and redox signaling
Introduction
An accolade of (mild) stress
Acute versus chronic stress
Control of aquaporin-facilitated transmembrane transport upon stress
Peroxiporins: Surfing between oxidative stress and redox signaling
References
12
Extracellular superoxide dismutase (SOD3): An antioxidant or prooxidant in the extracellular space?
Introduction
Functional regions of SOD3
Regulation of SOD3 activity
Transcriptional regulation of SOD3
Posttranslational regulation of SOD3
Spatiotemporal regulation of SOD3
SOD3: An antioxidant in the extracellular space
SOD3 as a sink for superoxide
SOD3 in oxidative distress
Pulmonary disease
Vascular disease
Cancer
SOD3: A prooxidant in the extracellular space
SOD3 as a source of hydrogen peroxide
SOD3 in oxidative eustress
SOD3 in inflammation
Spatial distribution of SOD3 in inflammation
Regulation of the inflammatory response
Conclusion
Acknowledgments
References
13
Protein S-glutathionylation and the regulation of cellular functions
Introduction
Reversible protein S-glutathionylation
How do S-glutathionylation reactions regulate proteins in response to physiological cues?
Nonenzymatic S-glutathionylation reactions do occur in cells
Protein S-glutathionylation reactions in the cytosol
Regulation of cytoskeletal dynamics
Glutaredoxin-1 and neural cell (dys)function
Apoptosis
Regulation of kinase signaling cascades
Role in muscle contraction and relaxation
Protein S-glutathionylation of mitochondrial proteins
The mitochondrial matrix favors S-glutathionylation reactions
Nutrient metabolism and oxidative phosphorylation
Regulation of the Krebs cycle
Modulation of complex I
Regulation of the other respiratory complexes and ATP synthase
Proton leaks and solute import
Mitochondrial fission/fusion
Chemical methods for detecting S-glutathionylated proteins
Sample treatment
35 S-labeling and immunoblot
Switch assays
Clickable glutathione analogs
Membrane permeable and intracellular glutathionylation probes
Conclusions
References
14
Dual stressor effects of lipid oxidation and antioxidants
Introduction
Oxidation of fatty acids and esters
Enzymatic oxidation of fatty acids and esters
Nonenzymatic oxidation of fatty acids and esters
Oxidation of cholesterol
Enzymatic oxidation of cholesterol
Nonenzymatic cholesterol oxidation
Adaptive response to lipid oxidation products: Distress or eustress ?
Effects of antioxidants
Summary
References
15
Oxidized phospholipid signaling: Distress to eustress
Introduction to redox signaling concepts
Oxidized phospholipids and their products
Oxidized lipid signaling versus damage
Overview of signaling mechanisms for lipid oxidation products: Noncovalent versus covalent interactions
Examples of the biological effects of oxidized lipids products
Studies of oxPL mixtures
Effects of individual oxidized lipids or oxidized lipid families
Eustress versus distress: A matter of concentration?
Oxidized lipid signaling: Parallels with hydrogen peroxide-based redox signaling
Acknowledgments
References
16
Redox regulation of protein kinase signaling
Introduction
Redox-mediated regulation of protein phosphatases
Protein tyrosine phosphatases (PTPs)
Serine/threonine phosphatases (PSPs)
Direct redox regulation of protein kinases
General concepts
AGC family
CAMK family
CMGC family
Tyrosine kinase (TK) family
Redox regulation of phosphoprotein binding
It is not always cysteine: Importance of other amino acids in redox regulation of kinase signaling
Achieving specificity in redox-dependent regulation of kinase signaling
The importance of location
How does cysteine oxidation affect kinase function?
Final considerations
References
17
FoxO transcription factors in the control of redox homeostasis and fuel metabolism
Introduction
FoxOs: General aspects
Molecular mechanisms underlying redox regulation of FoxO transcriptional activity
Role of FoxOs in the regulation of redox homeostasis and defense against oxidative stress
Role of FoxOs in the regulation of fuel metabolism
FoxOs and pancreas
FoxOs and the liver
FoxOs and skeletal muscle
FoxOs and adipose tissue
Concluding remarks
References
18
Oxidatively generated DNA base modifications: Relation to eustress and distress
Introduction
Oxidative distress at the DNA: Detrimental effects of oxidatively generated DNA damage
Oxidatively generated DNA damage and cancer risk: General considerations
DNA damage induced by ROS: Mechanisms and types of lesions
Reactivity of ROS and DNA damage spectra
Guanine oxidation products in DNA
DNA damage spectra by hydroxyl radicals and peroxynitrite
DNA damage spectra by bromate and tert -butoxyl radicals
DNA damage spectra by type I photosensitizers and singlet oxygen
Repair of oxidatively generated DNA base damage
Endogenously generated DNA damage: Basal levels
Endogenous sources of oxidatively generated DNA damage
Relevance of oxidatively generated DNA damage for carcinogenesis
Lessons from DNA repair defects in human cancers
Lessons from DNA repair defects in mice
Lessons from mutation spectra
Oxidative eustress at the DNA: Physiological effects of oxidatively generated DNA damage
Conclusions
References
19
Light-initiated oxidative stress
Introduction
Why is light important?
Some specifics about light
Light-initiated production of ROS
Singlet oxygen
What is ground state oxygen?
What is singlet oxygen?
The lowest energy singlet state, O 2 (a 1 Δ g)
The other singlet state, O 2 (b 1 Σ g +)
Transitions between states
How can singlet oxygen be produced by light?
Energy transfer from a photosensitizer
Direct irradiation of ground state oxygen
Dependence on the concentration of ground state oxygen
What reactions of O 2 (a 1 Δ g) are potentially pertinent to oxidative stress?
O 2 (a 1 Δ g) as a diffusible signaling agent
How can the reactions of O 2 (a 1 Δ g) modify/modulate cell redox states and cell response?
Examples with selected enzymes
Genetic regulation mediated by O 2 (a 1 Δ g)
Correlating cell response with the O 2 (a 1 Δ g) reaction in a given cellular location
Subcellular spatially dependent O 2 (a 1 Δ g)-mediated eustress response
Superoxide radical anion
Reactivity of superoxide
Photoinitiated production of superoxide as a complication
Selective photoinitiated production of superoxide as a mechanistic tool
Where does the field stand today? What does the future hold?
Selective production of O 2 (a 1 Δ g) and superoxide in space and time
Exploit the opportunity for better control of O 2 (a 1 Δ g) and superoxide dose
A plethora of new ways to monitor cells and cell response in space and time
Conclusions
References
20
Nutritional protection against photooxidative stress in human skin and eye
Introduction
Skin
Skin cancer
Erythema
Carotenoids
Vitamins E and C
Flavonoids
Eye
Cataract
Age-related macular degeneration (AMD)
Conclusion
References
Part 3: Exposome
21
Mechanisms integrating lifelong exposure and health
Introduction
Redox theory
Redox interface
Redox-responsive elements in complex systems
Multiomics approaches to understand redox systems
The redox code
Exposure memory
Omics and integrated approaches in oxidative stress research
Overview of omics technologies and bioinformatics tools for integrative omics
Biologic response to oxidative stress: Omics studies of low-level cadmium
Cadmium-epigenome
Cadmium-transcriptome
Cadmium-metabolome
Cadmium-proteome
xMWAS for integrated omics research
xMWAS
Case study: Integrated omics analysis of mouse lung response to cadmium and selenium
Dynamic responses over time
Variations in type and impact of exposures
Multiple omics gives deep phenotyping
Advantages of systems biology approaches based upon existing knowledgebase
Cumulative impact on redox networks
Measures of network flexibility and resilience
Summary and conclusion
Funding
Conflict of interest
References
22
Nutrient sensing, the oxidative stress response, and stem cell aging
Introduction
Nutrient sensing and the oxidative stress response
Nutrient sensing and the mitochondrial protein folding stress response
Oxidative stress, stem cell aging, and tissue degeneration
Therapeutic opportunities
Conclusion
Acknowledgments
References
23
How exercise induces oxidative eustress
Introduction: Exercise from oxidative stress to eustress
Worked example 1: NADPH oxidase-mediated O 2 • − production as an exercise-induced redox signal
Worked example 2: Exercise-induced lipid peroxidation as an exercise-induced redox signal
When does exercise eustress become oxidative distress?
Conceptual, technical, and methodological recommendations
Conclusion
Acknowledgments
References
24
Metabolomics as a tool to unravel the oxidative stress-induced toxicity of ambient air pollutants
Introduction
Ambient air pollution
Industrial air toxics
Traffic-related air pollution
Occupational exposures
Ozone
Discussion
References
25
Traffic-related environmental risk factors and their impact on oxidative stress and cardiovascular health
Global burden of pollution, noncommunicable disease, and role of oxidative stress
The pollutome: Risk factors in the physical environment
Air pollution: Number one environmental hazard
Traffic and occupational noise exposures: The underestimated environmental risk factor
Examples of mechanistic noise studies: Nonauditory effects of noise exposure on oxidative stress and cardiovascular health
Aircraft noise and translational studies in humans
Indirect, nonauditory vascular effects of ≤ 100 dB(A) noise exposure
Indirect, nonauditory pathway activation with aircraft noise exposure  ≤   85 dB(A)
Other environmental stressors may act in concert with traffic-related exposures by activation of similar oxidative stress a ...
Conclusions
Acknowledgments
Conflicts of interest
References
Further reading
Part 4: Oxidative stress in health and disease processes
26
Mitochondrial ROS production during ischemia-reperfusion injury
Introduction
Ischemia-reperfusion injury
Metabolic changes during ischemia
Mitochondrial superoxide production upon reperfusion following ischemia
The thermodynamic driving force for RET
The role of complex I in superoxide production during IR injury
Induction of the mitochondrial permeability transition pore
Conclusion
Funding
References
Further reading
27
Redox signaling in cellular differentiation
Introduction into cellular differentiation
General targets of ROS in differentiation
ER stress in differentiation
ROS in epigenetic mechanisms of cellular differentiation
DNA methylation
Histone methylation
ROS in methylation-controlled differentiation
Metabolism and ROS in differentiation
S-adenosylmethionine (SAM) and homocysteine
Glycolysis in stem cells
ROS in differentiation of embryonic stem cells
ROS in differentiation of adult stem cells
The niche of adult stem cells
Differentiation of adult stem cells
Interplay of ROS, Akt, and p38 in adult stem cells
Transdifferentiation: Epigenetic reprogramming?
Concluding remarks
References
28
Redox-regulated brain development
Oxidative eustress and distress in the brain
Brain development
H 2 O 2 signaling during development of the nervous system
H 2 O 2 production in physiological situations
Redox regulation of neurogenesis
Redox regulation of postmitotic neuronal development
Enzymatic regulation of oxidative eustress in the brain
Conclusion
References
29
Eustress, distress, and oxidative stress: Promising pathways for mind-body medicine
Introduction
General theories of psychological stress and health
The origin of stress: A brief history
Evidence for stressor specificity
The presence of threat versus the absence of safety
Distinguishing transdiagnostic distress from diagnosis
RDoC and neurocircuitry based frameworks
Eustress: Definition and discordance
Disentangling exposures and experiences from impact
Two paths to eustress: Hormesis versus stress buffering
The assessment of psychological distress
Standardized tools and tasks
The validity of self-reported psychological distress
Self-report measures of perceived stress
Self-report measures of depressive symptoms
Self-report measures of anxious symptoms
Acute laboratory psychological stress tasks
Neurocognitive tasks
Naturalistic exposures to stressful life events: Methods and assessment
Assessing objective stress exposures
Early adversity
Daily stressful exposures
The role of anticipation
Stress system biomarkers
Cortisol: Functions and measurement
Cortisol: No straightforward interpretation
Cortisol: From eustress to distress
The autonomic nervous system and oxidative stress
System allostasis: Robustness and resilience
Mind-body pathways and oxidative stress markers
Oxidative damage markers
Mitochondria
Nitric oxide and the nitric oxide synthase enzymes
Antioxidants
A brief overview of the literature
Depression, anxiety, and oxidative/nitrosative stress
PTSD and oxidative/nitrosative stress
A transdiagnostic perspective
Translational approaches to eustress
Oxidative stress as a mediator of psychosocial stress and aging
Oxidative stress: Explaining the stress-telomere connection
Cellular senescence: A mechanism of aging
Deficient autophagy: A promising frontier for eustress research
A causal role for oxidative stress in depression and anxiety?
Neuroinflammatory depression & anxiety
A neuroinflammatory/oxidative subtype of depression/anxiety
Repeated oxidative stress can trigger and sustain IL-1B
Evidence of potential clinical relevance
Inflammation and antidepressant nonresponse
Oxidative stress as a target for adjunctive treatment
Eustress, distress, and obesity
Adipose hypoxia
Brain-to-adipose pathways
Adipose-to-brain pathways
Neuroinflammation in obesity and metabolic disease
Clinical implications
Conclusions
References
30
Reactive oxygen species and cancer
Introduction
H2O2 promotes tumorigenesis
Cancer cells limit damaging lipid hydroperoxide accumulation
Targeting the redox biology for cancer therapy
Conclusion
Acknowledgments
References
31
Perspectives of TrxR1-based cancer therapies
Introduction
TrxR1 in relation to other cellular enzymatic reducing systems
Cellular and physiological functions of TrxR1
TrxR1 in health and disease
Potentially health-promoting effects of high TrxR1 activities
Potentially health-promoting effects of low TrxR1 activities
Potentially disease promoting effects of high TrxR1 activities
Potentially disease promoting effects of low TrxR1 activities
The intricate roles of TrxR1 in cancer
Genetic links of TrxR1 to cancer
Protection against carcinogenesis by TrxR1?
Promotion of cancer progression by TrxR1
TrxR1 as a selenoprotein oxidoreductase and the effects of its inhibition
Drugs targeting TrxR1 for use in cancer therapy
TrxR1-inhibiting drugs in clinical use for cancer therapy
Experimental compounds inhibiting TrxR1
Conclusions and future perspectives
Acknowledgments
COI Declaration
References
32
Oxidative/nitrosative stress and hepatic encephalopathy
Introduction
Astrocyte swelling in HE
Astrocyte swelling and oxidative/nitrosative stress in astrocytes in HE
Mitochondria and oxidative stress in HE
Functional consequences of osmotic and oxidative/nitrosative stress in HE
Oxidative/nitrosative stress and protein tyrosine nitration in HE
RNA oxidation in HE
Oxidative/nitrosative stress and gene expression changes in HE
Oxidative stress and astrocyte senescence in HE
O-GlcNAcylation and oxidative stress in astrocytes
Concluding remarks
Funding
References
33
ROS signaling in complex systems: The gut
Introduction
ROS-generating enzymes in the intestinal mucosa
ROS in maintenance of intestinal homeostasis
Intestinal disease: Too much ROS
Intestinal disease: Not enough ROS
Conclusions and future directions
Acknowledgment
References
34
Oxidative stress in skeletal muscle: Unraveling the potential beneficial and deleterious roles of reactive oxygen species
Introduction
Sites of ROS generation in skeletal muscle
Functions of physiological ROS in muscle
ROS in muscle degeneration
Age-related loss of skeletal muscle mass and function
Oxidative damage and defective redox signaling in muscle from old mice and humans
Modification of muscle ROS during ageing though knockout of key regulatory proteins
Role of ROS in denervation and link to ageing
Denervation of muscle leads to increased muscle mitochondrial generation of peroxides that stimulates muscle atrophy and lo ...
Implications of the increased mitochondrial peroxide production for prevention of muscle loss with ageing and following den ...
Future directions in dissection of the roles of redox signaling from oxidative stress in skeletal muscle
Summary
Acknowledgments
References
Further reading
35
Redox mechanisms in pulmonary disease: Emphasis on pulmonary fibrosis
Introduction
ER stress, mitochondrial dysfunction, and the age-associated enhanced susceptibility to lung fibrosis
Death of lung epithelial cells in fibrosis
Oxidative stress in lung fibrosis: Glutathione biochemistry
Protein S-glutathionylation, the death receptor FAS, and epithelial cell death
ER redox stress and tissue fibrosis
Concluding comments and challenges for future research and development
Acknowledgments
Conflict of Interest
References
36
Dicarbonyl stress and the glyoxalase system
Introduction
Reactive metabolites of dicarbonyl stress: Methylglyoxal, glyoxal, 3-deoxyglucosone, and other α -oxoaldehyde metabolites
Metabolic drivers of dicarbonyl stress
Proteins susceptible to modification by methylglyoxal: The dicarbonyl proteome
Activation of the unfolded protein response by dicarbonyl stress
Concluding remarks involvement of dicarbonyl stress in aging and disease
Acknowledgment
References
Further reading
37
Redox distress in organ fibrosis: The role of noncoding RNAs
Introduction
Fibrosis: Failed resolution of the repair response
Redox unbalance in fibrosis development
Oxidative stress in pulmonary fibrosis
Oxidative stress in kidney fibrosis
Oxidative stress in liver fibrosis
Oxidative stress in cardiac fibrosis
Oxidative stress in skin fibrosis
Noncoding RNAs in TGF- β /ROS-driven tissue fibrosis
Regulation of ROS production and antioxidant defense in the fibrotic response
Regulation of the EMT process in organ fibrosis
Regulation of TGF- β -induced signaling pathways
Regulation of other cellular mechanisms involved in organ fibrosis
Conclusion
Acknowledgments
References
Index
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Citation preview

Oxidative Stress

­Oxidative Stress Eustress and Distress

Edited by

Helmut Sies

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom © 2020 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-818606-0 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Andre Gerhard Wolff Acquisition Editor: Peter B. Linsley Editorial Project Manager: Samantha Allard Production Project Manager: Swapna Srinivasan Cover Designer: Greg Harris Typeset by SPi Global, India

Contributors Vikas Anathy Department of Pathology and Laboratory Medicine, University of Vermont, Larner College of Medicine, Burlington, VT, United States Elias S.J. Arnér Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden Kirstin Aschbacher Department of Medicine, Division of Cardiology; Center for Health and Community, Department of Psychiatry, UCSF, San Francisco, CA, United States Liam Baird Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Sendai, Japan Timothy E. Beach Department of Surgery and Cambridge NIHR Biomedical Research Centre, University of Cambridge, Cambridge, United Kingdom Vsevolod V. Belousov Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry; Pirogov Russian National Research Medical University, Moscow, Russia; Institute for Cardiovascular Physiology, Georg August University Göttingen, Göttingen, Germany Carsten Berndt Department of Neurology, Medical Faculty, Heinrich-Heine Universität Düsseldorf, Germany Alfonso Blázquez-Castro Department of Chemistry, Aarhus University, Aarhus, Denmark; Department of Physics of Materials, Faculty of Sciences, Autonomous University of Madrid, Madrid, Spain Mikkel Bregnhøj Department of Chemistry, Aarhus University, Aarhus, Denmark Thomas Breitenbach Department of Chemistry, Aarhus University, Aarhus, Denmark Nils Burger MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, United Kingdom Abigail Caron Department of Organismal Biology and Anatomy, University of Chicago, Chicago, IL; Department of Earth, Atmospheric and Planetary Sciences, Massachusetts Institute of Technology, Boston, MA, United States

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Contributors

Navdeep S. Chandel Department of Medicine, Division of Pulmonary and Critical Care; Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, Chicago, IL, United States Danica Chen Program in Metabolic Biology, Nutritional Sciences & Toxicology, University of California, Berkeley, CA, United States James Nathan Cobley Free Radical Research Group, Department of Biomedical Sciences, University of the Highlands and Islands, Inverness, United Kingdom Marcus Conrad Helmholtz Zentrum München, Institute of Developmental Genetics, Neuherberg, Germany Miriam M. Cortese-Krott Department of Cardiology, Pulmonology and Angiology, Medical Faculty, Heinrich Heine University of Düsseldorf, Düsseldorf, Germany Milene Costa da Silva INL—International Iberian Nanotechnology Laboratory, Braga, Portugal Andreas Daiber University Medical Center at the Johannes Gutenberg University Mainz, Center for Cardiology, Cardiology I, Mainz, Germany Kevin Davies Department of Clinical and Experimental Medicine, Brighton & Sussex Medical School, Brighton, United Kingdom Albert van der Vliet Department of Pathology and Laboratory Medicine, University of Vermont, Burlington, VT, United States Charles Diamond Department of Earth Sciences, University of California, Riverside, CA, United States Christopher M. Dustin Department of Pathology and Laboratory Medicine, University of Vermont, Burlington, VT, United States Bernd Epe Institute of Pharmacy and Biochemistry, University of Mainz, Mainz, Germany Emrah Eroglu Cardiovascular Medicine Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, United States Michael Etzerodt Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

Contributors

Martin Feelisch Clinical & Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, United Kingdom Greg Fournier Department of Earth, Atmospheric and Planetary Sciences, Massachusetts Institute of Technology, Boston, MA, United States Julia C Fussell NIHR Health Impact of Environmental Hazards HPRU, MRC Centre for Environment and Health, King’s College London, London, United Kingdom Pietro Ghezzi Department of Clinical and Experimental Medicine, Brighton & Sussex Medical School, Brighton, United Kingdom Virginia Ghiara Department of Philosophy, University of Kent, Canterbury, United Kingdom Robert Gill Department of Biochemistry, Faculty of Science, Memorial University of Newfoundland, St. John’s, NL, Canada Young-Mi Go Clinical Biomarkers Laboratory, Division of Pulmonary Medicine, Department of Medicine, Emory University, Atlanta, GA, United States Boris Görg Clinic for Gastroenterology, Hepatology, and Infectious Diseases, HeinrichHeine-University, Düsseldorf, Germany Anja V. Gruszczyk MRC Mitochondrial Biology Unit; Department of Surgery and Cambridge NIHR Biomedical Research Centre, University of Cambridge, Cambridge, United Kingdom Dieter Häussinger Clinic for Gastroenterology, Hepatology, and Infectious Diseases, HeinrichHeine-University, Düsseldorf, Germany David E. Heppner Department of Cancer Biology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, United States Lili Hu Department of Biomedicine, Aarhus University, Aarhus, Denmark Alan A. Jackson Human Nutrition, University of Southampton and University Hospital Southampton, Southampton, United Kingdom M.J. Jackson Department of Musculoskeletal Biology, MRC-Arthritis Research UK Centre for Integrated research into Musculoskeletal Ageing (CIMA), Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, United Kingdom

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Yvonne Janssen-Heininger Department of Pathology and Laboratory Medicine, University of Vermont, Larner College of Medicine, Burlington, VT, United States Dean P. Jones Clinical Biomarkers Laboratory, Division of Pulmonary Medicine, Department of Medicine, Emory University, Atlanta, GA, United States Frank J Kelly NIHR Health Impact of Environmental Hazards HPRU, MRC Centre for Environment and Health, King’s College London, London, United Kingdom Lars-Oliver Klotz Institute of Nutritional Sciences, Nutrigenomics Section, Friedrich Schiller University Jena, Jena, Germany Ulla G. Knaus The UCD Conway Institute of Biomolecular and Biomedical Research, School of Medicine, University College Dublin, Dublin, Ireland Hyewon Kong Department of Medicine, Division of Pulmonary and Critical Care; Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, Chicago, IL, United States Swenja Kröller-Schön University Medical Center at the Johannes Gutenberg University Mainz, Center for Cardiology, Cardiology I, Mainz, Germany Duvaraka Kula-Alwar Department of Medicine, University of Cambridge, Cambridge, United Kingdom Santiago Lamas Department of Cell Biology and Immunology, Centro de Biología Molecular “Severo Ochoa,” (CSIC-UAM), Madrid, Spain Laurel Y. Lee Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, United States Jos Lelieveld Atmospheric Chemistry Department, Max Planck Institute for Chemistry, Mainz, Germany Joseph Loscalzo Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, United States Timothy W. Lyons Department of Earth Sciences, University of California, Riverside, CA, United States

Contributors

Ryan J. Mailloux Department of Biochemistry, Faculty of Science, Memorial University of Newfoundland, St. John’s, NL; School of Human Nutrition, Faculty of Agricultural and Environmental Sciences, McGill University, Ste. Anne de Bellevue, QC, Canada Ashley E. Mason Center for Health and Community, Department of Psychiatry; Osher Center for Integrative Medicine, University of California San Francisco (UCSF), San Francisco, CA, United States A. McArdle Department of Musculoskeletal Biology, MRC-Arthritis Research UK Centre for Integrated research into Musculoskeletal Ageing (CIMA), Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, United Kingdom Iria Medraño-Fernandez Protein Transport and Secretion Unit, Division of Genetics and Cell Biology, IRCCS Ospedale San Raffaele/Università Vita-Salute San Raffaele, Milan, Italy Thomas Michel Cardiovascular Medicine Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, United States Verónica Miguel Department of Cell Biology and Immunology, Centro de Biología Molecular “Severo Ochoa,” (CSIC-UAM), Madrid, Spain Ditte J. Mogensen Department of Chemistry, Aarhus University, Aarhus, Denmark Wei-Chieh Mu Program in Metabolic Biology, Nutritional Sciences & Toxicology, University of California, Berkeley, CA, United States Thomas Münzel University Medical Center at the Johannes Gutenberg University Mainz, Center for Cardiology, Cardiology I, Mainz, Germany Michael P. Murphy MRC Mitochondrial Biology Unit; Department of Medicine, University of Cambridge, Cambridge, United Kingdom Etsuo Niki Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan Matthias Oelze University Medical Center at the Johannes Gutenberg University Mainz, Center for Cardiology, Cardiology I, Mainz, Germany Peter R. Ogilby Department of Chemistry, Aarhus University, Aarhus, Denmark

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Steen Vang Petersen Department of Biomedicine, Aarhus University, Aarhus, Denmark N. Pollock Department of Musculoskeletal Biology, MRC-Arthritis Research UK Centre for Integrated research into Musculoskeletal Ageing (CIMA), Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, United Kingdom Hiran A. Prag MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, United Kingdom Naila Rabbani Clinical Sciences Research Laboratories, Warwick Medical School, University of Warwick, University Hospital, Coventry, United Kingdom Jerome Santolini Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette Cedex, France Katrin Schröder Institute for Cardiovascular Physiology, Medical Faculty of the Goethe-University, Frankfurt am Main, Germany Helmut Sies Faculty of Medicine, Institute of Biochemistry and Molecular Biology I; Leibniz Research Institute for Environmental Medicine, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany Hadley D. Sikes Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, United States Roberto Sitia Protein Transport and Secretion Unit, Division of Genetics and Cell Biology, IRCCS Ospedale San Raffaele/Università Vita-Salute San Raffaele, Milan, Italy Mette Sørensen Danish Cancer Society Research Center, Copenhagen; Department of Natural Science and Environment, Roskilde University, Roskilde, Denmark Andrea Sorrentino Cardiovascular Medicine Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, United States Corinne M. Spickett Department of Biosciences, School of Life and Health Sciences, Aston University, Birmingham, United Kingdom Wilhelm Stahl Faculty of Medicine, Institute of Biochemistry and Molecular Biology I, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany

Contributors

C.A. Staunton Department of Musculoskeletal Biology, MRC-Arthritis Research UK Centre for Integrated research into Musculoskeletal Ageing (CIMA), Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, United Kingdom Holger Steinbrenner Institute of Nutritional Sciences, Nutrigenomics Section, Friedrich Schiller University Jena, Jena, Germany Sebastian Steven University Medical Center at the Johannes Gutenberg University Mainz, Center for Cardiology, Cardiology I, Mainz, Germany C. Stretton Department of Musculoskeletal Biology, MRC-Arthritis Research UK Centre for Integrated research into Musculoskeletal Ageing (CIMA), Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, United Kingdom Sarah Tauber Institute of Nutritional Sciences, Nutrigenomics Section, Friedrich Schiller University Jena, Jena, Germany Yannick J. Taverne Department of Cardiothoracic Surgery, Cardiovascular Research Institute COEUR, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands Marion Thauvin Center for Interdisciplinary Research in Biology (CIRB) Collège de France, Paris, France Paul J Thornalley Diabetes Research Center, Qatar Biomedical Research Institute, Hamad Bin Khalifa University, Qatar Foundation, Doha, Qatar Karan Uppal Clinical Biomarkers Laboratory, Division of Pulmonary Medicine, Department of Medicine, Emory University, Atlanta, GA, United States A. Vasilaki Department of Musculoskeletal Biology, MRC-Arthritis Research UK Centre for Integrated research into Musculoskeletal Ageing (CIMA), Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, United Kingdom Sophie Vriz Center for Interdisciplinary Research in Biology (CIRB) Collège de France, Paris, France Michael Westberg Department of Chemistry, Aarhus University, Aarhus, Denmark

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Christina Wilms Department of Neurology, Medical Faculty, Heinrich-Heine Universität Düsseldorf, Dusseldorf, Germany Steve A. Wootton Human Nutrition, University of Southampton and University Hospital Southampton, Southampton, United Kingdom Mingzhan Xue Diabetes Research Center, Qatar Biomedical Research Institute, Hamad Bin Khalifa University, Qatar Foundation, Doha, Qatar Masayuki Yamamoto Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Sendai, Japan Adrian Young Department of Biochemistry, Faculty of Science, Memorial University of Newfoundland, St. John’s, NL, Canada Elias D.F. Zachariae Department of Biomedicine, Aarhus University, Aarhus, Denmark

Preface Redox biology as a research field has provided new knowledge on the role and mechanisms of oxidation-reduction reactions in physiology and in disease processes. Molecular details on redox signaling and on the function of redox master switches have been elucidated. Oxidative eustress became noted as essential in physiological redox signaling and for steady-state maintenance of stress response systems, whereas oxidative distress is associated with molecular damage and initiation of subsequent damage repair processes. “Oxidative stress” as a concept was introduced in 1985,a and it has been recognized as a general principle of aerobic life. On Google Scholar, the term has several million entries, and on PubMed >210,000 publications have accumulated. The book “Oxidative Stress: Eustress and Distress” captures the state of development of this expanding field, covering many—but certainly not all—­ important topics from exposome-induced oxidative processes to intracellular signaling cascades, and from physiology to therapy. My thanks go to the contributing colleagues, experts in their diverse topics, for their effort to present knowledge and aspects for further research through various chapters in this book. It was a pleasure and a privilege working with each one of you: thank you again! I thank Wilhelm Stahl and Carsten Berndt for their helpful discussions. I would also like to thank Peter Linsley, senior acquisitions editor, for twisting my arm to embark on this project, and Rebeka Henry, Samantha Allard, and Swapna Srinivasan from the publishers for their help. Helmut Sies Düsseldorf, July 2019

a Sies, H. (1985) Oxidative Stress: Introductory Remarks. In: Oxidative Stress (Sies, H., ed.), 1–8, Academic Press, London.

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Oxidative eustress and oxidative distress: Introductory remarks

1 Helmut Siesa,b

a

Faculty of Medicine, Institute of Biochemistry and Molecular Biology I, Heinrich-HeineUniversity Düsseldorf, Düsseldorf, Germany b Leibniz Research Institute for Environmental Medicine, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany

Abstract In the research area of redox biology, the concept of oxidative stress encompasses redox imbalance, affecting redox signaling and leading to damage of biomolecules. Stress response systems can counteract, restoring redox balance. Normal metabolism and functions maintaining steady state require low levels of oxidants, denoted as oxidative eustress. Supraphysiological oxidant challenge is denoted as oxidative distress. Oxidative stress is generated endogenously (cell metabolism) and exogenously (exposome). Fundamental life processes and diseases have an oxidative stress component, opening the field for research on redox medicine. ­Keywords: Redox biology, Oxidants, Antioxidants, Stress response, Health, Disease, Metabolism, Exposome

­Introduction “Oxidative stress” is a global concept; it has been defined as “an imbalance between oxidants and antioxidants in favor of the oxidants, leading to a disruption of redox signaling and control and/or molecular damage” (Sies & Jones, 2007). This definition accommodates the significant advances in redox biology that occurred on understanding the role of redox signaling (D’Autreaux & Toledano, 2007; Finkel & Holbrook, 2000; Jones, 2006; Rhee, 2006; Stone & Yang, 2006; Winterbourn, 2008). The original definition was formulated in 1985 in the book Oxidative Stress, as “a disturbance in the prooxidant-antioxidant balance in favor of the former” (Sies, 1985), which describes the deviation from the steady state of reduction-oxidation (redox balance) in the system under consideration. The underlying biochemistry of oxidative stress was already known to quite some extent at the time (Sies, 1986). Research then focused on oxidative damage to biomolecules. Oxidative Stress. https://doi.org/10.1016/B978-0-12-818606-0.00001-8 © 2020 Elsevier Inc. All rights reserved.

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­What is new? Meanwhile, a substantial body of additional knowledge on the molecular basis of redox regulation identified an essential role of oxidants as second messenger. This revealed that oxidative stress is two-sided (Fig. 1): maintenance of a physiological (low) level of oxidant challenge is essential for governing life processes through redox signaling, termed oxidative eustress; excessive oxidant challenge causes damage to biomolecules, termed oxidative distress (see Sies, 2017; Sies, Berndt, & Jones, 2017) and discussion later). Oxidant signaling will address specific targets. These are targets that are the most redox sensitive and that are most closely positioned to the location of origin of the oxidant. Higher exposure to oxidants will “overflow” beyond the specific targets, reaching unspecific targets and potentially cause damage. Both these situations will elicit adaptive stress responses. A physiological “redox tone” is characterized by a physiological basal level of activity in the stress response systems, while excessive activation of stress response will lead to pathophysiological consequences such as inflammation and ultimately cell death.

FIG. 1 Oxidative stress and its relationship to redox signaling. Oxidants of different nature are produced by endogenous and exogenous (exposome) sources. Removal reactions (sinks) contribute to control of steady-state levels of the diverse oxidants. Low oxidant exposure allows for addressing specific targets in the use for redox signaling (oxidative eustress), while high exposure leads to disrupted redox signaling and/or damage to biomolecules (oxidative distress). Adaptive stress responses modulate and counteract. The outcome contributes to health and disease processes. Modified from Sies, H. (2017) Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: Oxidative eustress. Redox Biology 11, 613–619; see also Sies, H. (2019). Oxidative stress: Eustress and distress in redox homeostasis. In G. Fink (Ed.). Stress: Physiology, biochemistry and pathology. Amsterdam: Elsevier, pp. 153–163. Creative Commons License.

­On the development of stress response concepts

This, in a nutshell, is the concept. However, understanding the dichotomy between eustress and distress and characterizing the boundary between them is a current challenge in redox research, as expressed in this book in considerable diversity and detail by the chapters provided by experts in the respective fields.

­On the development of stress response concepts Formulation of the basic principle of stress and stress responses dates back to Selye (1936). Early use of the term “stress” came from different fields of applied research such as rubber chemistry, examining redox reactions related to vulcanization of rubber (see Ore, 1956). Selye enlarged the scope from the general adaptation response in ensuing years (Selye, 1976). Extending the stress topic to the whole organism, he introduced the terms eustress and distress (Selye, 1975). Focusing on oxidative eustress in molecular context, there has been considerable development. Bacteriologists revealed positive roles for redox-active metabolites in signaling and behavior, calling this “redox eustress” (Okegbe, Sakhtah, Sekedat, et al., 2012). Eustress, or “positive stress,” was viewed as an effector of epigenetic modulation in physical exercise (Sanchis-Gomar et  al., 2012). Investigating lipid peroxidation, Niki noted that this process could “either turn to positive stimulus, eustress, or deleterious insult, distress” (Niki, 2009) (see also Niki, 2016 and Niki, this book). Aschbacher, O’Donovan, Wolkowitz, et al. (2013) defined “eustress” as “manageable levels of life stress [that] may enhance psychobiological resilience to oxidative damage” (see also Aschbacher, this book). Lushchak introduced a classification of oxidative stress according to intensity (Lushchak, 2014). Steady-state levels of oxidants fluctuate within in a certain physiological range, and under challenge, these may increase or decrease, resulting in oxidative or reductive stress, respectively. There may be a return to initial conditions, but there can also be a new steady state as a result of adaptation. Acquisition of such new steady state has been called “allostasis” (Sterling & Eyer, 1988), literally meaning to achieve stability through change (McEwen, 2016). A similar term for this process is “adaptive homeostasis” (Davies, 2016). On the temporal scale, acute, chronic, and repetitive oxidative stresses have been examined (Pickering, Vojtovich, Tower, & Davies, 2013). Redox homeostasis has been phrased as the “golden mean of healthy living” (Ursini, Maiorino, & Forman, 2016). Some specific kinds of oxidative stress and related terms are listed in Table 1. The evolution of concepts of stress and stress system disorders in general has been followed (Chrousos & Gold, 1992; Goldstein & Kopin, 2007), and overviews on history of oxidative stress have been presented (Estevam, Nasim, Faulstich, et al., 2015; Sies, 2018; Sies, 2019). As mentioned, adaptive oxidative stress responses are a salient feature of oxidative challenge. They are activated by molecular redox switches that initiate the activation of gene expression of defense systems, thus counteracting the challenge. The result is a resetting of redox balance, or redox homeostasis. The bacterial OxyR regulon is the paradigm for such response system, first described in 1985 (Christman, Morgan, Jacobson, & Ames, 1985). Major such “master regulators” in eukaryonts are the Nrf2/ Keap1 (Itoh, Chiba, Takahashi, et al., 1997; Yamamoto, Kensler, & Motohashi, 2018)

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Table 1  Specific kinds of oxidative stress and some related terms. Term Oxidative eustress, physiological oxidative stress Oxidative distress, pathophysiological oxidative stress Oxidant stress, prooxidant stress Redox stress, electrophilic stress Hyperoxic stress Hypoxic stress Reductive stress Hypostress, hyperstress Environmental stress (sulfur dioxide, nitrogen dioxide, and ozone) Nutritional, dietary, postprandial oxidative stress Photooxidative stress (UV-B, UV-A, visible, and infrared-A) Light stress Radiation-induced oxidative stress Nitrooxidative, nitrosative, nitrative stress Endoplasmic reticulum (ER) stress Proteotoxic stress, disulfide stress Glycoxidative stress Dicarbonyl stress Nanoparticle-induced oxidative stress Shear stress Energy stress, metabolic stress See Sies (2015) and Sies et al. (2017) for details.

and the NF-kB/I-kB systems (Oliveira-Marques, Marinho, Cyrne, & Antunes, 2009; Schreck, Albermann, & Baeuerle, 1992). In plant research, the study of photosynthetic organisms suffering from photooxidative stress led to the distinction of distress and eustress of reactive electrophiles in acclimation to light stress (Roach, Stoggl, Baur, & Kranner, 2018). In addition to direct redox responses, there are a number of other fundamental response pathways (see Halliwell & Gutteridge, 2015). These include the heat shock response, the unfolded protein response, and the hypoxia-induced response, as well as important repair programs. The latter include programs for maintaining a healthy proteome (Reichmann, Voth, & Jakob, 2018) and lipidome (Tian, Sparvero, Blenkinsopp, et al., 2019) as well as powerful DNA repair programs. Upon increased oxidant challenge, damaged products accumulate, activating removal programs such as mitophagy, autophagy, apoptosis, necroptosis, and ferroptosis. These response programs are mediated by multiple intracellular second messengers (Zhou et al., 2019). Hormesis is the phenomenon of mounting defense patterns upon low-dose challenge (Calabrese, Bachmann, Bailer, et  al., 2007), relating to the concept of preconditioning. Mitohormesis is the response to mild mitochondrial oxidative stress, impacting on life span (Ristow & Schmeisser, 2014). Thus, there is an oxidative

­On the development of stress response concepts

e­ ustress aspect on longevity. The term “positive oxidative stress” was used in relation to aging and aging-related disease tolerance (Saldmann, Viltard, Leroy, & Friedlander, 2019; Yan, 2014). A decline in adaptive homeostasis has been linked to the aging process (Pomatto & Davies, 2017). Mild ozonization, activating the Nrf2 pathway, has been considered “eustress” (Galie, Costanzo, Nodari, et al., 2018). While it may suffice for most considerations to view distress as an increase in intensity of oxidative challenge beyond the eustress range, there also is a form of distress on the other side of the eustress range, that is, toward lower or even zero challenge. This has been called oxidative hypostress, as opposed to hyperstress (see Knaus, this volume; Fig.  2 therein). Indeed, there are conditions of compromised

FIG. 2 Synoptic view of oxidative stress and its relation to nutrition and redox medicine. Cofactors include micronutrients and metal ions. Redoxins are thioredoxin, glutaredoxin, and peroxiredoxin systems. Food and nutrition, depicted on the left side for the antioxidant systems, is also part of the exposome, therefore contributing to exogenous oxidant generation. Endogenous oxidant generation includes respiratory chain and lipid oxidation. Abbreviations: GPx, glutathione peroxidase; SOD, superoxide dismutase; ROS, reactive oxygen species; RNS, reactive nitrogen species; RSS, reactive sulfur species; RCS, reactive carbonyl species; RSeS, reactive selenium species. From Sies, H., Berndt, C., Jones, D.P. (2017). Oxidative stress. Annual Review of Biochemistry 86, 715–748, with permission.

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production of reactive oxygen species that relate to disease. For example, loss-offunction mutations in the NADPH oxidases NOX1 and DUOX2 are a risk factor for intestinal bowel disease (IBD) (Lipinski, Petersen, Barann, et al., 2019). Likewise, supraphysiological selenium intake, followed by higher selenoprotein levels and, consequently, lowered H2O2 concentrations, is associated with delayed insulin signaling and thus risk of diabetes (Steinbrenner, Speckmann, Pinto, & Sies, 2011). Mitochondrial reactive oxygen species are obligatory signals for glucose-induced insulin secretion (Leloup, Tourrel-Cuzin, Magnan, et al., 2009). Another striking example relates to low-background ionizing radiation (ultralow dose): when bacteria were grown at about 80 times less than normal background ionizing radiation, there was a decrease in growth rate, accompanied by upregulation of stress response genes (Castillo, Li, Schilkey, & Smith, 2018). As part of the exposome, nutrition relates to oxidative stress at multiple levels (Dennis, Go, & Jones, 2019). Dietary supply of micronutrients as building blocks for major protective enzyme systems is one major aspect, which also applies to the supply of low-molecular mass bioactives that include several vitamins and secondary plant products (Fig. 2). On the other hand, nutritional prooxidants or precursors of prooxidants contribute to dietary oxidative stress, notably postprandial oxidative stress (Sies, Stahl, & Sevanian, 2005).

­Merits and pitfalls: Usefulness of oxidative stress concept The combination of the basic chemical notion of oxidation-reduction reactions with the biological concept of stress and stress responses may be considered a merit. However, there are pitfalls, and constructive criticism has been voiced (Azzi, Davies, & Kelly, 2004; Gutteridge & Halliwell, 2018; Levonen, Hill, Kansanen, Zhang, & Darley-Usmar, 2014; Nathan & Cunningham-Bussel, 2013). A major issue relates to the fact that the global concept of “oxidative stress” cannot—and does not pretend to—provide detailed specific information for a given biological condition. It is obvious that many redox relationships between major enzyme systems and between the low-molecular mass redox components, which are summarily termed reactive species interactome (Cortese-Krott, Koning, Kuhnle, et  al., 2017), call for powerful capacity of counterregulation. Redundancy in coping with oxidative stress and stress response systems contributes to dampening the amplitude in redox fluctuations (Santolini, Wootton, Jackson, & Feelisch, 2019). The set of principles underlying the organization of redox metabolism constitutes the “redox code” (Jones & Sies, 2015). Oxidative challenge occurs from a pleiotropy of endogenous and exogenous sources. Attempts to identify redox biomarkers for oxidative stress led to the realization that not one single biomarker can be identified for disease states linked to oxidative stress (Frijhoff, Winyard, Zarkovic, et  al., 2015; Pinchuk, Weber, Kochlik, et  al., 2019). Focusing on disease-relevant sources and targets of reactive oxygen species may lead to precise therapeutic interventions (Casas, Dao, Daiber, et al., 2015; Elbatreek, Pachado, Cuadrado, Jandeleit-Dahm, & Schmidt, 2019).

­References

­Outlook These introductory remarks circumscribe a large area of research, spanning from the molecular interactions and reaction sequences to the organismic level of a future redox medicine. The delineation of the biological meaning of oxidative eustress versus oxidative distress will be a challenge for further research. With the advent of novel powerful tools for investigating these topics, impressively demonstrated by the expert contributions from leading scientists in this book, we can look forward to deeper understanding and to hopefully useful contributions to therapy of major redox-related diseases.

­Acknowledgments Helpful discussions with Wilhelm Stahl, Carsten Berndt, and Dean Jones are gratefully acknowledged. Thanks go to Deutsche Forschungsgemeinschaft (DFG), Bonn, Germany, and to National Foundation for Cancer Research (NFCR), Bethesda, MD, USA, for long-standing research support.

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Sies, H. (2018). On the history of oxidative stress: Concept and some aspects of current development. Current Opinion in Toxicology, 7, 122–126. Sies, H. (2019). Oxidative stress: Eustress and distress in redox homeostasis. In G. Fink (Ed.), Stress: Physiology, biochemistry and pathology (pp. 153–163). Amsterdam: Elsevier. Sies, H., Berndt, C., & Jones, D. P. (2017). Oxidative stress. Annual Review of Biochemistry, 86, 715–748. Sies, H., & Jones, D. P. (2007). Oxidative stress. In G. Fink (Ed.), Encyclopedia of stress (2nd ed., pp. 45–48). Amsterdam: Elsevier. Sies, H., Stahl, W., & Sevanian, A. (2005). Nutritional, dietary and postprandial oxidative stress. The Journal of Nutrition, 135, 969–972. Steinbrenner, H., Speckmann, B., Pinto, A., & Sies, H. (2011). High selenium intake and increased diabetes risk: Experimental evidence for interplay between selenium and carbohydrate metabolism. Journal of Clinical Biochemistry and Nutrition, 48, 40–45. Sterling, P., & Eyer, J. (1988). Allostasis: A new paradigm to explain arousal pathology. In S. Fisher & J. Reason (Eds.), Handbook of life stress, cognition and health (pp. 629–649). New York: Wiley. Stone, J. R., & Yang, S. (2006). Hydrogen peroxide: A signaling messenger. Antioxidants & Redox Signaling, 8, 243–270. Tian, H., Sparvero, L. J., Blenkinsopp, P., et  al. (2019). Secondary-ion mass spectrometry images cardiolipins and phosphatidylethanolamines at the subcellular level. Angewandte Chemie (International Ed. in English), 58, 3156–3161. Ursini, F., Maiorino, M., & Forman, H. J. (2016). Redox homeostasis: The golden mean of healthy living. Redox Biology, 8, 205–215. Winterbourn, C. C. (2008). Reconciling the chemistry and biology of reactive oxygen species. Nature Chemical Biology, 4, 278–286. Yamamoto, M., Kensler, T. W., & Motohashi, H. (2018). The KEAP1-NRF2 system: A thiolbased sensor-effector apparatus for maintaining redox homeostasis. Physiological Reviews, 98, 1169–1203. Yan, L. J. (2014). Positive oxidative stress in aging and aging-related disease tolerance. Redox Biology, 2, 165–169. Zhou, D. R., Eid, R., Miller, K. A., Boucher, E., Mandato, C. A., & Greenwood, M. T. (2019). Intracellular second messengers mediate stress inducible hormesis and programmed cell death: A review. Biochimica et Biophysica Acta, Molecular Cell Research, 1866, 773–792.

CHAPTER

Epistemological challenges of the oxidative stress theory of disease and the problem of biomarkers

2

Pietro Ghezzia, Virginia Ghiarab, Kevin Daviesa a

Department of Clinical and Experimental Medicine, Brighton & Sussex Medical School, Brighton, United Kingdom b Department of Philosophy, University of Kent, Canterbury, United Kingdom

Abstract The discovery of the formation of reactive oxygen species (ROS) in biological systems and their biological consequences in terms of oxidation of proteins, lipids and DNA constitute the basis of the concept of oxidative stress (OS), and there is good evidence that this occurs in many pathological conditions. However, the idea that OS is a cause of disease and, more importantly, its logical application of translational medicine, the use of low-molecular mass antioxidants, the so-called ROS scavengers, lags behind. As a result, the only market for low-molecular mass ROS scavengers is, so far, only if the field of alternative medicine and nutritional supplement, not that of approved medicines. This chapters deals with several issues with the “oxidative stress theory of disease” (OSTD) in terms of its epistemological challenges in drawing a causal link from the association of indicators of OS in disease. This is due not only to difficulty to extrapolate conclusions from basic observation to patients but also to the fact that the OSTD is often applied to multicausal diseases where OS may not be present and contribute to the disease in all individual patients. Finally, we elaborate on the fact that ROS, unlike other disease mediators (e.g., cytokines, prostaglandins, and bacteria) cannot be measured in patients due to their short half-life, and we need to rely on markers that are indirectly linked to ROS (“biomarkers of oxidative stress”) that can be ambiguous in their meaning and, for multicausal diseases, not be elevated in all patients. We also discuss the possibility that OS, when demonstrated in all patients with a pathology, may not be a causal component of the disease but a terminal metabolic derangement. A reflection on the epistemological basis of the OSTD could explain the lack of success of ROS-targeting therapies and possibly indicate other roles of OS in disease. ­Keywords: Evidence-based medicine, Epistemology, Causation, Biomarkers, ROS scavengers, Antioxidants, Oxidative stress, Nutritional supplements, Pharmacology, Disease mechanisms

Oxidative Stress. https://doi.org/10.1016/B978-0-12-818606-0.00002-X © 2020 Elsevier Inc. All rights reserved.

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Many of us working in the broader field of oxidative stress (OS) often wonder why we never achieved clinical application of the concept as there are no medicinal products based on their reactive oxygen species (ROS) scavenging properties approved by regulatory agencies for any indication (with the possible exception of edaravone). Despite the first formulation of the oxidative stress theory of disease (OSTD) in 1956, Harman’s free radical theory of aging (Harman, 1956), a recent review of successfully validated molecular drug targets does not list OS as a cause of diseases (Santos et al., 2017). There may be several reasons for this, such as the following: (1) There may have been not enough sponsor- or investigator-initiated clinical trials performed with low-molecular mass antioxidants, often called ROS scavengers, or they were not designed properly or targeted at the right patients. (2) Broadly acting ROS scavengers may interfere with regulatory processes mediated by ROS, as shown by the growing field of redox signaling that evolved from an earlier, oversimplified concept of OS (Sies, Berndt, & Jones, 2017; Sies, 2018). Other chapters in this book will highlight the need for more targeted molecules than broad-action scavengers. (3) Although OS is often associated with the presence of diseases, it may be difficult to establish that such diseases are caused by OS (Ghezzi, Jaquet, Marcucci, & Schmidt, 2016). This might be due to the lack of accepted evidence appraisal criteria or to the problem of multiple causes. (4) Finally, the way we measure OS also have some practical and theoretical problems, also leading to weak evidence, a problem that will be discussed in the section on biomarkers and was the subject of our earlier work (Ghezzi et al., 2018; Ghezzi, Davies, Delaney, & Floridi, 2018). In this chapter, we will focus on 3 and 4 in the preceding text (causation and biomarkers).

­Causation and the OSTD ­ ssociation versus causation: The problem of confounders and A reverse causation One prerequisite for hypothesizing that OS causes a specific disease is its association with that disease. This usually means that OS must be present in disease at higher levels than in healthy controls (or in an unrelated disease; but, as mentioned earlier, OS has been implicated in most of them, so this will seldom be the case). The theory of causation in medicine has been extensively studied, and several books are a good introduction to it, although most of them analyze it in the context of epidemiology or clinical recommendations, rather than lab-based research (Howick, 2011; Illari, Russo, & Williamson, 2011). We will try here to discuss some of its aspects in relation to the OSTD. Usually, in biomedical research, a robust, statistically significant, association is a prerequisite for making a causal claim. For instance, we can find an association

­Causation and the OSTD

b­ etween a disease and a biomarker of OS, and this would be the first step to hypothesize that OS has a causal role in that disease. Although in formal logic the symbol of an arrow has a different meaning (a material conditional), we usually indicate the causal hypothesis “A causes B” as an arrow pointing from A to B (A → B). Statistical associations between the putative cause and the outcome are, however, not enough to establish causation, since the correlation may have some noncausal explanation. For instance, the correlation between (A) and (B) might be caused by a confounder (C): if B is an inflammatory disease, it could be that both OS (A) and the disease (B) are caused by inflammation (C) (C → A and C → B). This would be a scientifically plausible hypothesis, because inflammation can cause OS, for instance, through activation of ROS production by phagocytes (Smith, 1994). Taking this “confounder” (C) into account makes the causal claim A → B disappear. Furthermore, there is also the possibility that (A) and (B) are associated, but not in the way we would expect. This phenomenon is known by the name of reverse causation: OS might be a result of the disease; consequently, the causal arrow should point in the opposite direction (A ← B). We will come back to this possibility in the final sections when discussing the statistical approach to the identification and validation of biomarkers.

­Using Koch’s postulates and Bradford Hill’s criteria in OSTD studies

Koch’s postulates expanded one of the most successful theories of disease, germ theory, and provided specific criteria that need to be met for establishing causality. Adapting them to other forms of diseases is not straightforward: The postulates assume the transmissibility of the disease, but numerous diseases develop without the transmission of bacteria or viruses. A recent review on the OSTD mentioned Koch’s postulates to exemplify the importance of discovering diseases’ causes but did not attempt to propose something similar for OS (Majima et  al., 2016). This was done, to our knowledge, only for identifying the causes of Alzheimer’s disease by de la Torre (de La Torre, 2011). In essence, the study proposed three criteria that should be met by causal hypotheses of disease: (1) The causal factor(s) should precede the symptoms of the disease; (2) targeting these factors should prevent or reverse the disease; and (3) targeting these factors should decrease the disease incidence. De la Torre argued that the OSTD would only meet the first of them, while other theories, such as the inflammatory or the cholinergic one, would not fit any (de La Torre, 2011). If we consider the abundant literature on the operation of these mechanisms, this conclusion might seem surprising. The problem, we argue, is that de la Torre mainly considered meta-analyses of evidence-based findings in clinical trials (de La Torre, 2011). His decision was aligned with the evidence hierarchies used in evidence-based medicine (EBM), whereby meta-analyses and randomized controlled trials (RCTs) are ranked more highly than observational studies, expert opinions and case reports. According to such hierarchies, furthermore, experimental, preclinical studies either do not count or are at the very bottom of the evidence pyramid, their importance being mainly in providing a rationale for initiating clinical studies.

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Applying EBM hierarchies to OS studies, however, involves not giving due importance to a massive amount of literature relating to oxidative stress in models, in  vitro and in  vivo, related also to disease. There are several reasons why EBM hierarchies can be considered inadequate when evaluating evidence in OS studies. One of the main limitations discussed so far in the literature (Blunt, 2015; Parkkinen et al., 2018) is the assumption that the quality of a piece of evidence can be determined straightforwardly by considering the methodology used to collect it. While these hierarchies may help to make decisions regarding the efficacy and approval of medicinal products, this limitation can weaken their usefulness, especially when used for the study of the pathogenesis of disease. Different types of studies can provide different types of high-quality evidence, which can be used to address the criteria that should be met in the case of causation, like those proposed by Koch. In addition to Koch’s postulates, furthermore, another set of criteria that might be used in in OSTD studies include the Bradford Hill’s criteria of causation: these are nine issues that Bradford Hill argued we should address when inferring causation from an observed association (Hill, 1965) (Table 1). In our view, these criteria, even if originally devised for epidemiological studies, could be useful in OSTD studies in assessing the causal effects triggered by OS.

Table 1  Bradford Hill’s criteria of causation. Criterion

Description

Strength

Extent (quantitative) of the effect, for example, an increase in mortality by a factor of 10 compared with a factor of 1.5 Reproducibility, both in terms of reproduced by different researchers (replicability) and in different cohorts/experimental setting (external validity, inferential reproducibility, and extrapolability) The cause is linked to a specific disease (even if smoking causes all types of disease, the strength of the association with lung cancer is far higher) The cause must precede the effect Such as a dose-dependent effect There is a theoretical basis for concluding that the relationship is causal (it helps but depends on scientific knowledge of biology at this time) Causal hypothesis should not conflict with known facts (this also depends on scientific knowledge at the time) Can we modify the disease if we modify the cause (e.g., will quitting smoking reduce cancer?) Are there analogous causal relations for the factor of the disease?

Consistency

Specificity

Temporality Biological gradient Biological plausibility

Coherence Experiment Analogy

Derived from Hill, A. B. (1965). The environment and disease: Association or causation? Proceedings of the Royal Society of Medicine 58, 295-300 and Holland, P. W. (1986). Statistics and causal inference. Journal of the American Statistical Association 81, 945-960.

­Causation and the OSTD

As for Koch’s postulates, furthermore, verifying if Bradford Hill’s criteria are met might require different types of evidence.

­Experimental approach to the study of the OSTD

We think that all evidence collected from studies related to the role of OS in disease, rather than being distinguished according to the methodology used to collect it, can be assigned to one of the typologies listed in Table 2. Broadly, the first two types of evidence somewhat relate to two of Koch’s postulates (isolation of the infectious agent and reproduction of the disease by inoculation of the infectious agent) and to two Bradford Hill’s criteria (strength and temporality). The third, to one of those proposed by de La Torre (2011) (targeting the causal factor should prevent or reverse the disease) and to Bradford Hill’s “experiment” criterion. Moreover, while the first type of evidence would provide information about the association between OS and diseases, the second and third types would offer insights into the mechanisms through which OS causes diseases. We might argue that all these types of evidence must be obtained for OS to be considered a causal factor in a disease. However, we would immediately encounter a problem because it will be difficult to find a disease for which at least one evidence of every type has been published. Consequently, this would lead us to conclude that OS is not a causal factor of specific disease, as we will discuss in the final section. Alternatively, we might accept an emerging approach in the literature and establish that, in order for OS to be considered a causal factor in a disease, both a correlation between OS and the disease and a mechanism linking OS and the disease should be identified (Clarke, Gillies, Illari, Russo, & Williamson, 2014; Gillies, 2011; Illari, 2011; Russo & Williamson, 2007). Even in this case, however, we would have to decide how to establish the presence of a correlation and of a mechanism and, consequently, how to evaluate the quality of the available evidence. There is a general agreement that, in OS studies, experimental evidence hierarchies have in  vitro studies on cell lines at the lowest level; then in vitro in primary cells or organotypic cultures; then ex vivo and in vivo Table 2  Types of experimental evidence in the OSTD. 1. Evidence of association of OS with disease 2. Effect of OS on diseaserelated outcomes/ processes 3. Effect of ROS scavengers on disease-related outcomes/processes

Measures of markers of OS in patients/animal models or in vitro models (e.g., macrophages exposed to inflammatory stimuli, neurons exposed to excitotoxins) Effect of inducing ROS (applying ROS or decreasing endogenous antioxidant enzymes or low-molecular weight ROS scavengers) on: expression of inflammatory genes in vitro or vivo; neurotoxicity, etc. For example, effect in disease models (animal or patients) on non-OS related markers (e.g., disease severity, surrogate markers of disease, disease-related processes such as levels of inflammatory mediators or signaling pathways)

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CHAPTER 2  Epistemological challenges of the oxidative stress theory

in animal models; and, at the highest level, in vivo in patients. This approach, however, would lead us to commit the same mistake made in EBM, where the evidence hierarchy is just based on the type of methodology employed. Another possible approach we might employ is based on the “Grading of Recommendations Assessment, Development, and Evaluation” (GRADE) (Guyatt et al., 2008; Guyatt et al., 2011). Although GRADE provides a hierarchy of evidence similar to the original EBM one, it also takes into account strengths and weaknesses of each evidence. In GRADE, the quality of each evidence is graded from high to very low based on criteria such as risk of bias, consistency across different studies, precision, magnitude of the effect, biological gradient, and possible confounders (Guyatt et al., 2008, Guyatt et al., 2011), many of which reflect some of the Bradford Hill’s criteria. GRADE is tailored for rating the quality of evidence for clinical recommendations, not for assessing the validity of a causal claim. Recently, however, Parkkinen et al. (2018) have developed a GRADE-style table for mechanistic evidence assessment, which is designed to help to determine the status of a causal claim. In our view, this offers a good starting point to examine the strength and consistency of key experimental results on OS effects. At the beginning of this chapter, we argued that the lack of evidence appraisal tools might prevent the identification of strong causal evidence from the growing volume of literature on the OSTD and the large number of diseases implicated. There are, however, further limitations influencing the collection of high-quality evidence that might contribute to the weakness of the OSTD. To begin with, the OS literature is still characterized by publication bias by which only positive findings (e.g., inhibitory effect of a ROS scavenger and increased levels of OS) are published and the ones that do not pass the P 30 smallmolecule drugs are in clinical use currently, amassing more than $30 million annually, with >150 clinical trials waiting FDA approval (Oprea et al., 2018). Perhaps, the same consideration should be given with respect to developing redox-based therapeutic strategies, which should be targeted at specific cellular sources of ROS or specific redox-based events, rather than attempting to suppress ROS in a nonselective manner. Interestingly, many of the currently developed kinase inhibitors are targeted to their ATP binding regions, and in a growing number of cases, these concern covalent drugs that target noncatalytic cysteines within their ATP binding site (Gushwa, Kang, Chen, et al., 2012; Liu et al., 2013; Zhao & Bourne, 2018). Oxidative modification of these cysteines would negatively impact on the efficacy of these inhibitors, especially when they are associated with enhance kinase activity, and approaches that selectively target these oxidized forms of, e.g., tyrosine kinases may represent an attractive alternative strategy.

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FoxO transcription factors in the control of redox homeostasis and fuel metabolism

17

Sarah Tauber, Holger Steinbrenner, Lars-Oliver Klotz Institute of Nutritional Sciences, Nutrigenomics Section, Friedrich Schiller University Jena, Jena, Germany

Abstract Transcription factors of the forkhead box, class O (FoxO) family play a key role in controlling cellular responses to oxidative and metabolic stress and help to maintain or restore cellular redox and fuel homeostasis. FoxOs stimulate and, in a few cases, repress the transcription of target genes that mediate antioxidant protection; cell fate decisions; and the availability of glucose, lipids, and proteins as sources of ATP production. FoxO target genes include antioxidant enzymes such as superoxide dismutases and peroxiredoxins, the plasma selenium transporter selenoprotein P, key gluconeogenesis enzymes, and autophagy proteins. Regulation of FoxO transcriptional activity occurs primarily through posttranslational modification, such as reversible phosphorylation and acetylation, and through interaction with various coactivators, corepressors, and other transcription factors. Redox signaling is implicated in many of those regulatory mechanisms in a direct or indirect manner, rendering ROS, namely, hydrogen peroxide, and major determinants of FoxO activity. Notwithstanding the many beneficial (homeostatic) actions of FoxOs, it is crucial to restrain FoxO activity under physiological and even more so under some pathological conditions, where FoxOs have been found to act as “double-edged swords.” This dual role is well known with respect to diabetes mellitus, as FoxO activation can elicit beneficial and detrimental responses in the major insulin target tissues and in the insulin-producing pancreatic β-cells. ­Keywords: FoxO1, DAF-16, Hydrogen peroxide, Akt, Insulin, Selenoprotein, Starvation, Diabetes, Gluconeogenesis

­Introduction Whereas oxidation of cellular macromolecules by reactive oxygen species (ROS) has been known for decades to occur and has been usually linked to damaging consequences, a role of radical (superoxide and nitrogen monoxide) and ­nonradical (H2O2, lipid hydroperoxides, peroxynitrite, and singlet oxygen) ROS as Oxidative Stress. https://doi.org/10.1016/B978-0-12-818606-0.00017-1 © 2020 Elsevier Inc. All rights reserved.

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­intracellular ­signaling messengers is being increasingly noticed (Brigelius-Flohe & Flohe, 2011; Klotz, 2002, 2015; Sies, Berndt, & Jones, 2017; Stone & Yang, 2006). Regarding carbohydrate and lipid metabolism, H2O2 was found to mimic and to enhance insulin signaling (Czech, Lawrence, & Lynn, 1974b; Mahadev et  al., 2001). Insulin receptor activation results in modulation of gene expression, in part mediated through inhibition of transcription factors of the forkhead box, class O (FoxO) family. Redox regulation of the latter is well established and occurs at several levels. The transcriptional activity of FoxOs is attenuated in the fed state, whereas metabolic stress (e.g., fasting, starvation, and diabetes mellitus) generally activates FoxO signaling (Eijkelenboom & Burgering, 2013; Gross, van den Heuvel, & Birnbaum, 2008).

­FoxOs: General aspects Since description of the first protein with a “forkhead box” motif (Weigel, Jurgens, Kuttner, Seifert, & Jackle, 1989), the winged helix/forkhead box (Fox) family of transcription factors has been demonstrated to comprise isoforms from several species, all sharing the characteristic DNA-binding domain consisting of three α-helices and two large loops (“wings”). Orthologs of the subclass O proteins (FoxO) were identified across animal phyla, including dauer formation-16 (DAF16) in Caenorhabditis elegans (C. elegans), which is associated with metabolic and developmental processes, insulin/insulin-like signaling (IIS) and longevity in the model organism (Kenyon, 2011; Murphy, 2006). DAF-16 activation enhances the biosynthesis of proteins that contribute to protection against various kinds of stress (e.g., exposure to oxidative, heat, and xenobiotic stress), while potentially deleterious genes are downregulated (Murphy, 2006). Mammals express four FoxO isoforms (FoxO1, 3, 4, and 6), encoded by distinct genes. FoxOs are transcriptional regulators, primarily activating, but in some cases also repressing gene expression, as seen in the reciprocal impact of FoxO1 on the gluconeogenic enzyme glucose6-phosphatase versus the glycolytic enzyme glucokinase in hepatocytes. In mammals, FoxOs are involved in the regulation of fuel metabolism, redox homeostasis, and defense against oxidative stress, as well as in the regulation of cell proliferation, differentiation, or death. All four isoforms are ubiquitously expressed and bind to the same FoxO-responsive sites (DAF-16-binding elements, DBEs) in the promotors of target genes, thus resulting in a certain degree of redundancy. Nevertheless, there are also cell- and tissue-specific effects of the four FoxO isoforms due to differences in their expression levels and regulation. A multitude of reversible posttranslational modifications (e.g., Ser/Thr phosphorylation and O-GlcNAcylation, Lys acetylation and ubiquitination, and Cys oxidation) and interactions with coregulators govern nucleo-cytoplasmic shuttling and transcriptional activity of FoxOs (Barthel, Schmoll, & Unterman, 2005; Eijkelenboom & Burgering, 2013; Klotz et al., 2015).

Molecular mechanisms underlying redox regulation

­ olecular mechanisms underlying redox regulation of FoxO M transcriptional activity Insulin is the major metabolic regulator of FoxO transcriptional activity. In response to binding of insulin to its receptor, protein kinase B (Akt) is phosphorylated and activated and, in turn, phosphorylates FoxO proteins, resulting in attenuation of their transactivating activity and, in the case of FoxOs 1, 3 and 4, in nuclear exclusion (Barthel et al., 2005). FoxO6, unlike the other three FoxO isoforms, does not undergo nuclear exclusion in response to insulin-induced phosphorylation (van der Heide, Jacobs, Burbach, Hoekman, & Smidt, 2005). Several proteins in the insulin signaling cascade possess Cys residues that are prone to reversible oxidation and contribute to redox regulation. These proteins include not only FoxOs themselves but also Akt and phosphatases that counterregulate insulin-induced phosphorylation: protein tyrosine phosphatase 1B (PTP-1B) and phosphatase and tensin homolog (PTEN) (Klotz et al., 2015; Szypowska & Burgering, 2011). Reactive oxygen species and H2O2, in particular, can both augment and attenuate IIS-induced FoxO phosphorylation (Fig. 1): (a) Insulin stimulates a burst of ROS (superoxide and H2O2) production through activation of membrane-bound NADPH oxidases, which occurs in parallel with signaling via the insulin receptor. H2O2 generated in response to insulin causes the reversible oxidation of redox-sensitive Cys residues at the active sites of PTP-1B and PTEN, thus provoking their transient inactivation. This is interpreted as a feed-forward regulation to enhance and extend insulin signaling (Mahadev et al., 2004; Steinbrenner, 2013; Szypowska & Burgering, 2011), and it provides a mechanism for how H2O2 may attenuate FoxO transcriptional activity, i.e., by increasing its insulin-induced phosphorylation and nuclear exclusion. Interestingly, H2O2 has already been reported in the 1970s to mimic effects of insulin on carbohydrate metabolism (Czech, Lawrence, & Lynn, 1974a). Xenobiotics such as arsenite may also mimic insulin-induced phosphorylation of FoxOs, probably through generation of ROS (Hamann & Klotz, 2013; Klotz et al., 2015). (b) Conversely, ROS have also been described to counteract growth factor–induced signaling to Akt: intramolecular disulfide formation between Akt2 residues Cys124 and either Cys297 or Cys311 results in inactivation of this Akt isoform. This oxidative inactivation of Akt2 is achieved in cultured cells exposed to PDGF in the presence of an inhibitor of catalase, indicating that it may occur under physiological conditions. Interestingly, oxidized Akt2 is inactive despite its activating phosphorylation (at Thr309, equivalent to Thr308 in Akt1) being increased. Moreover, inactivation seems to be isoform-specific, as Akt1 and Akt3 activities appear to be unimpaired by oxidative conditions, neither in cultured cells nor with isolated proteins exposed to hydrogen peroxide (Wani et al., 2011).

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FIG. 1 “ROS” and “non-ROS” routes for the modulation of FoxO activity along the insulin receptor/IGF-1 receptor dependent signaling cascade. Insulin or IGF1, via stimulation of their receptor (InsR or IGF1-R), via phosphoinositide 3′-kinase (PI3K)-induced generation of 3′-phosphorylated phosphoinositides and via the subsequent activation of the serine/ threonine kinase Akt, causes inactivation of FoxO transcription factors. This cascade is controlled by protein tyrosine phosphatases such as PTP1B and the lipid phosphatase PTEN. In addition to oxidation (A–D), the cascade can be interfered with by nonoxidants, such as zinc ions (B), or through Michael addition of the lipid peroxidation product 4-hydroxynonenal (HNE) to a susceptible cysteine residue (C). See text for further details. Figure taken from Klotz, L. O., & Steinbrenner, H. (2017). Cellular adaptation to xenobiotics: Interplay between xenosensors, reactive oxygen species and FOXO transcription factors. Redox Biology, 13, 646–654, with permission.

(c) Further kinases linked with IIS are also affected by ROS. For example, extracellular signal-regulated kinases (ERK) 1 and 2 are activated under oxidative stress to phosphorylate FoxO1 and FoxO3. Phosphorylation of FoxO1 by ERK1/ERK2 and by another mitogen-activated protein kinase (MAPK), p38-MAPK, affects the interaction of FoxO1 with transcription factor Ets-1 and thus regulates its function as a coactivator (Asada et al., 2007; Yang et al., 2008).

Molecular mechanisms underlying redox regulation

In addition, there is ample experimental evidence demonstrating that ROS affect FoxO transcriptional activity independent of insulin/insulin-like signaling: (a) FoxO4 possesses two threonine residues, Thr447 and Thr451, which can be phosphorylated by c-Jun N-terminal kinases (JNK). H2O2 activates JNK, which in turn may phosphorylate FoxO4 at Thr447 and Thr451. In contrast to Akt-mediated phosphorylation, phosphorylation by JNK results in nuclear accumulation and activation of FoxO4 (Essers et al., 2004). (b) H2O2-induced oxidative stress promotes hyperactivation of cyclin-dependent kinase 5 (Cdk5) in mouse cortical neurons. Cdk5, Cdk1 and Cdk2 were demonstrated to phosphorylate FoxO1 at Ser249, resulting in exclusion of FoxO1 from the nucleus and inhibition of its transcriptional activity (Huang, Regan, Lou, Chen, & Tindall, 2006; Liu, Kao, & Huang, 2008; Zhou et al., 2015). In contrast, and somewhat paradoxically, another study reported an increase in FoxO1 nuclear accumulation and transcriptional activity upon Ser249 phosphorylation by Cdk1 (Yuan et al., 2008), suggesting that cell type and exposure conditions direct the impact that Cdks have on FoxO1 function. (c) H2O2 exposure results in acetylation of FoxO3, which subsequently moves from the cytosol into the nucleus, where it is deacetylated by the nuclear sirtuin Sirt1. Deacetylated FoxO3 is trapped within the nucleus and stimulates the transcription of cell cycle arrest genes (e.g., cyclin-dependent kinase inhibitor p27Kip1, CDKN1B), DNA repair genes (e.g., growth arrest and DNA-damage-inducible protein 45, GADD45), and antioxidant enzymes (e.g., superoxide dismutase 2, SOD-2), whereas transcription of apoptotic genes is decreased. The authors of the corresponding study interpret this pattern as a shift in the capability of deacetylated FoxO3 to maintain cellular integrity instead of promoting cell death (Brunet et al., 2004). In support of this hypothesis, deacetylation of FoxO3 and its coactivator peroxisome proliferator activated receptor γ-coactivator 1α (PGC-1α) by Sirt1 stimulated the formation of a FoxO3/PGC-1α complex and increased the transcription of additional antioxidant enzymes such as catalase, peroxiredoxins (Prx) 3 and 5, and thioredoxin reductase 2 (TrxR2) (Olmos et al., 2013). Like FoxO3, FoxO1 and FoxO4 become acetylated in response to H2O2 exposure, which has been shown to be catalyzed by p300/CBP acetyltransferase. Treatment of HEK293 human embryonic kidney cells with H2O2 induced the formation of a heterodimer of FoxO4 and p300/CBP, linked through a disulfide bond between redox-sensitive Cys residues. Acetylation decreased the transcriptional activity of FoxO4 (Dansen et al., 2009). A bimodal regulation of FoxO1 in response to H2O2 exposure, consisting of p300-/CBP-mediated acetylation of cytosolic FoxO1 and subsequent Sirt1-mediated deacetylation upon FoxO1 nuclear translocation, has been proposed to contribute to protection of pancreatic β-cells against acute metabolic distress through increased transcription of FoxO1 target genes (Kitamura et al., 2005).

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(d) H2O2 exposure also enhances the physical and functional interaction between FoxOs and the Wnt-responsive transcription factor β-catenin. This evolutionarily conserved interaction has been demonstrated for mammalian FoxO1, 3 and 4 with β-catenin and for the respective C. elegans orthologs DAF-16 and BAR-1. FoxO/β-catenin interaction results in increased transcriptional activity of FoxOs, thus contributing to better cellular stress resistance through stimulation of the transcription of antioxidant enzymes such as SOD-2 (Essers et al., 2005). (e) Throughout the evolution of vertebrates, FoxO3 and FoxO4 have acquired isoform-specific redox-sensitive Cys residues, which appears to contribute to differences between the FoxO isoforms with respect to redox regulation (Putker et al., 2015). Both FoxO3 and FoxO4 form heterodimers with a number of other proteins through the formation of intermolecular disulfide bonds upon exposure to H2O2. A few overlapping binding partners of the two FoxO isoforms were identified, in particular peroxiredoxins, while the majority of binding partners was specific to either FoxO3 or FoxO4. As an example, redoxdependent heterodimerization with importin-7 and importin-8 is required for the H2O2-induced nuclear import of FoxO3 but not for FoxO4 translocation. Instead, FoxO4 forms a disulfide bond with another nuclear import protein, transportin-1, in response to H2O2 exposure (Putker et al., 2013; Putker et al., 2015). (f) Compared with FoxO3 and FoxO4, FoxO1 and FoxO6 have been much less studied with respect to their redox regulation. FoxO1 has the highest number of Cys residues (seven in human FOXO1) among the mammalian FoxO isoforms. Nevertheless, H2O2-induced heterodimers of FoxO1 with other proteins have not been identified yet. Recently, we reported that human FOXO1 interacts, through its most C-terminal Cys residue, Cys612, with its coregulators CBP and PGC-1α under basal (nonstressed) conditions. Cys612 was required for full basal transactivation activity of FoxO1 (Tsitsipatis, Gopal, Steinbrenner, & Klotz, 2018). We also showed that oxidative stress induced by the thiol-reactive α,β-unsaturated carbonyl compound diethyl maleate (DEM) caused trapping of FoxO1 in the nucleus; however, this was independent of FOXO1 cysteine residues, and the nuclear FOXO1 was inactive (Gille et al., 2019).

­ ole of FoxOs in the regulation of redox homeostasis R and defense against oxidative stress FoxO target genes in mammals comprise a full battery of antioxidant enzymes located in different cellular compartments and catalyzing the decomposition of various oxygen reduction states, such as superoxide and hydrogen peroxide (Fig.  2). For example, expression of mitochondrial SOD-2 was shown early on to be regulated by FoxOs (Kops et al., 2002); the same holds true for peroxisomal catalase (Nemoto & Finkel, 2002), mitochondrial thioredoxin reductase 2 (TrxR2) and peroxiredoxins

Role of FoxOs in the regulation of redox homeostasis

FIG. 2 FoxO target genes coding for antioxidant proteins: subcellular localization and functional significance of gene products. Abbreviations: CP, ceruloplasmin; GPx, glutathione peroxidase; GSH, glutathione; GSSG, glutathione disulfide; LPO, lipid peroxidation; MT, metallothionein; Prx, peroxiredoxin; SelP, selenoprotein P (also known as SELENOP); SOD, superoxide dismutase; Trx, thioredoxin, TrxR, thioredoxin reductase. Inset: color code to indicate subcellular localization of proteins. Figure taken from Klotz, L. O., Sanchez-Ramos, C., Prieto-Arroyo, I., Urbanek, P., Steinbrenner, H., & Monsalve, M. (2015). Redox regulation of FoxO transcription factors. Redox Biology, 6, 51–72, with permission.

Prx3 and Prx5 (Chiribau et al., 2008; Olmos et al., 2013), and probably also cytosolic glutathione peroxidase 1 (GPx1) (Marinkovic et al., 2007). Two of the aforementioned antioxidant enzymes, GPx1 and TrxR2, are selenoproteins, whose biosynthesis requires an adequate supply of the essential trace element and micronutrient selenium. Selenium is co-translationally incorporated into selenoproteins as selenocystein. In mammals, selenoprotein P (SELENOP) represents the major transport form of selenium in plasma. SELENOP is secreted from hepatocytes and supplies peripheral cells with selenium for the biosynthesis of selenoproteins, including antioxidant selenoenzymes (Steinbrenner, Speckmann, & Klotz, 2016). In this regard, we identified SELENOP as a FoxO1 target gene. FoxO1, together with its coactivator PGC-1α regulates SELENOP gene expression in hepatocytes, in ­response to insulin signaling (Speckmann et al., 2008; Walter, Steinbrenner, Barthel, & Klotz, 2008).

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Similar to selenoprotein P, the expression of another liver-derived protein involved in trace element homeostasis, ceruloplasmin (CP), is controlled by FoxOs (Leyendecker et  al., 2011; Sidhu, Miller, & Hollenbach, 2011). CP is the major copper-containing plasma protein in mammals, and it serves as a ferroxidase that converts Fe2+ exported from cells to Fe3+, thus allowing for iron loading of its plasma transport protein, transferrin. Moreover, Fe2+ oxidation prevents the generation of highly reactive radicals in Fenton-type reactions of Fe2+ with H2O2 or lipid hydroperoxides (Hentze, Muckenthaler, & Andrews, 2004). FoxOs may directly and indirectly affect transcriptional activity of nuclear factor erythroid 2-related factor 2 (Nrf2), a “xenosensor” well known for its role in xenobiotic metabolism that is involved also in the control of cellular redox homeostasis (Klotz & Steinbrenner, 2017). Nrf2 is usually being kept inactive and subjected to proteasomal degradation by binding to its negative regulator, Kelch-like ECH-associated protein 1 (Keap1). ROS may induce the oxidation of redox-sensitive cysteine residues in Keap1, causing release and subsequent nuclear translocation of Nrf2 (Fourquet, Guerois, Biard, & Toledano, 2010). This results in induction of Nrf2 target genes, including those encoding cytoprotective enzymes required for glutathione biosynthesis (e.g., γ-glutamylcysteine synthetase, GCS) and ROS elimination (e.g., TrxR1 and Prx1) (Yamamoto, Kensler, & Motohashi, 2018). Interaction between FoxOs and the Nrf2 system is evident also in C. elegans, where exposure to the Nrf2 activator, diethyl maleate, causes an enhanced stress resistance and life span extension. These positive effects rely both on the C. elegans ortholog of Nrf2, SKN-1 and on C. elegans FoxO, DAF-16 (Urban et al., 2017). Although the exact mode of interaction of these two transcriptional regulators is unknown at present, the SKN-1 promoter region contains two potential binding sites for DAF-16; thus, SKN-1 expression may be stimulated by DAF-16 (Tullet et al., 2017). Two possible ways of interaction between Nrf2 and FoxO systems in mammalian cells have been outlined so far (for review, see (Klotz & Steinbrenner, 2017)): (i) FoxOs may attenuate ROS-mediated Keap1 oxidation by mounting an antioxidative response and thus attenuate Nrf2 activation, and (ii) FoxO3 has been shown to stimulate transcription of Keap1 in human tumor cell lines (Guan et al., 2016), hence acting as a negative regulator of Nrf2.

­Role of FoxOs in the regulation of fuel metabolism Besides oxidative stress, metabolic stress represents a major environmental challenge that causes FoxO activation. During fasting and starvation, FoxOs stimulate the expression of genes coding for key proteins of gluconeogenesis and autophagy, to supply cells with glucose and amino acids and thus to cope with the cellular demand for nutrients and energy. In contrast, FoxO transcriptional activity is attenuated after food intake through insulin-induced FoxO phosphorylation. FoxO expression is highly abundant in the major insulin-responsive tissues and in the insulin-producing pancreatic β-cells, with highest levels of FoxO1 in the latter as compared with the

Role of FoxOs in the regulation of fuel metabolism

other FoxO isoforms. In addition to direct effects on fuel metabolism, FoxOs may affect the formation of skeletal muscle, adipose tissue, and pancreatic islets, through regulation of differentiation and/or proliferation of myocytes, adipocytes, and β-cells. Dysregulated activation of FoxOs under conditions of insulin resistance and diabetes mellitus contributes to the development of diabetic features such as hyperglycemia, dyslipidemia, and the loss of β-cell mass. On the other hand, the concomitant FoxO-mediated induction of antioxidant enzymes may help to preserve cellular integrity and function, as development and progression of diabetes mellitus have been linked to oxidative stress (Eijkelenboom & Burgering, 2013; Gross et al., 2008; Rains & Jain, 2011). This link between metabolic and oxidative stress is also observed during starvation, which may be associated with the formation of H2O2 (Scherz-Shouval et al., 2007). The key role of the FoxO ortholog DAF-16 in IIS signaling was first identified in C. elegans (for review, see Kenyon, 2011). Since then, distinct tissue-specific effects of FoxO activation on fuel metabolism caused by alteration of gene expression in pancreatic β-cells and insulin target tissues have been elucidated in mammals.

­FoxOs and pancreas In the adult pancreas, FoxO1 is exclusively expressed in β-cells of the islets, where it is mainly localized in the cytoplasm due to the autocrine action of secreted insulin, stimulating FoxO1 nuclear exclusion (Kitamura & Ido Kitamura, 2007). A similar regulation is observed during acute exposure of starved mouse islets and MIN6 insulinoma cells to high glucose, which resulted in rapid phosphorylation of FoxO1 and its cytoplasmic translocation, presumably mediated through glucose-induced insulin secretion (GSIS) (Martinez, Cras-Meneur, Bernal-Mizrachi, & Permutt, 2006). In contrast, chronic exposure to high glucose appeared to sustain FoxO activity in INS1 insulinoma cells: FoxO1 inhibited glucose-induced gene expression of thioredoxininteracting protein (TXNIP), a negative regulator of thioredoxin and promoter of β-cell apoptosis, by competing with the transcription factor carbohydrate response element-binding protein (ChREBP) (Kibbe, Chen, Xu, Jing, & Shalev, 2013). FoxO activation may thus contribute to protection of β-cells from damage induced by oxidative stress that is associated with hyperglycemia (Rains & Jain, 2011). Indeed, oxidative stress has been shown to induce nuclear translocation of FoxO1 in β-cells, and this resulted in protection from β-cell failure through upregulation of the transcription factors MafA and NeuroD, which are implicated in β-cell differentiation (Kitamura et al., 2005). Regarding fuel usage, FoxOs promote the use of glucose for generation of ATP in β-cells: mice with a triple knockdown of the FoxO isoforms 1, 3 and 4 preferentially utilized lipids rather than glucose as energy source, resulting in impaired GSIS (Kim-Muller et  al., 2014). However, FoxOs may also have detrimental effects on β-cells: FoxO1 acts as a repressor of FoxA2-dependent gene expression of the transcription factor pancreas/duodenum homeobox protein 1 (Pdx1), thus suppressing Pdx1-mediated β-cell proliferation and growth (Kitamura et al., 2002). FoxO1 nuclear translocation induced by exposure of INS1 cells to fatty

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acids and high glucose resulted in induction of the proapoptotic protein Bax and apoptotic cell death, thus contributing to glucolipotoxicity (Kim et al., 2005). Taken together, these data point to a dual role of FoxO1 in pancreatic β-cells: Tightly controlled FoxO1 activation supports β-cell differentiation, integrity, and function (e.g., GSIS), while dysregulation of FoxO1 may result in apoptosis and decreased proliferation and thus contribute to the loss of β-cell mass in diabetes mellitus.

­FoxOs and the liver The ability of the liver to appropriately respond to changes in nutrient supply through hormonal regulation of key enzymes involved in glucose utilization and production, respectively, is vital for the maintenance of systemic fuel homeostasis. FoxOs are transcriptional activators of glucose-6-phosphatase (G6PC) and phosphoenolpyruvate carboxykinase (PEPCK) as well as transcriptional corepressors of glucokinase in hepatocytes (Ganjam, Dimova, Unterman, & Kietzmann, 2009; Nakae, Kitamura, Silver, & Accili, 2001; Schmoll et al., 2000). FoxO activation in response to fasting/ starvation thus promotes a shift from hepatic glucose metabolism to glucose production (and subsequent release), to maintain blood glucose levels and glucose supply of extrahepatic tissues (Pajvani & Accili, 2015). Moreover, FoxO1 induces hepatic autophagy under conditions of nutrient deprivation (Liu et al., 2009). While FoxO1 represents the major FoxO isoform in hepatocytes, FoxO3, FoxO4, and FoxO6 have been shown to promote hepatic glucose production as well (Haeusler, Kaestner, & Accili, 2010; Kim et  al., 2011). Association of FoxOs with various transcription factors and coregulators may allow for fine-tuning of the transcriptional response, cross-talk with other signaling pathways and integration of other signals such as ROS (Eijkelenboom & Burgering, 2013), as it has been shown for the interaction of FoxO1 with β-catenin in the starved liver (Liu et al., 2011). As mentioned earlier, insulin-stimulated activation of protein kinase B (Akt) results in FoxO phosphorylation and inactivation; in the liver, Akt appears to rather restrain than to completely suppress FoxO transcriptional activity under physiological conditions (Lu et  al., 2012). In the insulin-resistant liver, unchecked FoxO activation results in dysregulated expression of gluconeogenic enzymes and thus contributes to the development of hyperglycemia, which is characteristic of diabetes mellitus (Dong et  al., 2008; Gross et al., 2008).

­FoxOs and skeletal muscle FoxOs are implicated in the adaptive response to fasting and starvation in the skeletal muscle, where FoxO activation promotes a switch from glucose to fatty acid oxidation as preferential mode of ATP generation and the breakdown of proteins (Eijkelenboom & Burgering, 2013; Gross et al., 2008). In myocytes, FoxO1 stimulates gene expression of pyruvate dehydrogenase kinase-4 (PDK-4), an enzyme that inhibits the pyruvate dehydrogenase-mediated conversion of pyruvate into

­Concluding remarks

a­ cetyl-CoA and thus the glycolytic flux (Bastie et al., 2005). Fatty acid uptake and metabolism are increased by FoxO1 in myocytes through directing the fatty acid translocase CD36 to the plasma membrane and through upregulating gene expression of lipoprotein lipase, which hydrolyzes plasma triglycerides, and peroxisomal fatty acid oxidase (Bastie et al., 2005; Kamei et al., 2003). FoxO3 promotes lysosomal proteolysis in myocytes, through upregulation of autophagic proteins, and to a lesser extent also proteasomal protein degradation (Zhao et al., 2007). This is thought not only to ensure the systemic supply of amino acids during starvation but also to contribute to the development of muscle atrophy. Interestingly, caloric restriction has been found to activate FoxO3 and FoxO4 in the skeletal muscle of humans and rats, resulting in upregulation of FoxO target genes implicated in autophagy, stress resistance and DNA repair (Mercken et al., 2013). FoxOs play also a role in skeletal muscle formation by affecting both myocyte differentiation and muscle fiber type specification. FoxO1 suppresses early myoblast differentiation, while it appears to be required during terminal differentiation to promote the fusion of myoblasts to myotubes (Xu, Chen, Chen, Yu, & Huang, 2017).

­FoxOs and adipose tissue The dual, stage-dependent role of FoxOs during myocyte differentiation is reminiscent of their impact on the differentiation of adipocytes, which occurs due to cell turnover and in response to a need for storage of excess fat in the adipose tissue. Even though mRNA and protein levels of FoxOs increase steadily in the course of differentiation from preadipocytes to mature adipocytes, they are transiently inactivated and excluded from the nucleus during the early stage of this process. Overexpression of constitutively active FoxO1 suppresses adipocyte differentiation (Nakae et al., 2003). This is mainly explained by the role of ROS as indispensable signaling molecules for initiation of adipocyte differentiation. Thus, upregulation of antioxidant enzymes including several FoxO target genes does only take place during terminal adipocyte differentiation, to prevent the cells from detrimental effects of unbalanced ROS generation (Higuchi et al., 2013; Klotz et al., 2015; Rajalin, Micoogullari, Sies, & Steinbrenner, 2014). In mature adipocytes, FoxO1 has been reported to interact with the adipose tissue-specific FoxO1 corepressor (FCoR). Knockout of FCoR resulted in glucose intolerance and insulin resistance of mice, thus highlighting the need for a physiological brake to restrain FoxO transcriptional activity in the adipose tissue (Nakae et al., 2012).

­Concluding remarks Regulation of FoxO activity as transcriptional regulators occurs primarily through posttranslational modification, such as reversible phosphorylation and acetylation, and through interaction with various coactivators, corepressors, and other transcription factors. Redox signaling is implicated at several stages, rendering ROS, such as

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H2O2, major determinants of FoxO activity. FoxOs were found to be “double-edged swords” in various situations, for example, eliciting beneficial and detrimental responses both in the major insulin target tissues and in the insulin-producing pancreatic β-cells. A modulation of ROS levels in cells, thereby affecting redox regulation of signaling processes, may therefore contribute to shifting FoxO activity in either direction.

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Szypowska, A. A., & Burgering, B. M. (2011). The peroxide dilemma: Opposing and mediating insulin action. Antioxidants & Redox Signaling, 15, 219–232. Tsitsipatis, D., Gopal, K., Steinbrenner, H., & Klotz, L. O. (2018). FOXO1 cysteine-612 mediates stimulatory effects of the coregulators CBP and PGC1alpha on FOXO1 basal transcriptional activity. Free Radical Biology & Medicine, 118, 98–107. Tullet, J. M. A., Green, J. W., Au, C., Benedetto, A., Thompson, M. A., et  al. (2017). The SKN-1/Nrf2 transcription factor can protect against oxidative stress and increase lifespan in C. elegans by distinct mechanisms. Aging Cell, 16, 1191–1194. Urban, N., Tsitsipatis, D., Hausig, F., Kreuzer, K., Erler, K., et al. (2017). Non-linear impact of glutathione depletion on C. elegans life span and stress resistance. Redox Biology, 11, 502–515. van der Heide, L. P., Jacobs, F. M., Burbach, J. P., Hoekman, M. F., & Smidt, M. P. (2005). FoxO6 transcriptional activity is regulated by Thr26 and Ser184, independent of nucleocytoplasmic shuttling. The Biochemical Journal, 391, 623–629. Walter, P. L., Steinbrenner, H., Barthel, A., & Klotz, L. O. (2008). Stimulation of selenoprotein P promoter activity in hepatoma cells by FoxO1a transcription factor. Biochemical and Biophysical Research Communications, 365, 316–321. Wani, R., Qian, J., Yin, L., Bechtold, E., King, S. B., et al. (2011). Isoform-specific regulation of Akt by PDGF-induced reactive oxygen species. Proceedings of the National Academy of Sciences of the United States of America, 108, 10550–10555. Weigel, D., Jurgens, G., Kuttner, F., Seifert, E., & Jackle, H. (1989). The homeotic gene fork head encodes a nuclear protein and is expressed in the terminal regions of the Drosophila embryo. Cell, 57, 645–658. Xu, M., Chen, X., Chen, D., Yu, B., & Huang, Z. (2017). FoxO1: A novel insight into its molecular mechanisms in the regulation of skeletal muscle differentiation and fiber type specification. Oncotarget, 8, 10662–10674. Yamamoto, M., Kensler, T. W., & Motohashi, H. (2018). The KEAP1-NRF2 system: A Thiolbased sensor-effector apparatus for maintaining redox homeostasis. Physiological Reviews, 98, 1169–1203. Yang, J. Y., Zong, C. S., Xia, W., Yamaguchi, H., Ding, Q., et al. (2008). ERK promotes tumorigenesis by inhibiting FOXO3a via MDM2-mediated degradation. Nature Cell Biology, 10, 138–148. Yuan, Z., Becker, E. B., Merlo, P., Yamada, T., DiBacco, S., et al. (2008). Activation of FOXO1 by Cdk1 in cycling cells and postmitotic neurons. Science, 319, 1665–1668. Zhao, J., Brault, J. J., Schild, A., Cao, P., Sandri, M., et al. (2007). FoxO3 coordinately activates protein degradation by the autophagic/lysosomal and proteasomal pathways in atrophying muscle cells. Cell Metabolism, 6, 472–483. Zhou, J., Li, H., Li, X., Zhang, G., Niu, Y., et al. (2015). The roles of Cdk5-mediated subcellular localization of FOXO1 in neuronal death. The Journal of Neuroscience, 35, 2624–2635.

CHAPTER

Oxidatively generated DNA base modifications: Relation to eustress and distress

18 Bernd Epe

Institute of Pharmacy and Biochemistry, University of Mainz, Mainz, Germany

Abstract Oxidative stress at the DNA, i.e., the generation of DNA damage by endogenously produced reactive oxygen species, is of particular concern as it can give rise to mutations and thereby an increased cancer risk. On the other hand, there is accumulating evidence that oxidized DNA bases, in particular 8-oxo-7,8-dihydroguanine (8-oxoG), are actively generated in mammalian cells as epigenetic marks and are involved in transcriptional regulation. To better understand this apparent paradox, this chapter first describes the types and mechanisms of DNA damage under conditions of exogenous and endogenous oxidative stress. It then summarizes the indications that oxidatively generated DNA damage and mutations contribute significantly to the overall human cancer risk, based on (i) the mutation spectra in cancer cells as obtained by whole genome sequencing, (ii) studies with repair-deficient mice, and (iii) the findings of repair defects in human cancers. Finally, the newly detected physiological role of 8-oxoG in the regulation of the transcription of certain genes is described and contrasted with the potential detrimental effects. ­Keywords: Oxidative stress, Reactive oxygen species, Oxidatively generated DNA damage, 8-Oxo-7, 8-dihydroguanine, OGG1, DNA repair, Mutation spectrum, Spontaneous DNA damage, Spontaneous cancer incidence, Carcinogenesis

­Introduction Among the various adverse effects of reactive oxygen species (ROS) in cells, the generation of DNA modifications (DNA damage) deserves special interest, not only because of the acute cytotoxic effects that can result from high numbers of DNA lesion but also because of the mutagenic risk that is inevitably associated with even low levels of DNA oxidation. The reaction of ROS with nuclear DNA, which will be in the focus of this review, is of particular concern, as the resulting oxidatively generated DNA modifications and mutations are expected to increase the cancer risk. The oxidation of nuclear DNA might also contribute to aging and degenerative disease, as concluded from the phenotypes associated with defects in DNA damage response Oxidative Stress. https://doi.org/10.1016/B978-0-12-818606-0.00018-3 © 2020 Elsevier Inc. All rights reserved.

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and DNA repair (Hasty, Campisi, Hoeijmakers, van Steeg, & Vijg, 2003; Lombard et  al., 2005). The oxidation of mitochondrial DNA has been implemented in the pathomechanism underlying various age-related degenerative diseases and aging process as well. It remains to be established, however, whether mitochondrial DNA damage and mutations are causally involved or rather represent a bystander effect of the regulatory function of the mitochondrial redox state in apoptosis and senescence (Balaban, Nemoto, & Finkel, 2005; Chocron, Munkacsy, & Pickering, 2019; Panieri et al., 2013; Sohal & Orr, 2012). The mutagenic risk associated oxidatively generated DNA damage implies that oxidative stress is always associated with adverse effects, at least if there is no threshold for the formation of DNA damage, as indicated by the observation that basal levels of oxidatively generated DNA modifications are observable in cells under all condition (see in the succeeding text). This notion is easy to accept as long as oxidative stress is regarded as detrimental anyhow (“distress”). It seems surprising, however, in view of the physiological role of ROS such as superoxide radicals and hydrogen peroxide (H2O2) as important second messengers (signaling molecules) in the regulation of numerous cellular pathways, which is now well established and sometimes addressed as “eustress” (Niki, 2016; Ray, Huang, & Tsuji, 2012; Sies, 2014, 2017; Sies, Berndt, & Jones, 2017). Even more confusing, recent evidence indicates that not only the oxidation of cysteine residues in proteins but also certain premutagenic oxidatively generated DNA modifications, in particular 8-oxo-7,8-dihydroguanine (8-oxoG), have a signaling function in certain sequences of the genome and possibly serve as epigenetic marks (Antoniali, Malfatti, & Tell, 2017; Ba & Boldogh, 2018; Fleming & Burrows, 2017a; Seifermann & Epe, 2017; Wang, Hao, Pan, Boldogh, & Ba, 2018). In this review, the conditions and mechanisms of the formation of premutagenic DNA modifications by ROS will be recapitulated and compared with the present knowledge of the generation of signaling DNA lesions by ROS. This should contribute to a better understanding of both the beneficial and adverse effects of ROS (eustress and distress) at the nuclear DNA.

­ xidative distress at the DNA: Detrimental effects O of oxidatively generated DNA damage ­ xidatively generated DNA damage and cancer risk: General O considerations As evident from numerous population studies, the overall human cancer risk is highly influenced by personal habits and environmental factors such as overweight, inflammation, and infections (Brown et  al., 2018; Coussens & Werb, 2002; Hussain & Harris, 2007; Luch, 2005; Schottenfeld, Beebe-Dimmer, Buffler, & Omenn, 2013). Under the assumption that (i) the malignant transformation of cells (carcinogenesis) virtually always originates from mutations in somatic cells (Lynch, 2010; Shendure & Akey, 2015) and (ii) mutations in mammalian cells are mostly caused by replication errors at DNA modifications (while replication errors at unmodified bases

­Oxidative distress at the DNA

are rare), it is an important question which type of DNA damage (caused by which agent) is responsible for the fact that one quarter of the population in developed countries dies of cancer. The high overall cancer incidence is difficult to explain by the average concentrations and estimated mutagenic potentials of the known exogenous (environmental) carcinogens, such as acrylamide in fried potatoes, aflatoxin in moldy peanuts, and so on, with some clear exceptions, in particular for smokers (Ding et al., 2008; Hainaut & Pfeifer, 2001; Pfeifer et al., 2002). Therefore, endogenously generated DNA modifications are suspected to contribute significantly to the initiating mutations responsible for human cancer (Lindahl, 1993). However, several types of the endogenous DNA lesions are spontaneously generated and unavoidable. Therefore, similar to spontaneous replication errors at unmodified bases, they cannot explain the observed environmental influence on the cancer risk. This applies not only to uracil or mispaired thymine residues generated in DNA by spontaneous or enzymatic deamination of cytosine or 5-methylcytosine (5mC), respectively (Barnes & Lindahl, 2004; Yonekura, Nakamura, Yonei, & Zhang-Akiyama, 2009), but also to sites of base loss (AP sites) produced by spontaneous depurination (Lindahl, 1993) and to methylated purines generated from S-adenosylmethionine (Rydberg & Lindahl, 1982). If these mechanisms would be responsible for most of the mutations that initiate malignant transformation, the observed strong environmental influence on the human cancer risk could only result from nongenotoxic effects, that is, from processes addressed by toxicologists as tumor promotion. In contrast, the oxidation of DNA by ROS is on the one hand an endogenous reaction that takes place in all cells but on the other hand is highly variable, that is, it depends on the cellular level of oxidative stress (defined by the rate of ROS generation and the concentrations of antioxidants), which is strongly influenced by various environmental and (patho) physiological conditions such as inflammatory response and nutrition, in accordance with the epidemiological findings for the cancer incidence (see earlier). The hypothesis that oxidatively generated DNA damage contributes significantly to the human cancer risk therefore appears attractive.

­DNA damage induced by ROS: Mechanisms and types of lesions ­Reactivity of ROS and DNA damage spectra

Most of our knowledge regarding the DNA damage caused by ROS and its consequences results from experiments with isolated (cell-free) DNA and from studies with high concentrations of exogenous oxidants, often under conditions associated with significant cytotoxicity (distress). In the following paragraphs, some of the major results from these investigations are summarized. The ability of ROS to react with DNA differs largely. Interestingly, H2O2 and superoxide, which are produced endogenously in many enzymatic reactions, do not modify cellular DNA directly but have to be converted to more reactive species, for example, hydroxyl radicals, in a metal-catalyzed Fenton or Haber-Weiss reaction (Aruoma, Halliwell, Gajewski, & Dizdaroglu, 1989; Blakely, Fuciarelli, Wegher, & Dizdaroglu, 1990). Also nitric oxide (NO), which is often addressed as a reactive

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nitrogen species, is poorly reactive toward DNA but can be converted to more reactive peroxynitrite (ONOO−) by reaction with superoxide, which takes place at a diffusion-controlled rate (Pacher, Beckman, & Liaudet, 2007). Remarkably, nitric oxide efficiently inhibits the cellular repair of oxidized guanines (Phoa & Epe, 2002). For most types of oxidants, DNA is not the preferential target in cells. This appears also true for lipid peroxidation products (Petersen & Doorn, 2004). Other constituents, in particular lipids and many proteins, are more readily oxidized and mediate most of the toxic effects. This is one of the reasons that genotoxicity tests with oxidants in mammalian cells often are positive only at highly toxic conditions. As an example, the carcinogenicity of ozone is difficult to demonstrate in vivo, although it clearly reacts with DNA and causes mutations (Jorge et al., 2002; Poma et al., 2017; Victorin, 1992). Important exceptions are those oxidants that bind noncovalently to DNA such as bleomycin (Bolzan & Bianchi, 2018). The spectrum of DNA modifications generated by various types of ROS and the reaction mechanisms have been well established in the last decades and described in comprehensive reviews (Cadet, Davies, Medeiros, Di Mascio, & Wagner, 2017; Fleming & Burrows, 2017b; Neeley & Essigmann, 2006; Ravanat, Cadet, & Douki, 2012). However, the quantification of the various types of lesions in cells and therefore the establishment of cellular DNA damage spectra is still a challenging problem for several reasons. (1) Not all oxidatively generated DNA lesions can be determined with a sufficiently high sensitivity, and spurious oxidation during work-up procedures can play an important role (ESCODD, 2003). (2) Some primary lesions, in particular 8-oxoG, are much more reactive toward ROS than the natural bases (Neeley & Essigmann, 2006). If high concentrations of oxidants are applied experimentally to increase the product yields, this gives rise to secondary oxidation products. These are not expected to be relevant if the level of primary lesions is low, for example, less than approx. 1 modification per 106 bp. They might, however, be generated if there is a repeated site-specific attack of an oxidant at certain DNA sequences (see “Oxidative eustress at the DNA: Physiological effects of oxidatively generated DNA damage” section). (3) Results obtained with isolated DNA or single nucleosides are difficult to translate into the cellular situation because the redox environment has an influence on the final product distribution (see in the succeeding text) and because in DNA—but not in nucleosides—charge transfer reactions (hole migration) play an important role (Hall, Holmlin, & Barton, 1996; Kanvah et al., 2010; Merino, Boal, & Barton, 2008; Nunez, Holmquist, & Barton, 2001). Primarily, the spectrum of DNA modifications generated by oxidants depends on the ultimately DNA-reactive species and can often be regarded as its fingerprint. Since, however, the primary reaction products are often unstable (e.g., DNA radicals), the redox environment (the presence of reductants such as thiols or potential oxidants such as superoxide) can influence the final product distribution (i.e., the damage spectrum) as well (Alshykhly, Fleming, & Burrows, 2015).

­Guanine oxidation products in DNA

Less reactive (mild) oxidants preferentially react with the guanine residues in DNA, that is, they generate oxidized purines in high excess of single-strand breaks (SSB),

­Oxidative distress at the DNA

AP sites, pyrimidine, and adenine modifications, not only because guanine has the lowest redox potential of all DNA base but also probably because guanines can act as a “sink” for DNA radicals by the aforementioned intramolecular charge transfer reactions (hole migration). As indicated before, the mechanisms of guanine oxidation by hydroxyl radicals, one-electron abstraction, and singlet oxygen have been thoroughly investigated in  vitro (Cadet et  al., 2017; Fleming & Burrows, 2017b; Neeley & Essigmann, 2006; Ravanat et al., 2012). The reactions are complicated and frequently involve rearrangements, since both the guanine radicals formed in the primary attack of hydroxyl radicals or one-electron oxidants and the endoperoxides generated in the direct reaction with singlet oxygen are short lived and stabilize by further oxidation or reduction. Among the final products detected, those resulting from two-electron oxidation and four-electron oxidation of guanine residues can be distinguished (Fig. 1). One of the long-known products generated by hydroxyl radicals, 2,6-diamino-4-­ hydroxy-5-formamidopyrimidine (Fapy-G), is even on the same oxidation level as guanine, since the primarily formed one-electron oxidation product is reduced again after the opening of the imidazole ring (Dizdaroglu, Kirkali, & Jaruga, 2008). The two-electron oxidation product 8-oxoG appears to be generated in c­ ellular DNA by

FIG. 1 Structures of some guanine modifications in DNA generated by ROS. 2Ih, 5-carboxamido-5-formamido-2-iminohydantoin; 8-oxoG, 8-oxo-7,8dihydroguanine; Fapy-G, 2,6-diamino-4-hydroxy-5-formamidopyrimidine; Gh, 5-guanidinohydantoin; Iz, 2,5-diamino-4H-imidazol-4-one; Sp, spiroiminodihydantoin; Z, 2,2,4-triamino-2H-oxazol-5-one.

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most oxidants in highest yields (Cui et al., 2013), although this has been questioned for the highly reactive hydroxyl radicals under certain conditions, for which the 5-carboxamido-5-formamido-2-iminohydantoin (2Ih) might dominate (Alshykhly et al., 2015). The four-electron oxidation product 2,5-diamino-4H-imidazol-4-one (Iz), which easily hydrolyzes to the oxazolone Z, can be generated if a C5 radical of guanine is trapped by superoxide, and the resulting 5-hydroperoxide rearranges before being reduced to the 5-hydroxy derivative, which would finally yield 2Ih. The 5-guanidinohydantoin Gh and the spiroiminodihydantoin Sp can be generated, at least in cell-free DNA, from singlet oxygen via the 4,8-endoperoxide of guanine. In cells, however, the peroxide might be preferentially reduced, generating 8-oxoG as the final product. In addition, Gh and Sp can be produced in the secondary ­oxidation of 8-oxoG. The mutagenic potential of 8-oxoG has long been known. It results from the ability of 8-oxoG to form a Hoogsteen base pair with adenine. This mispairing causes G:C to T:A transversions, which therefore are regarded as the “signature mutations” of 8-oxoG (Grollman & Moriya, 1993; Shibutani, Takeshita, & Grollman, 1991). The extent of adenine misincorporation opposite 8-oxoG depends largely on the type of DNA polymerase and probably the sequence context (Markkanen, 2017; McCulloch, Kokoska, Garg, Burgers, & Kunkel, 2009). When single-stranded viral DNA containing either 8-oxoG or the hydantoins Gh and Sp was transfected into repair-competent bacteria, the hydantoins were approx. 10-fold more mutagenic than 8-oxoG, possibly as a result of the efficient repair of mismatched 8-oxoG by MutY (Henderson et al., 2003). All four-electron oxidation products shown in Fig. 1 (Iz, Z, Gh, Sp) can give rise to G:C to C:G transversions (see in the succeeding text).

­DNA damage spectra by hydroxyl radicals and peroxynitrite

For the highly reactive—and therefore least selective—hydroxyl radicals, generated for example by ionizing radiation, the DNA damage spectrum is very broad. SSB, AP sites, and DNA base modifications are generated in comparably high yields, and among the base modifications, pyrimidine and purine products appear to be formed to a similar extent as well (Epe, Ballmaier, Adam, Grimm, & Saha-Möller, 1996; Pouget et al., 2002). The 3′- or 5′-ends of the SSB can be blocked by sugar residues, and the AP sites can be oxidized in the 1′- or 4′-position of the sugar moiety (Dedon, 2008; Pogozelski & Tullius, 1998). Moreover, tandem lesions (involving two adjacent DNA bases) and cyclonucleoside lesions (with a second covalent bond between base and sugar) have been described (Bellon, Ravanat, Gasparutto, & Cadet, 2002; Chatgilialoglu, Ferreri, & Terzidis, 2011). Also peroxynitrite, another quite reactive species, generates, in addition to guanine modifications, relatively high yields of SSB, but, in contrast to hydroxyl radicals, pyrimidine modifications and sites of base loss are relatively rare (Epe, Ballmaier, Roussyn, Briviba, & Sies, 1996). The reactions of peroxynitrite with guanine residues have been well investigated in  vitro (Burney, Caulfield, Niles, Wishnok, & Tannenbaum, 1999; Niles, Wishnok, & Tannenbaum, 2006). Besides oxidation, also nitration (the formation of 8-­nitroguanine) can take place.

­Oxidative distress at the DNA

In cell-free DNA treated with hydroxyl radicals generated from either ionizing radiation or photodecomposition of N-hydroxy-pyridine-2-thione, 40% of the DNA base modifications sensitive to the bacterial repair glycosylase Fpg, which recognizes a broad spectrum of oxidized guanines, were identified to represent 8-oxoG residues (Pflaum, Will, Mahler, & Epe, 1998). A similarly high percentage of 8-oxoG was also found after incubation of the DNA with 3-morpholinosydnonimine (SIN-1), a chemical source of peroxynitrite (Pflaum et al., 1998). In calf thymus DNA exposed to ionizing radiation or peroxynitrite, 8-oxoG was detected by HPLC and tandem mass spectrometry at more than 1000-fold higher levels than the oxazolone Z (Cui et al., 2013).

­DNA damage spectra by bromate and tert-butoxyl radicals

An instructive example for a less reactive oxidant is bromate (BrO3 − ), which is activated (rather than inactivated) in cells by thiols and probably gives rise to DNA modifications via a not yet identified bromine-derived radical. In rodents, bromate is an established carcinogen (DeAngelo, George, Kilburn, Moore, & Wolf, 1998; Wolf et al., 1998). In cell-free DNA treated with bromate in the presence of thiols, 8-oxoG accounts for the majority of the guanine modifications sensitive to the repair glycosylase Fpg, while the yield of SSB, AP sites, and oxidized pyrimidines is very low (Ballmaier & Epe, 1995, 2006). Accordingly, the spectrum of mutations induced by bromate in the transgenic gpt locus in the kidneys of mice was dominated by G:C to T:A transversions, the signature mutation of 8-oxoG (Arai, Kelly, Minowa, Noda, & Nishimura, 2002). Also photochemically generated tert-butoxyl radicals, which may be representative for several other organic radicals, induce 8-oxoG as the by far most frequent modification in cell-free DNA (Mahler et al., 2001). In agreement with this DNA damage analysis, G:C to T:A transversions are the most frequent type of mutation when plasmid DNA exposed to tert-butoxyl radicals is replicated in bacteria (Mahler et al., 2001). The intracellular generation of this type of radicals, however, has not been investigated so far.

­DNA damage spectra by type I photosensitizers and singlet oxygen

The excited states of so-called type I photosensitizers such as riboflavin can directly react with guanine residues by one-electron or hydrogen abstraction (Epe, 2012; Ito, Inoue, Yamamoto, & Kawanishi, 1993). As in the case of the tert-butoxyl radicals, the DNA damage spectrum observed under cell-free conditions is dominated by guanine modifications; the generation of SSB, AP sites, and pyrimidine modifications is rare (Schulz, Mahler, Boiteux, & Epe, 2000). In striking contrast to the situation observed with tert-butoxyl radicals, however, 8-oxoG in the case of photoexcited riboflavin accounts for only 20% of the guanine modifications sensitive to Fpg. The remaining guanine modifications most probably are responsible for the high number of G:C to C:G transversions (with the G mostly 5′ to another purine) that are observed when the DNA after the exposure to riboflavin and light was replicated in bacteria (Schulz et al., 2000). The guanine modification underlying this mutation was not identified,

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but misincorporation of guanine is known to take place opposite the imidazolone Iz (Kino & Sugiyama, 2001), its hydrolysis product, the oxazolone Z (Kino, HiraoSuzuki, Morikawa, Sakaga, & Miyazawa, 2017), and the hydantoin 2Ih (Alshykhly et  al., 2015). Interestingly, exactly this type of mutation was frequently observed by large-scale sequencing of breast cancer genomes (Pfeifer & Besaratinia, 2009). In a study using HPLC in combination with tandem mass spectrometry to analyze base modifications induced by photoexcited riboflavin in calf thymus DNA, similar amounts of 8-oxoG and the oxazolone Z were detected at early irradiation times, under conditions under which Iz was shown to be fully hydrolyzed to Z (Matter, Malejka-Giganti, Csallany, & Tretyakova, 2006). A last well-investigated oxidant is singlet oxygen (Di Mascio et al., 2019). It can be generated in cells by xenobiotic or endogenous photosensitizers such as porphyrins in the presence of light or UVA radiation and from lipid peroxide radicals by the so-called Russell mechanisms, albeit probably in low yields (Miyamoto, Martinez, Medeiros, & Di Mascio, 2003). In a direct reaction with guanine residues, singlet oxygen generates two unstable diastomeric 4,8-endoperoxides, which are converted to either Sp and Gh (via 5-hydoxy-8-oxoguanine as an intermediate) or to 8-oxoG (via reduction of the peroxide by an unknown agent) (McCallum, Kuniyoshi, & Foote, 2004; Neeley & Essigmann, 2006; Ye et al., 2003). In a study with calf thymus DNA and Rose Bengal as a singlet oxygen generating type II photosensitizer, Sp was shown to be most frequent among the four-electron oxidation products of guanine, albeit produced in much lower yields than 8-oxoG (Cui et  al., 2013). In another study with cell-free DNA, in which singlet oxygen was generated by thermal decomposition of the endoperoxide of 3,3′-(1,4-naphthylidene)-dipropionate (NDPO2), more than 50% of the guanine lesions sensitive to Fpg were modifications other than 8-oxoG (Schulz et al., 2000). Accordingly, after transfection of the damaged DNA into bacteria, only 50% of the mutations turned out to be G:C to T:A transversions characteristic for 8-oxoG, while the rest were G:C to C:G transversions. In contrast to the situation with type I photosensitizers, these mutations were not accumulated at positions 5′ to another purine (Schulz et al., 2000). Primer extension studies suggest that G:C to C:G transversions can result from both Sp and Gh (Kornyushyna, Berges, Muller, & Burrows, 2002; Zhu, Fleming, Orendt, & Burrows, 2016). In the DNA of cultured cells, 8-oxoG was found to be produced by chemically generated singlet oxygen (thermal decomposition of an isotope-labeled nonionic analogue of NDPO2) in a direct (!) reaction (Ravanat, Di Mascio, Martinez, Medeiros, & Cadet, 2000). Sp and Gh, however, were not analyzed.

­Repair of oxidatively generated DNA base damage The biological threat associated with DNA oxidation is emphasized by the existence of specific repair mechanisms for oxidized DNA bases in most or all bacterial and eukaryotic species. For 8-oxoG, the system to prevent detrimental consequences appears particularly sophisticated (Michaels & Miller, 1992; Michaels, Tchou, Grollman, & Miller, 1992; Russo et  al., 2004). The base modifications is excised from DNA in

­Oxidative distress at the DNA

e­ ukaryotic cells by a repair glycosylase called OGG1, which was first cloned from yeast by Boiteux in 1996 (van der Kemp, Thomas, Barbey, de Oliveira, & Boiteux, 1996). The functional analogue in bacteria, Fpg, was detected one decade earlier (Boiteux, O’Connor, & Laval, 1987). The mechanistic details of the excision and the subsequent steps of the so-called base excision repair pathway, which is coordinated by the platform protein XRCC1 (Marsin et al., 2003), are well understood (for reviews, see Boiteux, Coste, & Castaing, 2017; David, O’Shea, & Kundu, 2007; Wallace, 2014). It is worth mentioning that 8-oxoG is not removed by nucleotide excision repair (NER) (Larsen, Kwon, Coin, Egly, & Klungland, 2004) and that there is no clear “backup” glycosylase for OGG1 in mammalian cells. Interestingly, OGG1 does not excise 8-oxoG from the Hoogsteen mispair with adenine, since such an excision could cause a fixation of the mutation after a misincorporation of adenine opposite 8-oxoG by the replicative DNA polymerases ∂ and ε. Rather, another specialized glycosylase, MUTYH, similar to its bacterial analogue MutY, excises the adenine paired with 8-oxoG (Au, Clark, Miller, & Modrich, 1989; Banda, Nunez, Burnside, Bradshaw, & David, 2017; Woods et al., 2016). This gives another chance for the correct incorporation of cytosine opposite the modified guanine. A special repair polymerase, pol-λ, which faithfully pairs 8-oxoG with cytosine, might be involved in this step (Maga et  al., 2007; van Loon & Hübscher, 2009). Yet another player in the prevention of 8-oxoG-induced mutations is MTH1, also known as NUDT1, a sanitization enzyme that specifically hydrolyzes 8-oxoGTP generated in the nucleotide pool and thereby prevents its misincorporation (opposite adenine) by the replicative polymerases ∂ and ε (Markkanen, 2017; Nakabeppu, Ohta, & Abolhassani, 2017). The relevance of such a sanitization is underlined by the fact that the free nucleotide GTP is much easier oxidized than guanine in the DNA (Fleming, Muller, Dlouhy, & Burrows, 2012). Also most of the other oxidatively generated DNA base modifications described earlier are recognized by specialized repair glycosylases and therefore are subject to base excision repair, which again can be seen as an indication for their biological relevance during evolution. Oxidized pyrimidines such as thymine glycols, 5-­hydroxycytosine, and dihydrouracil are the main substrates of NTH1, an analogue of the bacterial glycosylase endonuclease III. The ring-opened guanine derivative Fapy-G is one of the few substrates of OGG1, besides 8-oxoG. The four-electron oxidation products of guanine, Gh and Sp, are substrates of NEIL1 in double-stranded and so-called quadruplex DNA, while NEIL2 appears preferentially active in singlestranded regions (Fleming & Burrows, 2017b; Hazra et al., 2007; Kino et al., 2017). These lesions, however, can also be removed by NER (Shafirovich et al., 2016). The oxazolone Z is a substrate of human NEIL1 and NTH1 (Kino, Takao, Miyazawa, & Hanaoka, 2012). For an overview of the recognition of various oxidized DNA bases by repair glycosylases from various species, see Bjelland and Seeberg (2003).

­Endogenously generated DNA damage: Basal levels An important indication pointing to the biological relevance of oxidatively generated DNA damage are the basal “background” levels of oxidized DNA bases detectable

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in apparently all types of cells, despite the existence of specific repair mechanisms to deal with these modifications (see earlier). These basal numbers of lesions are assumed to represent steady-state levels, which result from the balance between a continuous oxidation of cellular DNA by ROS generated in the cellular metabolism and the concurrent removal of the oxidation products by DNA repair. Without doubt, the best-studied oxidatively generated DNA modification in vivo is 8-oxoG. The absolute numbers of 8-oxoG lesions in the chromosomal and mitochondrial DNA of various types of tissues and cultured cells under physiological conditions have been a matter of debate, at least partially due to an easy artifactual generation during the isolation of DNA (Collins, Cadet, Moller, Poulsen, & Vina, 2004; ESCODD, 2003). Most probably, the correct basal levels in the nuclear DNA of untreated cells and tissues are well below one 8-oxoG residue per 106 bp. The levels in mitochondrial DNA have been reported to be higher and appear further increased in Ogg1−/− mice (de Souza-Pinto et al., 2001), although this had no significant influence on the mitochondrial functions (Stuart, Bourque, de Souza-Pinto, & Bohr, 2005). However, when only intact (supercoiled) mt-DNA was analyzed from the livers of repair-deficient mice, a level higher than the detection limit of approx. five 8-oxoG residues per 106 bp could be excluded (Trapp, McCullough, & Epe, 2007). There is relatively little information about endogenously generated levels of the other oxidized DNA bases described in “Guanine oxidation products in DNA” section. For the oxazolone Z, a frequency of 0.2 ± 0.1 lesions per 106 bp was found in the livers of untreated rats; approx. 10-fold lower than the level of 8-oxoG measured in parallel the same study (Matter et al., 2006). For Sp and Gh, levels between 0.02 and 0.14 lesions per 106 bp were detected in mouse liver and colon, more than 100-fold lower than the levels of 8-oxoG observed in parallel (Mangerich et al., 2012). No significant increase was observed in this study after an infection with Helicobacter hepaticus. It is interesting to compare these levels with the basal levels of other endogenously generated DNA modifications (lipid peroxidation products and alkylation products), as compiled, for example, by De Bont and van Larebeke (2004). AP sites are frequently generated by spontaneous depurination of unmodified and— more rapidly—alkylated purines in DNA but are also produced by hydroxyl radicals. Based on the use of so-called aldehyde-reactive probes for quantification, very high basal levels of aldehyde-reactive sites, which include not only most types of sites of base loss but also SSB with a sugar residue, have been reported (De Bont & van Larebeke, 2004; Nakamura & Swenberg, 1999). However, the method may suffer from considerable background problems resulting in a high overestimation (Wei, Shalhout, Ahn, & Bhagwat, 2015). The use of repair enzymes as probes revealed basal levels of AP sites in cultured mammalian cells that are much lower than those of 8-oxoG and close to the detection limit, that is, less than 0.1 abasic site per 106 bp (Andersen et  al., 2005; Sossou et  al., 2005). Also uracil residues, which emanate from spontaneous cytosine deamination or misincorporation of uracil during DNA replication, were much less frequent than 8-oxoG, as revealed by

­Oxidative distress at the DNA

the same ­technique (Andersen et al., 2005). For the sum of three etheno adducts, namely, 1,N6-etheno-dA, 3,N4-etheno-dC, and 1,N2-etheno-dG, a level of 0.7/106 bp was reported (Chen, Lin, & Lin, 2010). However, a much lower frequency, namely, 0.002–0.12 1,N6-etheno-dA per 106 bp, was observed in another study (Nair, Barbin, Velic, & Bartsch, 1999). A completely different type of endogenously generated oxidized DNA base is 5-hydroxymethylcytosine (5-hmC). It is an intermediate of the enzymatic oxidative demethylation of the 5mC, the most frequent epigenetic mark in mammalian DNA (He et al., 2011; Ito et al., 2011). 5-hmC is not miscoding. Since it was detected in very high levels in some tissues (Wagner et al., 2015), it might function as epigenetic mark that is distinct of 5mC. While it is clear that epigenetic dysregulation is associated with the malignant transformation of cells (Haffner et al., 2011; Ko et al., 2010), the underlying mechanisms and causal relationships remain to be established.

­Endogenous sources of oxidatively generated DNA damage There are many cellular enzymatic and nonenzymatic reactions in which ROS are generated as by-products or even main products (Sies et al., 2017). However, it has remained an open question which of these reactions contributes significantly (and predominantly) to the basal levels of oxidized DNA bases described earlier. There is little doubt that the largest part of the endogenously generated ROS (~90%) is produced in the mitochondrial electron transport chain, as about 0.2% of the mitochondrial oxygen consumption is funneled to ROS rather than water (Aguilaniu, Gustafsson, Rigoulet, & Nystrom, 2003; Balaban et  al., 2005; Staniek & Nohl, 2000). However, its deficiency or dysregulation in mammalian cultured cells had no significant influence on the observed level of oxidatively generated base modifications in the chromosomal DNA (Hoffmann, Spitkovsky, Radicella, Epe, & Wiesner, 2004). Possibly, the ROS produced in the mitochondria are not able to travel to the nucleus but are efficiently detoxified by mitochondrial SOD, cytosolic catalase, etc. However, this does not exclude that ROS from perinuclear mitochondria give rise to a very localized generation of chromosomal DNA damage, as suggested by AlMehdi et al. (2012). Similarly, experiments with cells overexpressing the inducible NO synthase suggest that the cellular NO synthesis is also not a major contributor to oxidatively generated DNA modifications (Phoa & Epe, 2002). The expression of the human NADPH-cytochrome P450 reductase in cultured V79 Chinese hamster cells, however, gave rise to elevated levels of oxidized purines in the nuclear DNA, detectable after depletion of glutathione (Heine, Glatt, & Epe, 2006). NADPH oxidases such as NOX4 are professional generators of ROS (Maghzal, Krause, Stocker, & Jaquet, 2012). After induction, for example, by cytokines such as TNF-α and INF-γ, the ROS generated by NOX4 activate various (patho)physiological signaling pathways, including a p53-mediated DNA damage response (Hubackova et al., 2016; Kim, Kang, Seu, Baek, & Kim, 2009). The influence of the NOX4 activation on the levels of 8-oxoG in the nuclear DNA, however, was not measured, and the induction of the DNA damage response may be indirect. Yet another professional

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generator of ROS is the oxidoreductase p66shc, which causes a generation of H2O2 in the mitochondrial intermembrane space (Gertz, Fischer, Wolters, & Steegborn, 2008; Giorgio et al., 2005; Pinton et al., 2007) and mediates general oxidative stress (Francia et al., 2004; Nemoto & Finkel, 2002; Trinei et al., 2002). Interestingly, the deletion of p66shc in mice had only little influence on the overall mutation rates in the nuclear DNA of liver, small intestine and cultured embryonic fibroblasts but increased the frequency of big deletions/insertions (genome rearrangements), probably as a consequence of a suppression of p66shc-mediated apoptosis of damaged cells (Beltrami et al., 2013). There is no doubt that the endogenous oxidation of DNA is increased under oxidative and nitrosative stress. For example, 8-oxoG was increased in the aorta and heart of rats after treatment with nitroglycerin (Mikhed et al., 2016), and the oxidative stress induced in the kidneys of mice treated with angiotensin gave rise to increased numbers of DNA strand breaks (Schmid, Stopper, Schweda, Queisser, & Schupp, 2008; Zimnol, Amann, Mandel, Hartmann, & Schupp, 2017).

­Relevance of oxidatively generated DNA damage for carcinogenesis ­Lessons from DNA repair defects in human cancers

Important support for the notion that the malignant transformation of cells is initiated by (unrepaired) DNA damage comes from the finding that mutations of genes involved in DNA repair or DNA damage response are frequently observed in tumors (Pfeifer & Besaratinia, 2009). Mutations in repair-related genes act as “driver mutations” because they increase the chance for further mutations required for the malignant transformation (“mutator phenotype”). Well-known examples are the ­tumor-suppressor gene TP53, which codes for a transcription factor that mediates cell cycle arrest or apoptosis in cells bearing a high load of DNA modifications and which is mutated in many different tumors (Petitjean, Achatz, Borresen-Dale, Hainaut, & Olivier, 2007), the BRCA1 and BRCA2 genes, which are involved in recombinational repair and therefore, if mutated, predispose to various cancer types including breast cancer (Friedenson, 2007) and mismatch repair genes such as MSH2, which are defective in 15% of colorectal cancers (Jiricny & Nystrom-Lahti, 2000; Kolodner, 1995). The relevance of a defective NER, which removes bulky DNA modifications from the genome, for the human cancer risk is also well established (Marteijn, Lans, Vermeulen, & Hoeijmakers, 2014). For genes involved in the specific removal of oxidatively generated DNA modifications, however, the evidence for an impact on cancer incidence is rarer (Nemec, Wallace, & Sweasy, 2010; Sweasy, Lang, & DiMaio, 2006). Inherited defects of MUTYH, the repair glycosylase that specifically removes adenine when mismatched with 8-oxoG, are responsible for a MUTYH-associated colorectal polyposis and an increased risk of colorectal cancer, associated with a high incidence of G:C to T:A transversions (Al-Tassan et al., 2002; Cleary et al., 2009; Jones et al., 2002; Nielsen, Morreau, Vasen, & Hes, 2011). A common OGG1 polymorphism, Ser263Cys, appears to be associated with an increased risk for several types of c­ ancer, possibly

­Oxidative distress at the DNA

as a result of a reduced repair activity of the variant under conditions of oxidative stress (Bravard et  al., 2009; Moritz et  al., 2014). Moreover, decreased activities of OGG1 were found in the lymphocytes of lung cancer patients when compared with healthy controls (Paz-Elizur et al., 2003). However, the sequencing of cancer genomes did not indicate that the loss of function mutations of the OGG1 gene and the associated mutator phenotype (see in the succeeding text) is a frequent event in human carcinogenesis (Lawrence et  al., 2014; Martincorena & Campbell, 2015). The situation therefore is similar to that of most other genes directly involved in DNA repair.

­Lessons from DNA repair defects in mice

As a reciprocal approach, the consequence of a deletion of repair proteins on spontaneous tumor formation can be studied experimentally in knockout mice. Results obtained for various types of BER proteins have been summarized (Larsen, Meza, Kleppa, & Klungland, 2007). The defective removal of 8-oxoG in Ogg1−/− mice gives rise to an approx. threefold increase of the overall spontaneous mutation frequency in two different indicator genes in the liver (Klungland et al., 1999; Minowa et al., 2000; Osterod et al., 2001). As expected, most of the additional mutations were G:C to T:A transversions. Despite the elevated mutation frequency, the animals do not show an overt cancer-prone phenotype. However, an increased formation of preneoplastic liver foci was observed in Ogg1−/−/Csb−/− mice, which accumulate 8-oxoG even stronger than Ogg1−/− mice (Osterod et al., 2002), when liver cell proliferation was stimulated by administration of a peroxisome proliferator (Trapp, Schwarz, & Epe, 2007). The finding is a clear indication for the important role of continuous cell proliferation (promotion) in malignant transformation. Importantly, the spontaneous tumor incidence is extremely increased when the 8-oxoG-A specific mismatch repair protein MUTYH is knocked out in addition to OGG1. Tumors in lung, ovary, and lymphatic cells develop in two thirds of the double knockout animals (Xie et  al., 2004). An increased tumor incidence was also observed in single Mutyh−/− knockout mice, and the carcinogenicity of bromate applied in the drinking water, which generates 8-oxoG (see earlier), was drastically enhanced (Sakamoto et al., 2007). Also Mth1 deficiency causes an elevated spontaneous tumor incidence (Nakabeppu et al., 2017; Tsuzuki et al., 2001). In contrast, no increased cancer incidence was observed in Neil1−/−/Neil2−/−/Neil3−/− triple knockout mice, and neither the basal levels of 8-oxoG in kidney, spleen, and liver nor the spontaneous mutation rates (measured for the Piga gene) were elevated (Rolseth et al., 2017). The relevance of these repair glycosylases to decrease the risk of malignant transformation associated with the oxidation of DNA bases therefore remains to be established. It is interesting to note that in bacteria not only the defective repair of oxidized DNA bases, for example, in fpg mutants (Boiteux et  al., 2017; Cabrera, Nghiem, & Miller, 1988), but also a defective regulation of ROS-detoxifying enzymes, for example, in oxyR mutants (Storz, Christman, Sies, & Ames, 1987) has long been known to increase the spontaneous mutation rates. The findings clearly demonstrate the genotoxicity of endogenously generated ROS under regular growth conditions.

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­Lessons from mutation spectra

The whole genome sequencing of cancer cells was originally carried out to identify new cancer genes the mutation of which can stimulate malignant transformation, but the huge number of cancer genomes that is available today has also contributed a lot to our molecular understanding of the origins of cancer (Alexandrov et al., 2013; Bozic et al., 2010; Martincorena & Campbell, 2015; Pfeifer & Besaratinia, 2009; Tomasetti & Vogelstein, 2015; Tomasetti, Vogelstein, & Parmigiani, 2013). Thus, 21 “signature mutations” could be identified, which contribute to different extent to the total point mutations (mostly “bystander” mutations) observed in a given cancer type (Alexandrov et al., 2013). Data revealed that the most frequent type of mutation in virtually all tumor types is a C to T transition at NpCpG trinucleotides. Since the number of these mutations is strongly correlated with the age at cancer diagnosis, they are probably generated in the cells already before the malignant transformation and are caused by spontaneous deamination of 5mCs in the DNA. G:C to T:A transversions, which would be the consequence of replication errors at 8-oxoG, are observed in high frequency in various cancers that are known to be caused by tobacco smoking. However, in this case, they are more likely to result from bulky guanine adducts generated by polycyclic hydrocarbons, as supported by an observed transcriptional strand bias: the mutated G is more frequently located in the untranscribed strand than in the transcribed strand, which points to a clear role of transcription-coupled nucleotide repair in the removal of the lesions, as expected for bulky guanine adducts, but not for 8-oxoG (Pfeifer et al., 2002). Interestingly, G:C to T:A transversions were also very frequent in neuroblastomas, for unknown reasons (Alexandrov et al., 2013). G:C to C:G transversions, which, as indicated earlier, could result from several oxidized guanines other than 8-oxoG, were elevated in breast cancer (Alexandrov et  al., 2013; Pfeifer & Besaratinia, 2009). Significant frequencies of a signature dominated by A:T to C:G transversions, which would result from a misincorporation of 8-oxoGTP from the nucleotide pool opposite adenine, are observed in cancers of the liver, the esophagus, the stomach, and in B-cell lymphomas, with the highest contribution (27%) in esophageal cancer (Alexandrov et  al., 2013). Interestingly, esophageal cancer is correlated with the Ser326Cys polymorphism of human OGG1 (Wang, Gan, Nie, & Geng, 2013), and an accumulation of 8-oxoG was observed in the affected tissue (Hagiwara, Kitajima, Sato, & Miyazaki, 2005; Kubo et al., 2014; Rasanen, Sihvo, Ahotupa, Farkkila, & Salo, 2007). It is interesting to note that the recently reported spontaneous mutation spectrum in human pluripotent stem cells cultured in  vitro (obtained by whole genome sequencing of single-cell derived subclones of “control cells”) is dominated by G:C to T:A transversions, pointing to the high relevance of 8-oxoG generation under these conditions (Kucab et al., 2019). It has also to be noted, however, that the risk associated with single-strand breaks, AP sites, and complex lesions generated by ROS is difficult to assess from mutation spectra since there are no clear signature mutations.

­Oxidative eustress at the DNA

­ xidative eustress at the DNA: Physiological effects O of oxidatively generated DNA damage As indicated in the introduction, the function of ROS as second messengers in various signal transduction pathways and the associated role of oxidative generated protein modifications as physiologically relevant posttranslational modifications are well established (Ray et al., 2012; Sies, 2014). Oxidative stress, which has been addressed as eustress in this context, not only triggers a protective response, mediated by release of the transcription factor NRF2 from the redox-regulated signaling molecule KEAP1 (Schmidt, 2015), but also stimulates various other redox-sensitive transcription factors such as the most important proinflammatory factor NF-κB (Brasier, 2006; Jamaluddin, Wang, Boldogh, Tian, & Brasier, 2007). Moreover, it inactivates various regulatory tyrosine phosphatases by oxidation of particularly sensitive cysteine residues (Cross & Templeton, 2006; Salmeen & Barford, 2005). The oxidation of DNA, in contrast to that of proteins, was long seen as an accidental and detrimental side effect of ROS, because of the long-term risks associated with DNA damage, as described earlier. First indications that oxidatively generated DNA base modifications, in particular 8-oxoG, could also have a physiological function were obtained from a closer examination of the phenotype of Ogg1−/− mice, which, as indicated earlier, are deficient in the removal of 8-oxoG from the genome. Although these mice do not suffer from major developmental defects and have a normal life span, some metabolic disturbances were evident after high-fat diet, apparently due to a failure to suppress gluconeogenesis in the well-fed state (Sampath et  al., 2012; Scheffler et  al., 2018). In addition, a reduced immune response was observed after endotoxin-induced organ dysfunction, oxazolone-induced contact hypersensitivity, and streptozotocin-induced destruction of beta cells (Mabley et  al., 2005). The inflammation caused by infection with Helicobacter pylori (Touati et al., 2006) or an airway allergen (Li et al., 2012) was reduced. Downregulation of OGG1 in the airway epithelium ameliorated the allergic lung inflammation. Recently, a newly developed small-molecule inhibitor of OGG1 was shown to suppress proinflammatory gene expression and inflammation (Visnes et al., 2018). As a mechanistic explanation for the observed consequences of an OGG1 deficiency, OGG1 was shown to act as an auxiliary transcription factor by binding to 8-oxoG residues in the regulatory (promoter) sequences of genes (Fig. 2A). The direct influence of OGG1 on gene expression was first demonstrated by Perillo et al. for the estrogen receptor-regulated transcription of BCL-2 (Perillo et al., 2008) but later also observed for genes regulated by hypoxia-activated HIF-1α (Pastukh et al., 2015), MYC (Amente et al., 2010), the retinoic acid receptor (Zuchegna et al., 2014), the androgen receptor (Yang et al., 2015), and NF-κB (Ba et al., 2014; Pan et al., 2016). The corresponding promoter and regulator sequences appear always rich in guanines and have the propensity to form so-called G-quadruplex structures when single-stranded (Fleming, Ding, & Burrows, 2017; Redstone, Fleming, & Burrows, 2019). As an explanation for the binding of OGG1 to the promoter sites, a local accumulation of Fpg-sensitive DNA modifications, which include 8-oxoG, was

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(A)

(B)

FIG. 2 (A) Suggested role of 8-oxoG in DNA as an epigenetic mark, with the histone demethylase LSD1 as potential inducer (“writer”) and the repair glycosylase OGG1 as an auxiliary transcription factor (“reader”). (B) Suggested role of the free base 8-oxoGua (excised from DNA) in a stoichiometric complex with OGG1 as an activator of small GTPases such as RAS and RAC1 in signal transduction. For details, see text and recent reviews (Ba & Boldogh, 2018; Fleming & Burrows, 2017a; Seifermann & Epe, 2017; Wang et al., 2018). The graphs are adopted from Seifermann, M., & Epe, B. (2017). Oxidatively generated base modifications in DNA: Not only carcinogenic risk factor but also regulatory mark? Free Radical Biology & Medicine, 107, 258–265, with modifications.

d­ emonstrated after hypoxia induction in the VEGF promoter sequence, by comparing the efficiency of PCR amplification with and without preceding incubation with the repair enzyme Fpg (Pastukh, Ruchko, Gorodnya, Wilson, & Gillespie, 2007). In a recently developed genome-wide sequencing technique for 8-oxoG residues, the preferential localization of the lesion in gene promoters and regulatory regions was verified (Ding, Fleming, & Burrows, 2017). How the repair glycosylase OGG1 influences the gene expression after binding to the promoter regions remains to be established. Interestingly, the enzymatic activity of OGG1 is redox dependent and reduced under conditions of oxidative and nitrosative stress (Eiberger et al., 2008; Jaiswal, LaRusso, Nishioka, Nakabeppu, & Gores, 2001; Phoa & Epe, 2002), which is unexpected under the assumption that increased rather than reduced activity would be needed to protect the DNA in this situation. In the context of transcription regulation, however, an inactivated OGG1 trapped at its substrate for a prolonged time might facilitate the recruitment of the transcription factor (Ba et al., 2014; Kim et al., 2016; Pan et al., 2016). Alternatively, the generation of an AP sites by the glycosylase activity of OGG1 in a quadruplex structure might cause a structural switch, which allows the binding of the AP endonuclease APE1 and subsequent recruitment of transcription factors (Redstone et al., 2019). APE1 has long been known as a redox-sensitive transcriptional coactivator for various genes including those regulated by NF-κB and HIF-1α and in this context

­Oxidative eustress at the DNA

was also named REF-1 (Evans, Limp-Foster, & Kelley, 2000; Tell, Quadrifoglio, Tiribelli, & Kelley, 2009). Another major question regarding the gene regulation by 8-oxoG as an epigenetic mark (and OGG1 as its “reader”) is the mechanism underlying the site-directed generation of 8-oxoG, that is, the question of the “writer” of the supposed epigenetic mark. Intriguingly, Perillo et  al. (2008) observed that the lysine-specific histone demethylase LSD1 is responsible for a localized generation of 8-oxoG after treatment of cultured cells with an estrogen and required for the OGG1-mediated activation of transcription. The enzyme, similar to other flavin-dependent amine oxidases, produces H2O2 as a stoichiometric by-product of its catalytic activity (Forneris, Binda, Vanoni, Mattevi, & Battaglioli, 2005). A dependence on LSD1 of the OGG1-mediated induction of transcription was also observed for other genes including the NF-κB-regulated transcription of TNF-α (Seifermann et al., 2017). It remains to be established, however, how an only stoichiometric generation of H2O2 during histone demethylation in an unknown distance from DNA can give rise to the extent of site-specific 8-oxoG residues required for gene activation, in particular because, as described earlier, H2O2 cannot oxidize DNA directly, but causes the formation of various types of DNA modifications via hydroxyl radicals produced in a Fenton reaction. To some extent, however, the guanine rich sequences in the promoter regions can act as sinks during DNA oxidation via intrahelical charge transfer reactions (hole migration), as mentioned in “Reactivity of ROS and DNA damage spectra” section. The generation of 8-oxoG via LSD1-mediated co-oxidation does not necessarily require cytosolic oxidative stress. As an alternative source of ROS, perinuclear clustered mitochondria have been suggested (Al-Mehdi et al., 2012). Such a cellular oxidative stress-related site-specific generation of 8-oxoG would be in line with the well-established regulation of NF-κB by oxidative stress (Brasier, 2006; Hirota et al., 1999; Jamaluddin et al., 2007). It deserves mentioning that the role of OGG1 as auxiliary transcription factor at 8-oxoG is not the only explanation for an OGG1-dependent regulation of genes. Thus, it was shown that a stoichiometric complex of OGG1 and the excised “free base” 8-oxo-7,8-dihydroguanine (8-oxoGua), but not OGG1 alone, binds to and activates small GTPases such as Ras, Rho, and Rac, which are involved in the signal transduction of an immune response by activating MAP kinases (Aguilera-Aguirre et al., 2014; Boldogh et al., 2012; Hajas et al., 2013; Luo et al., 2014) (Fig. 2B). Yet, another possibility is an oxidative stress induced demethylation of silenced genes: after treatment of cultured MCF-10A mammary epithelial cells with H2O2, the oxidation of guanine residues at methylated CpG islands attracts the repair glycosylase OGG1, which subsequently physically interacts with the methylcytosine dioxygenase TET1 to initiate active demethylation and increases gene expression, as demonstrated for the BACE1 gene (Zhou et al., 2016). In this context, it is interesting to note that the affinity of methyl-CpG binding proteins, which mediate the silencing, to oligonucleotide duplexes is significantly reduced when a guanine is replaced by 8-oxoG or a 5mC by 5-hmC (Valinluck et al., 2004).

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­Conclusions The data summarized in this review indicate that the generation of ROS in cells is always associated with the generation of DNA damage and, in replicating cells, with the risk of mutations, which potentially initiate the malignant transformation of cells. Major finding in support of this conclusion are (i) the observation that basal levels of oxidatively generated DNA modifications can be detected in apparently all types of cells and tissues even under physiological conditions, pointing to an equilibrium between a continuous generation and repair of the DNA lesions and the absence of a threshold for their generation (see “Endogenously generated DNA damage: Basal levels” section) and (ii) the fact that a defect in the specific repair of oxidized bases such as 8-oxoG and its mismatch with adenine in mice directly translates into an accumulation of mutations and an increased risk of malignant transformation (see “Lessons from DNA repair defects in mice” section). However, there are also good indications that the absolute cancer risk associated with the basal levels of 8-oxoG and other oxidatively generated DNA base modifications under physiological conditions, which are estimated to be well below one lesion per 106 base pairs, is low and does not contribute very much to the high general cancer risk in the human population. Support for this assumption comes from the sequencing of human tumor genomes, which, with only few exceptions, only rarely carry the signature mutations characteristic for oxidized DNA bases, in particular 8-oxoG (see “Lessons from DNA repair defects in mice” section). Similarly, mutations in caretaker genes such as MUTYH, which are relevant for the specific prevention of 8-oxoG-induced mutations, were observed in only a few types of cancer in humans (see “Lessons from DNA repair defects in human cancers” section). It appears possible, therefore, that the established high influence of inflammation and oxidative stress on the cancer risk is predominantly caused by tumor promotion, that is, nongenotoxic mechanisms such as stimulation of cell proliferation. The accumulating evidence that oxidized DNA bases, in particular 8-oxoG, are actively generated as epigenetic marks and participate in the regulation of transcription remains puzzling, not only because of the expected mutagenic risk associated with the lesion and its repair but also in view of the mechanism of damage generation and the overall relevance (see “Oxidative eustress at the DNA: Physiological effects of oxidatively generated DNA damage” section). So far, the mild phenotype of mice deficient in the removal of the guanine modifications (Ogg1−/− and Neil1−/−/Neil2−/−/Neil3−/− mice) points to an only auxiliary function, which might become important only under special physiological conditions. It appears possible, however, that the term “eustress” is justified also with respect to some consequences of ROS generation at the nuclear DNA.

­References Aguilaniu, H., Gustafsson, L., Rigoulet, M., & Nystrom, T. (2003). Asymmetric inheritance of oxidatively damaged proteins during cytokinesis. Science, 299(5613), 1751–1753.

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Light-initiated oxidative stress

19

Alfonso Blázquez-Castroa,b, Michael Westberga, Mikkel Bregnhøja, Thomas Breitenbacha, Ditte J. Mogensena, Michael Etzerodtc, Peter R. Ogilbya a

b

Department of Chemistry, Aarhus University, Aarhus, Denmark Department of Physics of Materials, Faculty of Sciences, Autonomous University of Madrid, Madrid, Spain c Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

Abstract The production of reactive oxygen species, ROS, plays an important role in light-mediated oxidative stress. As a tool to study general mechanisms of stress and eustress, and associated cellular signaling pathways, light allows for spatial and temporal control of ROS production in the heterogeneous environment of a living cell. Light is particularly suited for the selective initial production of singlet molecular oxygen at the expense of the superoxide radical anion and vice versa. With fluorescent probes, light is also a valuable tool used to monitor the space- and time-dependent effects of stress on cell physiology, as well as selected reactive intermediates pertinent to oxidative stress. ­Keywords: Singlet oxygen, Superoxide anion, Reactive oxygen species (ROS), Photosensitizer, Optogenetic actuator

­Introduction Light can initiate production of reactive oxygen species (ROS) and, as such, influence oxidative stress. This is important given that we live in a world of oxygen, sunlight, and molecules susceptible to oxidation/oxygenation. Light can also be a mechanistic tool to investigate general ROS-dependent processes. For example, pulsed lasers can probe for and characterize reactive intermediates in both space- and time-resolved subcellular experiments. Although the palette of ROS relevant to oxidative distress and eustress is diverse (Halliwell & Gutteridge, 2015; Sies, Berndt, & Jones, 2017), we focus on two species important in light-induced processes: singlet molecular oxygen, O2(a1Δg), and the superoxide radical anion, O2•− (hereafter denoted as superoxide). However, the reactions of these two species spawn other ROS. Thus, through the selective and Oxidative Stress. https://doi.org/10.1016/B978-0-12-818606-0.00019-5 © 2020 Elsevier Inc. All rights reserved.

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spatially localized production of O2(a1Δg) or superoxide, one can provide insight on ROS action in general. Even within the confines of light-induced oxidative stress, an extensive amount of information has accumulated. As such, we cannot provide a comprehensive review. Rather, biased by our own experiments, we summarize recent work.

­Why is light important? The word “light” is often associated only with radiation over the range ~400–700 nm (i.e., visible to the human eye). However, an appreciable amount of nonvisible UV and near-IR light is present to influence living organisms. Although high-energy light (i.e., ionizing gamma and x-ray radiation) is not routinely encountered in our ambient environment, such exposure is relevant for selected medical treatments (Kirakci et al., 2018; Larue et al., 2018). Light can initiate ROS production. The processes involved vary according to the wavelength and the molecules that absorb this light. For many molecules, the excited electronic state produced may initiate electron transfer reactions that involve oxygen. Alternatively, energy transfer from the light-absorbing molecule to oxygen may kinetically compete to produce O2(a1Δg) and, ultimately, a different set of ROS. These two processes are the foundation for the often-used monikers of Type I and Type II photooxidation reactions, respectively (Foote, 1991). The literature is replete with examples of light-initiated ROS-dependent oxidative distress and eustress. One example is photodynamic therapy, PDT, commonly used in cancer treatments, in which ROS initiate cell death (Agostinis et al., 2011). Another is the phenomenon of Low-Level Laser (Light) therapy, LLLT, in which it is inferred that ROS initiate proliferative events in cells (Chung et al., 2012). However, for many such processes, the mechanisms of ROS-based action are still far from being resolved. Light is also a useful mechanistic tool to study ROS-dependent stress. The combination of pulsed lasers, fluorescent probes, and optical microscopes enables subcellular time-resolved spectroscopic studies. Indeed, when combined with optogenetic actuators, as discussed further in the succeeding text, the use of light in this way defines one of the frontiers in the field (Trewin et al., 2018; Westberg, Etzerodt, & Ogilby, 2019).

­Some specifics about light A desirable feature of light, certainly as a source to initiate reactions (i.e., so-called actinic light), is that one can accurately control the dose delivered to the sample. With control over the wavelength, duration of exposure, and incident intensity, one controls the concentration of reactive species initially produced. This alone justifies the use of light as a mechanistic tool; the response to ROS (distress or eustress) depends strongly on dose (Westberg, Bregnhøj, Blázquez-Castro et al., 2016).

­Some specifics about light

With light, one also has the opportunity to control the subcellular location of ROS production. Even with the limiting laws of light diffraction, the volume of excited states obtained simply by focusing the light can be useful (e.g., ~500–700 nm diameter at the beam waist for visible light) (Westberg, Bregnhøj, Blázquez-Castro, et al., 2016). However, the excitation volume can be decreased appreciably, and the effects of scattered light precluded, if excitation occurs via a two-photon process in which light is only absorbed where the photon flux is sufficiently high (Fig. 1) (Gollmer, Besostri, Breitenbach, & Ogilby, 2013; Pimenta et  al., 2012; Westberg, Bregnhøj, Banerjee, et al., 2016). Control over the site of excitation can also be exerted by localizing the lightabsorbing molecule to a specific intracellular organelle. This can be achieved by exploiting (a) solubility parameters and (b) molecular targeting mechanisms (e.g., organelle-specific peptide conjugates) (Celli et al., 2010; Mahon et al., 2007). At the limit, one can use optogenetics to localize the light-activated ROS source on a specific protein (Rodriguez et al., 2017; Trewin et al., 2018; Westberg et al., 2019). For some optogenetic systems, photoinitiated electron transfer reactions quantitatively result in the production of superoxide (Lee, Kim, & Rhee, 2018; Pletnev et al., 2009).

FIG. 1 Fluorescence image of HeLa cells based on a mitochondrial localized dye (green) that illustrates the extent to which actinic light can be controlled. The white dots in the image illustrate the location specificity and spatial resolution accessible through two-photon excitation of a photosensitizer. Reprinted with permission Westberg, M., Bregnhøj, M., Banerjee, C., Blázquez-Castro, A., Breitenbach, T., & Ogilby, P. R. (2016). Exerting better control and specificity with singlet oxygen experiments in live mammalian cells. Methods, 109, 81–91; Westberg, M., Bregnhøj, M., Blázquez-Castro, A., Breitenbach, T., Etzerodt, M., & Ogilby, P. R. (2016). Control of singlet oxygen production in experiments performed on single mammalian cells. Journal of Photochemistry and Photobiology A: Chemistry, 321, 297–308.

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In contrast, different optogenetic actuators can be used to efficiently and selectively produce O2(a1Δg) via energy transfer to ground state oxygen (Westberg et al., 2019; Westberg, Bregnhøj, Etzerodt, & Ogilby, 2017a). Although one can now achieve remarkable control over light-initiated processes, this may not have been the case for experiments performed in the past. As such, given the stated importance of dose, location, and selectivity, many published studies of photoinitiated processes may be “less than conclusive.”

­Light-initiated production of ROS Although light can generate different ROS, the processes most common to living organisms result in the nascent production of O2(a1Δg) and/or superoxide. For example, although irradiation of hydrogen peroxide results in the production of the hydroxyl radical (Okabe, 1978), this occurs only at wavelengths shorter than 300 nm where, in the least, other molecules in a cell will effectively compete for the incident light. In contrast, many molecules, including oxygen itself, absorb light over the range 400–800 nm to produce O2(a1Δg) and/or superoxide. The selective initial production of either O2(a1Δg) or superoxide does not preclude the subsequent formation of other ROS. For example, O2(a1Δg) oxygenates lipids through the “ene” reaction to make an allylic hydroperoxide (Clennan & Pace, 2005). In turn, hydroperoxides readily cleave to yield the hydroxyl radical and an alkoxyl radical (Foote, Valentine, Greenberg, & Liebman, 1995), which propagate to form a plethora of reactive species that can influence the system (Walling, 1995). Similarly, superoxide can be protonated to yield the hydroperoxyl radical that can likewise propagate to yield other ROS. The dismutation reaction of superoxide forms hydrogen peroxide, which, because it is not as reactive as other ROS, can diffuse over greater distances and act as a unique signaling agent (Halliwell & Gutteridge, 2015; Redmond & Kochevar, 2006). Despite the complexity of these reactions, indeed because of the complexity of these reactions, it is mechanistically useful to selectively produce only one ROS at a given initial time.

­Singlet oxygen Molecular oxygen has three low-energy electronic states (Fig. 2). These states are most properly denoted by their term symbols, O2(X3Σg−), O2(a1Δg), and O2(b1Σg+), which also clearly distinguish one state from the other (Atkins, De Paula, & Keeler, 2018; Paterson, Christiansen, Jensen, & Ogilby, 2006). In the past, it has been sufficient to use the moniker “singlet oxygen” with the understanding that this refers to the O2(a1Δg) state. However, recent experiments establish the need to distinguish between the two singlet states of oxygen, O2(a1Δg) and O2(b1Σg+), and we refer to both of these states when using the moniker “singlet oxygen.”

­Singlet oxygen

FIG. 2 Selected features of the three lowest energy electronic states of molecular oxygen: ground state, O2(X3Σg−), first excited state, O2(a1Δg), and second excited state, O2(b1Σg+). (Right side) Crude illustration of the orbital occupancy of the respective states. We only consider the highest occupied orbitals: the degenerate πg orbitals. (Left side) Light can be absorbed by O2(X3Σg−) to directly produce O2(a1Δg) and, independently, O2(b1Σg+). The “wavy” arrow indicates that, once formed, O2(b1Σg+) will decay to produce O2(a1Δg).

­What is ground state oxygen? The ground electronic state of oxygen, O2(X3Σg−), is a spin triplet, as noted by the superscript 3 in the term symbol. This state is a classic example of Hund’s rule of maximum spin multiplicity. When distributing the 16 electrons of O2 into the available molecular orbitals, the last two electrons are placed in degenerate (i.e., equal energy) π antibonding orbitals. The configuration of lowest energy is such that one electron goes into each orbital (Fig. 2), and the spin moments of these electrons are the same yielding a triplet state (Atkins et al., 2018; Paterson et al., 2006). The X in the term symbol indicates that this is the ground state. Of arguably less importance in the present context is the remaining information in the term symbol that characterizes the orbital angular momentum (Σ) and the symmetry (−) and parity (g) of the molecule (i.e., how the wavefunction responds to selected symmetry operations). Although O2(X3Σg−) is not normally considered a ROS, we will deviate somewhat from tradition. As a spin triplet, O2(X3Σg−) is effectively a biradical. In this way, it is a wonderful “radical trap” (i.e., a molecule that will readily react with other free radicals). Thus, if a carbon-based free radical is present, O2(X3Σg−) can trap this radical to form an alkyl peroxide. This now starts well-established propagating processes that result in the formation of other ROS (Walling, 1995). The trapping of free radicals by O2(X3Σg−) can thus be important in oxidative stress.

­What is singlet oxygen? ­The lowest energy singlet state, O2(a1Δg)

The lowest excited electronic state of oxygen, O2(a1Δg), is a spin singlet and results from a change in orbital occupancy relative to O2(X3Σg−). The letter “a” in the term symbol indicates that this is the state next in energy above the ground state, and

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because the spin is different, this letter is written in lower case. Following our description of how the electrons in O2(X3Σg−) are distributed, O2(a1Δg) is often represented as a state in which the final two electrons occupy only one of the degenerate π antibonding orbitals (Fig.  2) (Halliwell & Gutteridge, 2015; Krumova & Cosa, 2016). This can only occur when the spin moments of the respective electrons are opposed (i.e., the Pauli exclusion principle) and, thus, yield a state of net singlet spin. Although this way of illustrating the difference between the O2(X3Σg−) and O2(a1Δg) states is easy to comprehend, it is inaccurate. The proper expression of the more general Pauli Principle is that the total wavefunction for the molecule must change sign (i.e., be antisymmetric) upon interchanging electrons (Atkins et al., 2018). The illustration of orbital occupancy that accurately represents this becomes more complicated (Paterson et al., 2006) and thus is rarely used. The energy difference between the O2(X3Σg−) and O2(a1Δg) states, ~94.3 kJ/mol, depends slightly on the solvent, with the spectroscopic transition falling in the wavelength range ~1270–1280 nm (Bregnhøj, Westberg, Minaev, & Ogilby, 2017; Ogilby, 1999). With few exceptions, light-absorbing molecules of biological importance have excited state energies that exceed the ~94 kJ/mol excitation energy of O2(a1Δg). This is important for one method to produce O2(a1Δg): photosensitization. Although the study of O2(a1Δg) has long been important in quantum mechanics and spectroscopy (Herzberg, 1950; Mulliken, 1928), the reactions of O2(a1Δg) have arguably thrust this molecule into the limelight (Clennan & Pace, 2005; Foote, 1968). Many of these reactions occur with molecules of biological importance (DiMascio et al., 2019) and, as such, are pertinent to oxidative stress.

­The other singlet state, O2(b1Σg+)

The second excited electronic state of oxygen, O2(b1Σg+), is also a spin singlet. The crude way to represent the orbital occupancy in this case is to put the last two of oxygen’s 16 electrons in each of the degenerate antibonding π orbitals and to have their spin moments opposed (Fig. 2) (Paterson et al., 2006). The O2(b1Σg+) - O2(X3Σg−) spectroscopic transition occurs at 765 nm (Fig.  2). Once formed in solution-phase systems, O2(b1Σg+) rapidly decays to produce the O2(a1Δg) state with almost unit efficiency (Schweitzer & Schmidt, 2003). In particular, there is no evidence to indicate that O2(b1Σg+) undergoes chemical reactions in the same way as O2(a1Δg) (Scurlock, Wang, & Ogilby, 1996). Nevertheless, O2(b1Σg+) is important because it is a convenient precursor to O2(a1Δg).

­Transitions between states When discussing transitions between electronic states of a given molecule, the term symbols are important in ascertaining whether the transition is probable (i.e., “allowed”) or improbable (i.e., “forbidden” or weak). Thus, for the aforementioned states of oxygen, we say that the transition between the O2(X3Σg−) and O2(a1Δg) states is “spin forbidden,” among other things; a transition involving a change from triplet to singlet spin is generally not probable.

­Singlet oxygen

­How can singlet oxygen be produced by light? ­Energy transfer from a photosensitizer

The most common and efficient way to produce singlet oxygen using light is via a photosensitized process. In this case, the light is absorbed by a separate molecule, the photosensitizer, and the energy of excitation is transferred to O2(X3Σg−) to produce either O2(a1Δg) or O2(b1Σg+) (Fig. 3A). This process of energy transfer requires a collision between oxygen and the sensitizer and generally occurs with the longer-lived sensitizer triplet state via the reaction 3Sens1 + O2(X3Σg−) → 1Sens0 + O2(a1Δg)/O2(b1Σg+)

FIG. 3 Relevant processes that can occur upon irradiation of a photosensitizer, xSensy, where x denotes the electronic spin and y distinguishes one state from another. Green arrows represent one-photon excitation, whereas red arrows represent two-photon excitation. Depending on the sensitizer used, these transitions can initially populate the same or different states. The “wavy” lines represent nonradiative transitions between states. (A) The sensitized production of O2(a1Δg). (B) A sensitized electron transfer reaction in which an amino acid, AA, donates an electron to the excited state sensitizer to form a radical anion. The latter then reduces O2(X3Σg−) to superoxide in a second electron transfer reaction.

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(Schweitzer & Schmidt, 2003). Although not immediately apparent, a ­collision complex between two triplet states has a component of net singlet spin. Thus, this reaction is spin allowed. A process that kinetically competes with energy transfer to produce singlet oxygen is electron transfer from or to the excited state sensitizer (Fig. 3B). This process also plays an important role in ROS production. The production of the excited state sensitizer can occur through both one- and two-photon processes (Fig. 3). One-photon excitation is most commonly employed and encountered, and absorption can occur with low light intensities. Two-photon excitation is a nonlinear process and occurs only with high light intensities (i.e., lasers) (Ogilby, 2010; Ogilby, 2016). The two-photon process has several advantages: (a) the incident photons are of longer wavelength and generally occur in the so-called “biological window” where many endogenous biological molecules do not absorb, and (b) the required high photon flux can be achieved by focusing the incident light, thereby facilitating spatial control and localization (Fig. 1). Many molecules can act as a singlet oxygen sensitizer, including molecules endogenous to mammalian cells (Redmond & Gamlin, 1999). Exogenous sensitizers can be added, such as the light-absorbing compound used in PDT (Agostinis et al., 2011). Although most molecules of relevance are energetically capable of generating singlet oxygen (vide supra), energy transfer does not always occur with unit efficiency. The site of singlet oxygen production in, on, or near a cell can have significant stress-related consequences (Blázquez-Castro, Breitenbach, & Ogilby, 2018; Gollmer et al., 2013; Kessel, 2004; Redmond & Kochevar, 2006; Rubio, Fleury, & Redmond, 2009). The localization of exogenous sensitizers is often achieved based solely on solubility parameters (e.g., hydrophobic vs hydrophilic domains). This can be undesirable because the stress response may result from a small amount of sensitizer in site A rather than the larger amount in site B, and data can thus be misinterpreted. To address these issues, there is a range of options by which (a) targeting schemes are used to better localize the sensitizer (Celli et al., 2010; Mahon et al., 2007), and (b) location-specific molecular processes are used to activate the sensitizer (e.g., local pH or protein binding) (Callaghan & Senge, 2018). One recent approach is the use of genetically encoded proteins that encapsulate the sensitizer (Trewin et al., 2018; Westberg et al., 2019). A distinct advantage of the latter is that one can achieve molecular level specificity for the site of singlet oxygen production. The design and characterization of photosensitizers that selectively make singlet oxygen at the expense of superoxide, and vice versa, has been extensive (e.g., the kinetic competition illustrated in Fig. 3). Indeed, this issue has been the focus of recent work on genetically encoded sensitizers (Trewin et al., 2018; Westberg et al., 2019).

­Direct irradiation of ground state oxygen

Selective O2(a1Δg) production has also been achieved by directly irradiating O2(X3Σg−) (Fig.  2) (Blázquez-Castro, 2017). Both the O2(X3Σg−)-O2(a1Δg) transition at 1275 nm (Anquez, Yazidi-Belkoura, Randoux, Suret, & Courtade, 2012; Krasnovsky, Roumbal, & Strizhakov, 2008) and the O2(X3Σg−)-O2(b1Σg+) ­transition

­Singlet oxygen

at 765 nm (Blázquez-Castro et  al., 2018; Bregnhøj, Blázquez-Castro, Westberg, Breitenbach, & Ogilby, 2015) have been exploited in this regard, including in live cells. Although these transitions are extremely weak (e.g., they are spin forbidden), one can nevertheless control the O2(a1Δg) dose such as to cover the range from distress to eustress (Blázquez-Castro et al., 2018; Bregnhøj et al., 2015; Westberg, Bregnhøj, Blázquez-Castro, et al., 2016). The O2(X3Σg−)-O2(b1Σg+) transition at 765 nm has favorable attributes in that other compounds common to biological systems, particularly water, do not absorb at this wavelength (i.e., this transition occurs in the “biological window”). Although this is a one-photon experiment, the light can still be sufficiently localized for subcellular irradiation (Fig. 4). In this way, progress through the cell cycle can be accelerated upon low-dose irradiation into the cytoplasm, whereas proliferation is delayed upon analogous irradiation into the nucleus (Blázquez-Castro et al., 2018).

­Dependence on the concentration of ground state oxygen

Both methods described earlier for producing O2(a1Δg) depend on the concentration of O2(X3Σg−) in the system. This can be an important variable for experiments with cells, where there are appreciable location-dependent differences in the O2(X3Σg−) concentration. Specifically, oxygen is more soluble in hydrocarbons than in aqueous media; the concentration of O2(X3Σg−) in air-saturated water is ~10 times smaller than in an air-saturated hydrocarbon solvent (Battino, Rettich, & Tominaga, 1983). Thus, there is less dissolved O2(X3Σg−) in the cytosol than in a membrane.

FIG. 4 Focused 765 nm light can be used to primarily create O2(a1Δg) in the cytoplasm and, independently, the nucleus of a cell. The O2(X3Σg−) → O2(b1Σg+) absorption profile is sufficiently narrow that irradiation with 775 nm light will not produce an appreciable amount of O2(a1Δg) (Bregnhøj et al., 2015). The green fluorescence from a dye localized in the mitochondria of a HeLa cell was used for this image. Reprinted with permission Blázquez-Castro, A., Breitenbach, T., & Ogilby, P. R., 2018. Cell cycle modulation through subcellular spatially resolved production of singlet oxygen via direct 765 nm irradiation: Manipulating the onset of mitosis. Photochemical & Photobiological Sciences, 17, 1310–1318.

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To illustrate salient features of this point, we modeled the O2(X3Σg−) concentration dependence of O2(a1Δg) production for three cases (Fig. 5). Production of O2(a1Δg) upon one-photon excitation of oxygen at 765 nm should depend linearly on the O2(X3Σg−) concentration, as expected from the Lambert–Beer law, and this has been confirmed (Bregnhøj et al., 2015). Given the extended focal volume used thus far in cell-based 765 nm experiments (i.e., beam waist diameter of ~2 μm) (Blázquez-Castro et al., 2018), it is clear that O2(X3Σg−) in both hydrocarbon and aqueous domains is irradiated. Despite the small absorption coefficient for this transition, appreciable and controllable amounts of O2(a1Δg) can still be made using this technique with air-equilibrated cells (Blázquez-Castro et  al., 2018; Westberg, Bregnhøj, Blázquez-Castro, et al., 2016). A key parameter in a photosensitized O2(a1Δg) experiment is the lifetime of the sensitizer triplet state, τT, as measured in a deoxygenated sample. The magnitude of τT is a measure of the deactivation pathways against which quenching by O2(X3Σg−) must kinetically compete. For a freely dissolved sensitizer, a rough guideline is that if τT is longer than ~5–10 μs, then most of the sensitizer triplet states will be quenched by O2(X3Σg−) in an air-saturated aqueous solution. In short, one sees a plateau effect in the O2(a1Δg) yield as [O2(X3Σg−)] is increased beyond an air-saturated solution (Fig. 5) (Hatz, Poulsen, & Ogilby, 2008; Kristiansen, Scurlock, Iu, & Ogilby, 1991). This is a desirable feature if one is concerned that differences in the intracellular O2(X3Σg−)

FIG. 5 A model of how oxygen concentration affects the yield of O2(a1Δg) for three cases: direct irradiation at 765 nm (bottom, green line), and sensitized formation with a shortlived sensitizer triplet state, τT = 1 μs (middle, red line) and with a long-lived sensitizer triplet state, τT = 50 μs (top, blue line). As noted in the text, τT refers to the lifetime in a deoxygenated sample. For the quenching of the sensitizer triplet state by O2(X3Σg−), we used the typical rate constant of 4 × 109 s−1 M−1. The point is to illustrate how, for a given case, the O2(a1Δg) yield responds to a change in the oxygen concentration. Thus, emphasis should not be placed on the yields between cases.

­Singlet oxygen

concentration will influence the dose of O2(a1Δg); the plateau effect ­precludes this problem. In contrast, if τT is comparatively short, then unimolecular triplet state deactivation channels will still compete with quenching by O2(X3Σg−), and the plateau may not be reached even in an oxygen-saturated system (Fig. 5). It is appropriate to carry this discussion over to the photosensitized production of superoxide (Fig. 3B). Superoxide formation is often preceded by electron transfer reactions involving the sensitizer and other molecules (e.g., proteins). As such, the final electron transfer reaction from a radical anion of an organic molecule to O2(X3Σg−) can depend on the O2(X3Σg−) concentration in a convoluted way. Thus, when a sensitizer can produce both O2(a1Δg) and superoxide, the ratio of the amounts of O2(a1Δg) to superoxide may change as a function of the local oxygen concentration.

­ hat reactions of O2(a1Δg) are potentially pertinent to oxidative W stress? The reactions of O2(a1Δg) with organic molecules of biological relevance have been studied for decades (Clennan & Pace, 2005; Davies, 2003; DiMascio et al., 2019; Foote, 1968; Girotti, 2001; Wilkinson, Helman, & Ross, 1995). Classic reactions include the formation of hydroperoxides (e.g., the “ene” reaction with olefins in lipids, Fig. 6), endoperoxides (e.g., 2 + 4 cycloaddition reaction with the dienes in histidine and guanine), dioxetanes (e.g., 2 + 2 cycloaddition with tryptophan), and sulfoxides from sulfides such as methionine. Most importantly, the product initially formed in these reactions is generally just a precursor to other oxidation products, often formed by thermal reactions in the absence of light. Hydroperoxide decomposition to form

FIG. 6 Two O2(a1Δg) reactions of biological importance. Lipids such as oleic acid react via the “ene” reaction to produce a hydroperoxide. The hydroperoxide OO bond is readily cleaved, thermally and photolytically, to yield the hydroxyl radical and an alkoxyl radical, both of which are reactive species. GSH reacts to yield GSSG and other oxidized products.

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alkoxyl and hydroxyl radicals is a good example (Fig. 6) (Walling, 1995). Thus, the O2(a1Δg)-mediated oxidation of a specific amino acid in a protein can have appreciable consequences, particularly if that residue is part of an enzyme’s active site or a site for posttranslational modification (Davies, 2003). Glutathione, GSH, reacts with O2(a1Δg) to predominantly form a dimer, GSSG, linked by a disulfide bridge (Fig. 6) (Devasagayam, Sundquist, DiMascio, Kaiser, & Sies, 1991). It is likely that GSSG is formed via a O2(a1Δg)-mediated electron transfer reaction. Because O2(a1Δg) can influence the GSSG/GSH ratio, it follows that this is one reaction of O2(a1Δg) that can certainly be pertinent to oxidative stress. In this reaction with O2(a1Δg), it is the thiolate, RS−, rather than the thiol, RSH, that is more reactive. This has broad environment-dependent intracellular ramifications for a number of thiol-containing molecules, not just GSH, and is consistent with the fact that O2(a1Δg) is an electrophile. The O2(a1Δg)-influenced redox state of a cell can also be modulated by other antioxidants. It is known from solution-phase experiments that members of the carotenoid family efficiently deactivate O2(a1Δg) to O2(X3Σg−) with rate constants close to the diffusion-controlled limit (DiMascio, Kaiser, & Sies, 1989; Edge, McGarvey, & Truscott, 1997; Foote & Denny, 1968). This process involves energy transfer from O2(a1Δg) to produce the carotenoid triplet state. Carotenoids are unique in this regard because, unlike almost all other organic molecules, they have a triplet state energy that is slightly lower than the ~94 kJ/mol excitation energy of O2(a1Δg). Most importantly, this interaction requires a collision between the respective molecules. Thus, if the system precludes this collision within the lifetime of O2(a1Δg) (e.g., by compartmentalization or by a viscosity that adversely affects diffusion), then the carotenoid is benign as a O2(a1Δg) quencher. However, the carotenoid may still act as a radical trap for secondary/downstream oxidation products of a O2(a1Δg) reaction. With this in mind, it is relevant to note that, when added to a mammalian cell, β-carotene does not quench O2(a1Δg) produced in a photosensitized reaction, but it still protects against O2(a1Δg)-mediated cell death (Bosio et al., 2013).

­O2(a1Δg) as a diffusible signaling agent

Having established that O2(a1Δg)-mediated oxygenation or oxidation reactions can alter the structure and function of biomolecules, we must consider how these reactions kinetically compete with other processes for O2(a1Δg) removal. This analysis provides an estimate for the diffusion distance of O2(a1Δg) from its point of production in a cell. The dimensions of O2(a1Δg)‘s “sphere of activity” help define its role as a diffusible signaling agent capable of influencing stress. We first revisit the phrase “reactive oxygen species.” In the strict chemical sense, O2(a1Δg) is not a “reactive” intermediate; it is a “selective” intermediate. For most biomolecules of significance, the rate constants, k, for interaction with O2(a1Δg) are no greater than ~5 × 107 s−1 M−1, and many are on the order of ~105–106 s−1 M−1 (Wilkinson et  al., 1995). In short, these reactions occur well below the diffusion-­ controlled limit that characterizes “reactive intermediates” such as the hydroxyl radical (i.e., where k ~1010 s−1 M−1) (Redmond & Kochevar, 2006).

­Singlet oxygen

The abundance of organic material in a cell is dominated by proteins, and only selected amino acids have rate constants for reaction with O2(a1Δg) on the order of ~107 s−1 M−1 (e.g., tryptophan, tyrosine, histidine, cysteine, and methionine). If we consider GSH, 10 mM is a representative intracellular concentration, and the rate constant for reaction with O2(a1Δg) is  50 y Plasma

Brower et al. (2016)

Mixed vehicle exhaust: 100 or 300 μg/m3 or filtered air for 6 h

Male, 10-week-old C57/BL6 mice; n = 6 per group

Huang et al. (2015)

PM2.5 sampled from Xiamen City, China

A549 cells

38 pathways identified related to Inflammation Endothelial function mitochondrial bioenergetics Amino acid metabolism Energy production Oxidative stress Lipid metabolism

Nitrogen metabolism, citrate cycle Aminoacyl-tRNA biosynthesis Phenylalanine, tyrosine, and tryptophan biosynthesis Glutathione, glyoxylate, and dicarboxylate metabolism

Occupation air pollutants Kuo et al. (2012)

Welding fumes

Acetone, betaine/ trimethylamine N-oxide, creatine, creatinine, glycine, gluconate, serine, S-sulfocysteine, taurine, hippurate

Amino acid metabolism Carbohydrate metabolism Oxidation reduction pathways Urea metabolism Continued

469

35 male welders and 16 male office workers Age: 45–64 Urine

­Industrial air toxics

318 metabolic features associated with lipid peroxidation, endogenous inhibitors of nitric oxide, and vehicle exhaust exposure biomarkers including Glutamate and linolenic acid Notable changes 2-Methylbutyrylglycine, 2-hydroxy-3-methylvalerate, 3-methyl-2-oxobutyrate, alphahydroxyisocaproate, creatine, taurine, cysteine, lactate, mannose, glycerate, branched chain amino acid catabolites, butyrylcarnitine, fatty acids Oxidized glutathione, cysteine-glutathione disulfide, 13-HODE, 9-HODE, 12,13-diHOME 16 metabolites including: cis-Aconitate, malate, pantothenate, adenosine diphosphate Glutamate, NAcGlu, phenylalanine, tryptophan, glutathione

470

Study

Pollutant exposure

Population Sample for analysis

Notable metabolite signal

Mainly affected metabolic pathways

Ozone Mathews, Kasahara, et al. (2017)

Ozone: 2 ppm for 3 h

Lean wild-type and obese db/db female mice Lung

Cheng et al. (2018)

Ozone: 0.3 ppm for 2 h while undergoing intermittent exercise

23 adults Bronchoalveolar lavage fluid

Items in bold reflect changes in metabolic outcomes associated with oxidative stress.

Bacterial/mammalian cometabolites, citrulline, heme, hypotaurine (db/ db), monoglycerides (WT), PC lysolipids (WT), branched chain amino acid metabolites, lysolipids, glutathione Allantoin, alphaketoglutamate, alanine, arginine, aspartate, cyanoamino acid, glutamate, glutamine, glycerophospholipid, glycine, proline, serine, sphingolipid, threonine, threonate

Amino acid metabolism Lipid metabolism Microbiome

Aldarate metabolism Amino acid metabolism Aminoacyl-tRNA biosynthesis Ascorbate metabolism Ether lipid metabolism Lipid metabolism

CHAPTER 24  Metabolomics as a tool to unravel the oxidative stress

Table 1  Metabolomic studies characterizing changes in low-molecular-weight metabolites, indicative of oxidative stress, in response to gaseous and particulate air pollution exposure—cont’d

­Industrial air toxics

decadienylcarnitine, hydroxydodecenoylcarnitine, dodecadienylcarnitine, and dodecenoylcarnitine) were significantly increased. Uric acid was the only metabolite that was significantly decreased. Several of these observations are indicative of an increased PAH and metal exposure causing various oxidative stress-related effects in that the following metabolites, other than uric acid, were all significantly increased in the exposed populations: • Pyroglutamic acid—an oxidative product of proline and a metabolite of the γ-glutamyl cycle through which glutathione is synthesized and degraded (Pederzolli et al., 2007). Human urinary pyroglutamic acid has also been identified as a potential biomarker of oxidative stress induced by chronic cadmium (Gao et al., 2014). • 3-Methylhistidine—a key constituent of muscle proteins and a marker of muscle protein breakdown (Aranibar et al., 2011), which in turn can be promoted by oxidative stress (Powers, Smuder, & Criswell, 2011). • Azelaic acid, decenedioic acid, and hydroxytetradecanedioic acid—by-products of lipid peroxidation and indicators of oxidative stress (Maes, Mihaylova, & Leunis, 2006). • Decenedioylglucuronide—catalyzed by UDP-glucuronosyltransferases (UGTs), which catalyze the conjugation of xenobiotics or their reactive metabolites with glucuronic acid and as such act as indirect antioxidants (Kalthoff, Ehmer, Freiberg, Manns, & Strassburg, 2010). The expression of UGTs can be induced by oxidative stress, metals, and xenobiotic exposure (Jennings, Limonciel, Felice, & Leonard, 2013; Kalthoff et al., 2010). • Several unusual medium-chain acylcarnitines—synthesized with l-carnitine and the corresponding medium-chain fatty acids (Mentlein, Reuter, & Heymann, 1985) and in the main stem from incomplete mitochondrial β-oxidation of longchain fatty acids and/or lipid peroxidation (Chen et al., 2009). • Uric acid (significantly decreased in the exposed children and elderly nonsmokers)—a powerful antioxidant and end product of purine metabolism. Taken together, these results prompted the authors to surmise that the perturbation of metabolites reflected the oxidative stress-related biological effects induced by ambient PAH and metal exposure, including the depletion of antioxidants, accelerated muscle proteolysis, elevated UGT activity, increased lipid peroxidation, and mitochondrial lipid metabolism dysfunction. Despite increases in benzene being higher than those for PAHs and metals, significant associations between benzene exposure and the metabolic biomarkers were not observed (Wang et al., 2019). Two possible explanations relate to a weaker oxidation capacity of benzene compared with that of PAHs and metals and a much lower exposure level to benzene such that its involvement in oxidative stress was great enough to be detected. Another study to identify potential metabolites linking industrial air toxic exposures to oxidative stress through plausible exposure-related pathways focused on 252 children and elderly subjects living at varying distances from oil refineries and coal-fired power plants within a petrochemical complex in Taiwan

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(Chen et al., 2017). The pollution-affected area was characterized by elevated ambient concentrations of vanadium and PAHs, and its residents had increased urine concentrations of 1-OHP, vanadium, nickel, copper, arsenic, strontium, cadmium, mercury, thallium, and biomarkers of oxidative stress (8-hydroxy-2′-deoxyguanosine [8-OHdG], 4-­hydroxy-2-nonenal-mercapturic acid [HNE-MA], 8-isoprostaglandin F2α [8-isoPF2α], and 8-nitroguanine [8-NO2Gua]) compared with low-exposure subjects. Untargeted urine metabolomics identified age-dependent potential metabolites responsible for the separation between high- and low-exposure groups, while pathway analysis revealed four biological pathways (tryptophan metabolism; phenylalanine metabolism; glycine, serine, and threonine metabolism; and alanine, aspartate, and glutamate metabolism) that could associate multiple exposures with increased oxidative stress. Specifically, tryptophan, the metabolism of which has been shown to be involved with increased oxidative stress and cancer, neurodegenerative diseases, rhinitis, and asthma (Chen & Guillemin, 2009; Ciprandi, De Amici, Tosca, & Fuchs, 2010; Gostner, Becker, Kofler, Strasser, & Fuchs, 2016; Stoy et al., 2005) was downregulated in the high-exposure compared with low-­exposure group in children and correlated with 8-OHdG, HNE-MA, and 8-isoPGF2α. A downstream metabolite of tryptophan metabolism, 1H-indole-3-acetamide, was also identified and associated with 8-NO2Gua. Altered phenylalanine metabolism was also identified in children. Phenylalanine, which has been used as a biomarker of oxidative damage (Orhan, Vermeulen, Tump, Zappey, & Meerman, 2004), was significantly correlated with exposures as well as 8-OHdG and HNE-MA, while its downstream metabolites, hippuric acid, 4-hydroxy benzoic acid, and succinic acid, correlated with HNE-MA, 8-isoPGF2α, and 8-NO2Gua. In elderly subjects, glycine, serine, and threonine metabolism, a pathway closely related to oncogenic transformation and the biosynthesis of the antioxidant glutathione (Amelio, Cutruzzola, Antonov, Agostini, & Melino, 2014), was identified, with threonine correlating with 8-NO2Gua; serine with 8-OHdG, HNE-MA, and 8-NO2Gua; and glyceric acid with HNE-MA and 8-NO2Gua. Altered alanine, aspartate, and glutamate metabolism was identified in both children and elderly subjects. Aspartic acid was downregulated in high-­exposure subjects and associated with 8-NO2Gua and HNE-MA in children and elderly participants, respectively. The relevance here is that studies have shown that aspartic acid could increase glutathione levels and decrease lipid peroxidation in animal models (Sivakumar, Babu, & Shyamaladevi, 2011). Finally, threonate was upregulated in both age groups, indicative of deregulation in its antioxidant precursor, ascorbic acid (Gao et al., 2012), associating multiple exposures with HNE-MA and 8-NO2Gua in children and all four oxidative stress biomarkers in elderly participants. The investigators drew from these results, a complicated web illustrating the association between exposures and different oxidative stress-induced health effects through age-dependent diverse biological pathways (Fig. 1). The industrial city of Taiyuan in China suffers from particularly serious PM2.5 pollution owing to a large number of coal combustion and active industrial activities (Cao et al., 2014). In the winter of 2016, the average concentration of PM2.5 was 175 μg/m3—significantly higher than concentrations of approximately 11–18 μg/m3

­Industrial air toxics

FIG. 1 Exposure pathways of petrochemical of air pollution and the effects on urine metabolic profile changes and increased oxidative stress. Reproduced from Chen, C. S., Yuan, T. H., Shie, R. H., Wu, K. Y., & Chan, C. C. (2017). Linking sources to early effects by profiling urine metabolome of residents living near oil refineries and coal-fired power plants. Environment International, 102, 87–96.

measured in European cities during the cold period (Jedynska et  al., 2015). Furthermore, the large-scale combustion of coal in Taiyuan generates high concentrations of particularly toxic organic chemicals (Li, Kou, Geng, Dong, & Cai, 2014). Nontargeted and targeted metabolomics combined with multiple cytotoxicity assays (indices of oxidative stress, inflammation, mitochondrial respiration, and glycolysis and the expression levels of key genes) has been used to investigate the overall metabolic changes and relevant toxicological pathways caused by Taiyuan winter total PM2.5 and its water-soluble and organic-soluble fractions in human lung ­bronchial epithelial cells (BEAS-2B) (Song et  al., 2019). The exposure concentrations were chosen according to the Taiyuan winter actual PM2.5 concentration. Significant metabolome alterations were observed after exposure to total PM2.5 or its organic-­soluble fraction, and moreover, the higher the exposure concentrations, the more number of components changed, indicating that PM2.5 affected the bronchial epithelial cells in a dose-dependent manner. The most influenced pathways were glutathione metabolism, purine metabolism (upregulated cluster of metabolites), and arginine and proline metabolism (downregulated cluster of amino acids).

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In ­addition, the ­tricarboxylic acid (TCA) cycle (decreased) and glycolysis (increased) were affected in cells exposed to total PM2.5 samples and its organic-soluble fraction, indicating that the energy metabolism was affected. The specific perturbations in metabolites indicative of antioxidant-oxidant balance disruption included the oxidation of GSH to GSSG, and this was reinforced by gene expression data showing a large increase in the expression of nuclear factor erythroid 2-related factor 2 and its downstream genes heme oxygenase 1 and quinone oxidoreductase. While purine metabolism, which produces reactive oxygen species (ROS) via the xanthine oxidase (XO) pathway, was one of the most significantly affected pathways following total PM2.5 exposure, gene expression data revealed increased concentrations of XO. Exposure also disrupted arginine metabolism. One well-described pathway of arginine metabolism involves its conversion to nitric oxide, the accumulation of which could generate reactive nitrogen species. A speculative scenario as a result of the interplay between increased oxidative stress and inflammation, mitochondrial dysfunction, energy metabolic reprogramming, oxidative stress, and inflammation and energy metabolic reprogramming from oxidative phosphorylation to glycolysis is illustrated in Fig. 2. Another group (Wang, Jiang, et al., 2017) have investigated the pulmonary metabolome responses to PM2.5 sampled from Tangshan, China’s top steelmaking city and one of the country’s most polluted urban areas (Luo, Hanb, & Liu, 2017). Adult male rats (n = 6) were treated by intratracheal instillation with PM2.5 suspension once a week at the dose of 1 mg/kg/week for 3 consecutive months. The control group (n = 6) was treated with saline. In an attempt to understand the comprehensive pulmonary response to PM2.5, a nontargeted metabolomics strategy was adopted together with biochemical analyses of oxidant (malondialdehyde and thiobarbituric acid reactive substances) and antioxidant (catalase, glutathione peroxidase, and superoxide dismutase) imbalance to characterize the overall metabolic changes and relevant toxicological pathways. Along with the biochemical findings of a significant increase in oxidative stress, significant metabolome alterations were observed in the lung tissues of the treated rats. A variety of identified metabolites are mainly involved in the metabolism of lipid and nucleotides. Abnormal lipid metabolism has been associated with the activation of oxidative and inflammatory pathways (Zhao et al., 2015), and in this study, the reduction in the integral surfactant phospholipids (e.g., phosphatidylcholine [PC]) and elevation in lysophospholipid PC suggests increased hydrolysis, possibly owing to oxidative stress-induced activation of phospholipase A2 (PLA-2). Sphingolipid is another important component of cell membrane. Sphingosine, which is synthesized from sphinganine, and sphingomyelin form primary parts of sphingolipids and play a pivotal role in the cellular responses to oxidative stress. Wang, Jiang, et  al. (2017) observed significantly reduced concentrations of sphinganine and sphingomyelin levels in treatment group. Oxidative stress indicators identified in the pulmonary metabolome of treated rats included significant up- and downregulations in 8-hydroxyguanosine and spermidine, respectively. Spermidine maintains membrane potential and controls intracellular pH and volume through synchronizing many biological processes and possesses antioxidant

­Traffic-related air pollution

FIG. 2 Schematic overview of the disturbed metabolic pathways in BEAS-2B cells upon PM2.5 exposure. Molecules marked in red represented the upregulated metabolites and in green represented the downregulated metabolites. Reproduced from Song, Y., Li, R., Zhang, Y., Wei, J., Chen, W., Chung, C. K. A., & Cai, Z. (2019). Mass spectrometry-based metabolomics reveals the mechanism of ambient fine particulate matter and its components on energy metabolic reprogramming in BEAS-2B cells. The Science of the Total Environment, 651, 3139–3150.

­activity (Kim, 2017). Combining the results may suggest that PM2.5-induced pulmonary ­toxicity through disturbing pro-oxidant/antioxidant balance, causing changes of phospholipid, sphingolipid, and purine metabolism and DNA damage in lung tissues.

­Traffic-related air pollution Traffic-related air pollution has been linked to numerous adverse health effects, but the specific constituents responsible for these effects and how they contribute to corresponding biological responses are poorly understood (HEI, 2010). This uncertainty is, in part, due to the chemical and physical heterogeneity of this pollutant source and the numerous biological factors and pathways that may mediate responses (Bates et al., 2015; Brown et al., 2012; Zanobetti, Austin, Coull, Schwartz, & Koutrakis, 2014; Zora et al., 2013). It is not surprising therefore that to address these research gaps and uncertainties, workers have adopted a m ­ etabolomic ­approach to

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gain further insights into the toxicological mechanisms u­ nderlying TRAP exposurerelated diseases in human panel-based protocols (Ladva et al., 2018; Liang et al., 2018; Walker et al., 2019), animal models (Brower et al., 2016), and in vitro studies (Huang et al., 2015). Ladva et al. (2018) used measurements collected as part of a longitudinal panel (Atlanta Commuters Exposure [ACE] study) of car commuters, to examine in-vehicle air pollution concentrations, targeted inflammatory and vascular injury biomarkers, and metabolomic profiles associated with on-road traffic exposures during morning rush hour commutes in Atlanta (Ladva et  al., 2018). Sixty adults participated in a crossover study, in which each participant conducted a highway commute and was randomized to either a (a) side-street commute or (b) clinic exposure session. The metabolomics analyses detected 10-h perturbations in features associated with in-vehicle, particulate metal exposures (aluminum, lead, and iron), which reflected changes in arachidonic acid, leukotriene, and tryptophan metabolism. Other TRP parameters including PM2.5 mass and particle number concentration had no association. The association of tryptophan metabolism with lead exposure is consistent with the evidence of this metal, despite being redox inactive, participating in the depletion of antioxidants (Matovic, Buha, Ethukic-Cosic, & Bulat, 2015; Valko, Jomova, Rhodes, Kuca, & Musilek, 2016). Furthermore, while aluminum is also redox inactive, it has demonstrated ability to shift biological systems into a state of oxidative stress (Verstraeten, Aimo, & Oteiza, 2008). In the Dorm Room Inhalation to Vehicle Emission (DRIVE) study, the ability of metabolomic profiling to reflect internal exposures to complex traffic-related air pollution mixtures was assessed (Liang et  al., 2018). Here, untargeted metabolomics was applied to a 12-week field protocol incorporating repeated biomonitoring (plasma and saliva) in a panel of 54 college students living in dormitories either near (20 m) or far (1.4 km) from a major highway. In parallel, a suite of TRAP (black carbon, carbon monoxide, nitrogen oxides, and PM2.5) were measured at multiple ambient and indoor sites at varying distances from the highway artery. Workers observed 1291 unique metabolic features significantly associated with at least one ­traffic indicator and confirmed the chemical identities of 10 metabolites indicative of endogenous metabolic signals, including arginine, histidine, gamma linolenic acid, and hypoxanthine. Pathway analysis indicated elicitation of inflammatory and oxidative stress-related pathways, and among these, those involved in leukotriene and vitamin E metabolism consistently showed the strongest associations with TRAPs in both saliva and plasma samples. Tentatively matched metabolites in the latter pathway included dehydrogenation precursors of tocopherols and tocotrienols. Of note, 11 metabolic features were matched to vitamin E metabolites, and the intensities of these antioxidants decreased with higher TRAP exposures. Other identified metabolites worthy of discussion include arginine and histidine and hypoxanthine. In a study examining the serum amino acid profiles in obese and nonobese women, both histidine and arginine were found to be negatively associated with inflammation and oxidative stress (Niu et  al., 2012). Hypoxanthine, a purine derivative that protects against cellular oxidative injury by inhibiting the activation of nuclear

­Traffic-related air pollution

poly(ADP-ribose) polymerase (Durkacz, Omidiji, Gray, & Shall, 1980; Szabo, 1998; Szabo & Dawson, 1998), was positively associated with both indoor and outdoor CO levels. Vlaanderen et al. (2017) has also identified hypoxanthine as associated with a 5-h exposure to real-world ambient air pollution (Vlaanderen et al., 2017) as did Song et al. (2019) and Wang, Jiang, et al. (2017) in their in vitro and rat metabolomic profiling studies with PM2.5. Further efforts to delineate biological response mechanisms associated with a year-long averaged personal ultrafine particle (UFP) exposure have incorporated untargeted metabolomics to profile plasma from 59 participants enrolled in the Community Assessment of Freeway Exposure and Health (CAFEH) study. Metabolic variations associated with UFP exposure were assessed using a cross-sectional study design based upon low (mean 16,000 particles/cm3) and high (mean 24,000 particles/ cm3) annual average UFP exposures. Prior to this, nonmetabolomic characterization of amino acids, lipid/fatty acid metabolites, cofactors, and cellular respiration identified five metabolites that were differentially expressed between low and high exposures, including arginine, aspartic acid, glutamine, cystine, and methionine sulfoxide. Elevated methionine sulfoxide and cystine are consistent with increased oxidative stress. In humans, the major extracellular thiol/disulfide redox couple is cysteine and its disulfide form, cystine (Go & Jones, 2011). Increased oxidation of the couple and thus a shift toward a more positive redox potential leads to an activation of proinflammatory cytokines (Iyer et al., 2009), regulates initial atherosclerotic events (Go & Jones, 2005), and is associated with cardiovascular disease and endothelial function (Ashfaq et al., 2008). Methionine sulfoxide is the oxidation product of methionine with reactive oxygen species and a recognized marker of oxidative stress (Mashima, Nakanishi-Ueda, & Yamamoto, 2003; Moskovitz, Berlett, Poston, & Stadtman, 1997). Analysis of the metabolome identified 316 features associated with UFP exposure, consistent with increased lipid peroxidation, endogenous inhibitors of nitric oxide, and vehicle exhaust exposure biomarkers. Identified decreases in linolenic acid and glutamate provide additional evidence of oxidative stress in that glutamate is a precursor for glutathione and linoleic acid is a polyunsaturated fatty acid susceptible to lipid peroxidation (Yin, Xu, & Porter, 2011). Network correlation analysis and metabolic pathway enrichment identified 38 pathways and included variations related to inflammation, endothelial function, and mitochondrial bioenergetics. Experimental studies aimed at unraveling the toxicity of urban combustion emissions have also highlighted complex metabolomic alterations (Brower et  al., 2016; Huang et  al., 2015). For example, investigating the temporal (immediately and 18 h post) effects of acute exposure (100 or 300 μg/m3 for 6 h) to mixed (gasoline and diesel) vehicle emissions (MVE) on metabolites in the serum of C57Bl/6 mice highlighted both concentration-dependent and concentration-independent changes in numerous serum biochemicals (Brower et  al., 2016). Among the more profoundly altered metabolites immediately after the MVE exposure were elevations in oxidized glutathione and cysteine-glutathione disulfide. Additional evidence for oxidative stress included an increase in taurine that can effectively scavenge ROS at

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FIG. 3 Schematic overview of the disturbed metabolic pathways in mitochondria of A549 cells on PM2.5 exposure. Molecules marked in red represent the differential metabolites detected by metabolomics. Reproduced from Huang, Q., Zhang, J., Luo, L., Wang, X., Wang, X., Alamdar, A., Peng, S., Liu, L., Tian, M., & Shen, H. (2015). Metabolomics reveals disturbed metabolic pathways in human lung epithelial cells exposed to airborne fine particulate matter. Toxicology Research, 4, 939–947 with permission from The Royal Society of Chemistry.

p­ hysiological concentrations (Oliveira et al., 2010); in cysteine, an intermediate in ­taurine and glutathione synthesis; and in 13-HODE, 9-HODE, and 12,13-DiHOME. By 18 h post exposure, serum metabolite differences between animals exposed to MVE versus those exposed to FA were less pronounced. Another group investigated the pulmonary metabolome responses to PM2.5 in human lung epithelial cells (A549) and again demonstrated that oxidative stress was the most important factor to cause metabolism dysfunction after exposure (Huang et al., 2015). The PM2.5 extract was sampled from a suburban region with rapid urbanization in Xiamen City, China. PM2.5 significantly changed the abundance of 16 metabolites in a dose-dependent manner, and the citrate cycle, amino acid biosynthesis, and glutathione metabolism were the three major metabolic pathways disturbed. In addition, changes in the expression of several key genes involved in these pathways further validated the metabolic alterations. Notably, GPX1 and SOD2 (a specific antioxidant enzyme in mitochondria) were both elevated in cells following PM2.5 exposure (Fig. 3).

­Occupational exposures The increased susceptibility to acute disorders and chronic diseases in several working groups following prolonged exposure to poor air quality has prompted a small number of studies investigating associations between occupational air ­pollutants and

­Ozone

metabolite disturbances (Kuo et al., 2012; Pradhan, Das, Meena, Nanda, & Rajamani, 2016; Wei et al., 2013). The impact of air pollutants from welding processes is a major concern in occupational medicine and public health (Hobson, Seixas, Sterling, & Racette, 2011). Welding fumes contain large amounts of various gases and ultrafine and fine particles containing metals and their oxides (Antonini et  al., 2005; Kim et al., 2003; Tessier & Pascal, 2006). They can be viewed as oxidative pollutants (Liu, Wu, & Chen, 2007), producing adverse health effects on welders through increased oxidative stress (Han et al., 2005). Kuo et al. (2012) characterized a series of urinary metabolomic changes associated with the long-term, complex effects of welding fume exposure in workers at a Taiwanese shipyard (Kuo et al., 2012). Compared with 16 male office workers, the metabolomic patterns of 35 male welders exhibited higher concentrations of glycine, taurine, betaine/trimethylamine N-oxide, serine, S-sulfocysteine, hippurate, gluconate, creatinine, and acetone and lower levels of creatine. Among these are metabolites that play a role in modulating oxidation/reduction pathways. In addition to increases in taurine, in agreement with Brower et al. (2016), glycine is able to inhibit ROS production in human neutrophils (Giambelluca & Gende, 2009), and betaine has been shown to restore glutathione concentrations and quench free radicals to lessen liver injury (Varatharajalu, Garige, Leckey, Gong, & Lakshman, 2010). The increased concentrations of these metabolites may be indicative of a self-protective mechanism against increased oxidative stress in welders. Similarly, the significant decrease in creatine concentrations among the welders may be explained by ROS and free radical induced decreases in creatine kinase activity (Genet, Kale, & Baquer, 2000; Koufen et al., 1999).

­Ozone Exposure to ozone can result in mild pulmonary irritation and infection, exacerbation of lung disease, and increased risk of mortality in people with underlying cardiorespiratory disease, effects that have set the WHO guideline value for ozone at 100 μg/m3 (~0.05 ppm) for an 8-h daily average (WHO, 2005). Controlled human studies have shown that ozone initially reacts with lipid-rich components in the lung lining fluid and the membranes of epithelial cells lining the respiratory airways forming stable lipid peroxides. This results in oxidative stress, depletion of antioxidant reserves, damage to cells, and release of proinflammatory mediators followed by cellular repair (Bromberg, 2016; Devlin, Raub, & Folinsbee, 1997; Leikauf et al., 1995). To gain a greater understanding of this series of mechanistic events in the airways, Cheng et  al. (2018) undertook a targeted metabolomics evaluation of bronchoalveolar lavage fluid (BALF) following controlled human exposure to ozone in two-arm crossover study (Cheng et  al., 2018). Healthy adult volunteers were randomly exposed to filtered air and to 0.3-ppm ozone for 2 h while undergoing intermittent exercise with a minimum of 4 weeks between exposures. Although these concentrations are higher than those found on average in ambient air in the United States (0.059 ppm), the United Kingdom (0.044 ppm), and China (0.068 ppm)

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(HEI, 2019), a large number of Chinese cities experience high-ozone events, with hourly maximum concentrations frequently exceeding 0.15 ppm (Wang, Xue, et al., 2017; Li et  al., 2017). Bronchoscopy was performed and BALF obtained at 1 or 24 h post exposure. At 1-h post exposure, metabolite changes were associated with increased glycolysis and antioxidant responses, suggesting rapid increased energy utilization as part of the cellular response to oxidative stress. Changes in the metabolic signals at 24 h indicated a broader set of responses consistent with tissue repair. Changes associated with increased lipid membrane turnover were also observed. At 1-h but not 24-h post ozone exposure, significant increases in allantoin and threonate were observed. These are metabolites of the antioxidants urate and ascorbate, respectively, and the early-phase increases are consistent ozone-induced oxidative stress. The lack of elevation at 24 h suggests a transition from active oxidative stress to repair of damage. Glutathione was not significantly altered, possibly owing to its relatively high concentrations (1–10 μM) compared with those of urate (200 μM) and ascorbate (50 μM) (van der Vliet et al., 1999). Although sample numbers were small, GSTM1 null but not GSTM1 positive subjects had statistically significant elevations of glucose and glutamine (as well threonate) levels in BALF samples after ozone exposure, which is consistent with increased sensitivity to ozone-induced oxidative stress (Holloway, Savarimuthu Francis, Fong, & Yang, 2012; Peng et al., 2016; Zhang et  al., 2014). The altered energy-related metabolism (increases in α-ketoglutarate, glutamate, and glutamine) led the authors to suggest that mitochondrial function may be impaired during ozone-induced oxidative stress and that elevated TCA cycle replenishment from an alternative source is used to support cellular repair. Global metabolomic profiling of the lungs of female obese db/db and lean wildtype (WT; C57BL/6J) mice 24 h following a 3-h exposure to ozone (2 ppm) was performed to identify metabolites that could contribute to the augmented pulmonary responses to ozone observed in obese mice (Mathews, Kasahara, et al., 2017; Mathews, Krishnamoorthy, et al., 2017; Johnston et al., 2008). Ozone differentially affected the lung metabolome in obese versus lean mice. These included biochemicals related to carbohydrate and lipid metabolism, which were each increased in the lungs of obese versus lean mice. Of relevance to the present review, the osmolyte hypotaurine that acts as an antioxidant in the mammalian reproductive tract (Yancey, 2005) was significantly increased after ozone in db/db but not WT mice. Since reductions in blood hypotaurine in db/db versus WT mice have been reported by others (Yun et al., 2013), the increase in pulmonary hypotaurine in db/db mice exposed to ozone may have occurred to protect the lungs from oxidative damage.

­Discussion Environmental metabolomics has emerged as a means for sensitively quantitating thousands of chemical signals in a biological sample, providing a broad spectrum of measurements of human metabolism that may reveal biological effects and associated toxicological mechanisms associated with an exposure (Ladva et al., 2017).

­Discussion

Within the environmental science arena, this approach is proving to be useful in exploring the association between exposure to poor air quality and global metabolic disruptions and, as such, is beginning to contribute to our understanding of the molecular events in the exposure-disease continuum. Indeed, the studies reviewed in this chapter demonstrate the utility of metabolomics in combination with various statistical models to identify complex changes in several key biological pathways associated with real-world exposures of complicated air pollutants such as those present in occupational exposures, industrial aerosols, and urban environments (Table 1). Moreover, the metabolomic observations sometimes backed up with additional cytotoxicity assays from these studies are providing additional support for a role of oxidative stress in ill health elicited by poor air quality. Notwithstanding the interpretative challenges associated with the complicated web of metabolic outcomes that have been observed in studies utilizing different air pollutants, methods, and models, many findings, feasibly the result of an oxidative imbalance and a subsequent antioxidant response, have been discussed. For example, lipid peroxidation, protein breakdown, mitochondrial dysfunction, antioxidants depletion, purine metabolism interruption, and citric acid cycle perturbation were observed in human populations exposed to ambient PAHs and metals (Wang et al., 2019, 2015). Similarly, mitochondrial disturbance was found in humans after ozone exposure (Cheng et al., 2018) and in in vitro PM2.5 models (Huang et al., 2015; Song et al., 2019). The latter three studies have also all observed reprogramming of energy metabolism, possibly as a possible means to support cellular repair. In addition, rat and mice studies revealed changes in lipid metabolism, particularly in phospholipids and sphingolipids, that occurred after PM2.5 or vehicle emissions exposure, possibly through disturbing pro-oxidant/antioxidant balance (Wang, Jiang, et al., 2017; Zhao et al., 2019; Brower et al., 2016). Studies of metabolomics and human health have mainly considered human blood and urine however noninvasive sampling of exhaled breath condensate (EBC), and saliva may serve as less-intensive alternatives. Indeed, Ladva et  al. (2017) demonstrated largely comparable metabolic profiles in plasma, EBC, and saliva when metabolomics is used to identify features associated with exposure to complex road traffic-related air pollution mixtures. The analysis by Liang et  al. (2018) reviewed in this chapter extracted 20,766 metabolic features from plasma samples and 29,013 from saliva samples. Roughly 45% were detected in both types of samples. Moreover, 12 significant metabolic pathways were shared by both saliva and plasma. The advantages of saliva as a matrix have recently been reviewed by Bessonneau, Pawliszyn, and Rappaport (2017), focusing on not only the simplicity of collection to both improve sample repeatability and participation rates in cohort studies and thus enlarge (repeated) sets of biospecimens but also the rich and dynamic molecular source of this media (Bessonneau et al., 2017). The ultimate goal of such research is to identify molecular events in an exposureeffect continuum to provide valuable information to intervene and change the outcome. Detecting and monitoring markers of adverse outcomes following exposure to different air pollutants in large human cohorts could divulge differential toxicities

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and thereby help to target regulatory efforts to those pollutants that pose the greatest risk to public health. Metabolomic phenotyping of large cohorts in epidemiological studies is limited at present to the work reporting strong associations between shortterm exposure to NO2 and changes in long-chain fatty acid concentration (WardCaviness et al., 2016) and another study where findings in at-risk individuals have potential to uncover and clarify air pollution-metabolomics associations in a population particularly susceptible to the health effects of air pollution (Breitner et al., 2016). Continued development of this field, in combination with complementary “omics” technologies such as genomics and proteomics, and traditional hypothesisled research will be crucial to help strengthen the causal basis for the epidemiological findings that associate air pollution with an ever-growing number of diseases. Achieving this will bring us significantly closer, via evidence-based mitigations strategies, to reduce the public health burden attributable to this complex and insidious environmental pollutant.

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Traffic-related environmental risk factors and their impact on oxidative stress and cardiovascular health

25

Andreas Daibera, Jos Lelieveldb, Sebastian Stevena, Matthias Oelzea, Swenja Kröller-Schöna, Mette Sørensenc,d, Thomas Münzela a

University Medical Center at the Johannes Gutenberg University Mainz, Center for Cardiology, Cardiology I, Mainz, Germany b Atmospheric Chemistry Department, Max Planck Institute for Chemistry, Mainz, Germany c Danish Cancer Society Research Center, Copenhagen, Denmark d Department of Natural Science and Environment, Roskilde University, Roskilde, Denmark

Abstract The adverse effects of the environment on health are increasingly recognized. The WHO estimates that noise accounts for 1 million annually lost healthy life years in Western Europe due to increased incidence of hypertension, heart failure, myocardial infarction, and stroke. An even more severe health impact was reported for air pollution (e.g., PM2.5) accounting for up to 800,000 annual excess deaths in Europe. Adverse effects of air pollution are mechanistically better characterized, but there is still a great need to understand the pathophysiology of air pollution-induced cardiovascular disease, especially the potential synergistic effects together with noise. With the present book chapter, we discuss the most recent data on noise/ air pollution-induced stress responses that increase blood pressure, heart rate, stress hormone levels, and oxidative stress leading to vascular dysfunction and worsening of cardiovascular prognosis. The impact of these environmental risk factors on redox signaling and oxidative stress is discussed in detail. ­Keywords: Environmental risk factors, Traffic noise exposure, Air pollution, Stress hormones, Oxidative stress, eNOS uncoupling, NADPH oxidase (Nox2), Reduced nitric oxide bioavailability

­ lobal burden of pollution, noncommunicable disease, G and role of oxidative stress With the world population estimated to reach 9 billion by 2050, we face a distressing health crisis as a result of striking effects of worsening environmental pollution. The Lancet Commission on pollution and health concluded that “Pollution is the largest Oxidative Stress. https://doi.org/10.1016/B978-0-12-818606-0.00025-0 © 2020 Elsevier Inc. All rights reserved.

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environmental cause of disease and premature death in the world today. Diseases caused by pollution were responsible for an estimated 9 million premature deaths in 2015—16% of all deaths worldwide—three times more deaths than from AIDS, tuberculosis, and malaria combined and 15 times more than from all wars and other forms of violence. In the most severely affected countries, pollution-related disease is responsible for more than one death in four” (Landrigan, Fuller, Acosta, et al., 2018). The WHO underscores this assertion with the estimate that 12.6 million global deaths in 2012 were due to living in unhealthy environments (Online-Link5, 2016; OnlineLink6, 2016). The Global Burden of Disease (GBD) study estimates that in total, all forms of pollution were responsible for 268 million disability-adjusted life years (DALYs). F. Collins crystallized the relationship of environmental risk factors with health with the statement, “Genetics load the gun, but environment pulls the trigger.” The incidence of communicable, maternal, perinatal, and nutritional diseases is reduced as a result of modern medical advances but is being supplanted by noncommunicable diseases like atherosclerosis and metabolic diseases. Though scientific efforts are often focused on traditional cardiovascular risk factors (e.g., diabetes, smoking, and hypertension), the GBD data indicate air pollution (ambient and household) and other environmental risks as detrimental factors facilitating the development of chronic noncommunicable disease and as contributors to global mortality (Lim, Vos, Flaxman, et al., 2012; Murray, Ezzati, Flaxman, et al., 2012). Also, behavioral factors (e.g., smoking, diet, and physical activity) belong to the environmental factors that clearly influence our health. The four leading health risk factors and diseases that contribute to global mortality and impaired life quality (in the form of disability-adjusted life years [DALYs]) are high blood pressure, smoking, ischemic heart disease, and cerebrovascular disease (Fig. 1) (Lim et al., 2012; Murray et al., 2012), all related to cardio−/cerebrovascular complications. The ranking of ambient air pollution by particulate matter (PM2.5) to the fifth place as a global risk factor of chronic noncommunicable diseases and global mortality (Cohen, Brauer, Burnett, et al., 2017) and occupational noise is now mentioned as a significant environmental health risk factor (Collaborators, 2017). Using new hazard ratio functions based on an updated global exposure mortality model, the most recent calculations estimate a dramatically higher contribution of PM2.5 to global deaths (8.79 million per year) and disease burden (Lelieveld, Klingmuller, Pozzer, et al., 2019), which will further increase its significance among the leading health risk factors and diseases. With the present overview, we want to provide evidence that oxidative stress plays a role in pathophysiological processes triggered by these environmental factors. Oxidative stress, for us, represents an imbalance between the formation of reactive oxygen species (ROS, e.g., superoxide and hydrogen peroxide) and their detoxification or efficient repair of the resulting damage (e.g., as envisaged by accumulation of oxidative DNA damage such as 8-oxo-dG or oxidative protein damage such as sulfonic acid and 3-nitrotyrosine residues). However, also adverse redox signaling events may contribute to oxidative stress conditions (e.g., uncontrolled H2O2-driven smooth muscle proliferation or the lack of apoptosis control by oxidative caspase denitrosation) (Egea, Fabregat, Frapart, et al., 2017). Of note, oxidative stress is a

­The pollutome: Risk factors in the physical environment

FIG. 1 Global burden of disease and related deaths. Comparison of the magnitude of the leading diseases, injuries, and risk factors based on the percentage of global deaths and the percentage of global disability-adjusted life years (DALYs). The leading four diseases and risk factors (see red markup) are all associated with increased risk for vascular disease and mortality. LRI, lower respiratory infections; COPD, chronic obstructive pulmonary disease; HAP, household air pollution from solid fuels; BMI, body mass index; FPG, fasting plasma glucose; PM2.5 Amb, ambient particulate matter pollution. Orange color indicates lifestyle or behavioral risk factors, whereas red color indicates environmental risk factors. Redrawn from Murray, C. J., Ezzati, M., Flaxman, A. D., et al. (2012). GBD 2010: Design, definitions, and metrics. Lancet, 380, 2063–2066.

two-edged sword: On one hand, basal formation of ROS is essential for cellular functions such as cell differentiation, proliferation, and migration and for providing basic antioxidant defense to protect the organism from future life-threatening events by a process called (ischemic) preconditioning in which a low and timely limited burst of ROS causes sustained induction of protective, antioxidant enzymes (Egea et al., 2017; Sies, 2015; Sies, Berndt, & Jones, 2017). On the other hand, chronic excess ROS formation leads to the aforementioned described accumulation of oxidative damage that ultimately will lead to development and progression of various diseases and contribute to premature mortality (Daiber, Kroller-Schon, Frenis, et al., 2019; Daiber, Oelze, Steven, et al., 2017).

­The pollutome: Risk factors in the physical environment The pollutome accounts for the sum total of all forms of pollution that could potentially impact human health (Landrigan et al., 2018) and, as such, is a fully nested

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subset of the exposome (Wild, 2005; Wild, 2012). Besides air pollution as leading health risk factor, there are other important risk factors in the physical environment, including water pollution, soil pollution, heavy metal, and chemical occupational exposure as well as other nonchemical environmental health risk factors such as mental stress and noise exposure.

­Air pollution: Number one environmental hazard Air pollution, in the form of solid particles (e.g., ambient particulate matter with a diameter of  4Δp is the requirement for the forward reaction to occur. The red arrow in complex I indicates forward electron transport. SDH, succinate dehydrogenase. (B) Reverse electron transport (RET) by complex I. When the Δp is large and/or the ΔEh across complex I is low such that 4Δp > 2ΔEh, electrons can be driven backward from the CoQ pool onto the flavin mononucleotide (FMN) of complex I, reducing the FMN that can donate a pair of electrons to NAD+ to form NADH or pass one electron to oxygen to generate superoxide. The red arrow in complex I indicates reverse electron transport.

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there is a dramatic and steep increase in O2•– production by RET (Robb et al., 2018). RET requires a sufficient thermodynamic force to drive RET at complex I and further shows the steep dependence of this O2•– production on this driving force. A particularly intriguing aspect of RET is that it does not require damage to or inhibition of the respiratory chain (Murphy, 2009; Pryde & Hirst, 2011). As RET responds sensitively to physiological variables, O2•– production by complex I can be modulated under physiological conditions (Murphy, 2009, Pryde & Hirst, 2011). More generally, RET at complex I occurs as a redox signaling pathway in inflammation (Mills et al., 2016), contributes to life span in flies (Scialo et al., 2016), and is part of the oxygen sensing mechanism of the carotid body (Fernandez-Aguera et al., 2015). The production of ROS by mitochondria acts as a signal from the mitochondrion to the rest of the cell (Collins et al., 2012; Finkel, 2011; Holmstrom & Finkel, 2014; Janssen-Heininger et al., 2008). The appeal of RET as a mitochondrial redox signal is due to the tremendous sensitivity of RET to Δp and the redox status of the CoQ pool. The magnitude of Δp is directly linked to demand on mitochondria to make ATP, while the redox state of the CoQ pool reflects electron supply to the respiratory chain. Hence, there is considerable interest in understanding the mechanisms by which mitochondria regulate O2•– production at complex I by RET as a physiological signaling process. A further point to note is that the term RET is often interpreted as requiring NAD+ reduction at the FMN site of complex I. This is not the case, as O2•– production by RET at complex I occurs when the matrix NAD+/NADH pool is highly reduced and there is no net electron flow from complex I into this pool.

T­ he role of complex I in superoxide production during IR injury The earlier analysis suggests that complex I is a major site of the ROS production by RET upon reperfusion. Consistent with RET at complex I driving IR injury, many inhibitors of complex I are protective against IR injury such as rotenone (Chouchani et al., 2016; Lesnefsky et al., 2004) or with S1QELs (Brand et al., 2016). Furthermore, other factors that decrease the driving force for RET such as mitochondrial uncouplers that will decrease the Δp and thus the driving force for RET are also protective against IR injury (Hoerter et al., 2004; Korde et al., 2005). Furthermore, inhibitors of SDH are also protective against IR injury (Chouchani et al., 2014; Valls-Lacalle et al., 2016; Valls-Lacalle et al., 2018). Two sites have been proposed for O2•– production by complex I during RET, the flavin mononucleotide (FMN) of the complex I NADH binding site (Hirst, King, & Pryde, 2008; Hirst & Roessler, 2016; Kussmaul & Hirst, 2006; Pryde & Hirst, 2011) and the CoQ binding site (Lambert, Buckingham, Boysen, & Brand, 2008; Lambert, Buckingham, & Brand, 2008). The FMN site is a plausible source of O2•– production by complex I during RET. This is because the penetration of O2 to the CoQ site is difficult to envisage from the structure of complex I (Agip et al., 2018; Efremov, Baradaran, & Sazanov, 2010; Fiedorczuk et al., 2016; Zhu, Vinothkumar, & Hirst, 2016). In addition, the generation of the negatively charged O2•– from the CoQ site would require

­The role of complex I in superoxide production during IR injury

its generation within the hydrophobic core of the membrane bilayer that is likely to be thermodynamically unfavorable (Agip et al., 2018; Efremov et al., 2010; Fiedorczuk et al., 2016; Zhu et al., 2016). In contrast, the FMN site is well established as a source of O2•– production by rotenone-inhibited complex I reacting with NADH, which reduces the FMN to FMNH−, which is then readily accessible to O2 to form O2•– (Hirst et al., 2008, Hirst & Roessler, 2016, Kussmaul & Hirst, 2006, Pryde & Hirst, 2011). Furthermore, any O2•– formed at this site is released directly into the mitochondrial matrix (Birrell, Yakovlev, & Hirst, 2009; Kussmaul & Hirst, 2006; Pryde & Hirst, 2011). The relationship between [O2] and RET was linear over the physiological [O2] range (Robb et al., 2018), consistent with O2•– production being driven by the second-order reaction between O2 and FMNH− on complex I when it is free from bound NAD+ or NADH (Birrell et al., 2009, Kussmaul & Hirst, 2006, Pryde & Hirst, 2011). When the CoQ binding site of complex I is inhibited by rotenone, the FMN/FMNH− ratio is set by a rapid pre-equilibration with the matrix NAD+/NADH pool and thereby determines the rate of O2•– production (Kussmaul & Hirst, 2006; Pryde & Hirst, 2011). We favor FMNH− as the donor of an electron to O2 for O2•– production by complex I during RET. However, this is not yet conclusive because if the FMN site is the source of RET O2•–, then the greater O2•– production during RET compared with rotenoneinhibited complex I has to be explained, as under both situations O2•– production is determined by the FMN/FMNH− ratio (Lambert & Brand, 2004a, 2004b; Lambert, Buckingham, Boysen, & Brand, 2008; Pryde & Hirst, 2011). One possible reason why RET-driven O2•– production is greater is that the large thermodynamic driving force for electron movement backward through complex I during RET holds the FMN/FMNH− ratio at a more negative Eh than is possible by equilibrium with the NAD+/NADH pool (Pryde & Hirst, 2011). The more negative midpoint potential at pH 7.5 (Em7.5) of the FMN/FMNH− couple (−380 mV (Sled, Rudnitzky, Hatefi, & Ohnishi, 1994)) compared with that of the NAD+/NADH couple (−335 mV) is consistent with this hypothesis. Other possible factors that could contribute to the elevated O2•– production during RET compared with that after rotenone inhibition include differential access of O2 to the FMNH− due to alterations in the NADH and NAD+ binding and differences in the activity of peroxidases within the mitochondrial matrix that degrade H2O2. One further consideration is that during O2•– production, FMNH− is a one-electron donor to O2 to form a semiquinone radical FMN•, which is then thought to rapidly redistribute its unpaired electron throughout the iron sulfur centers on complex I (Birrell et al., 2009). However, the FMN• radical also reacts very rapidly with O2 to form O2•–, so if this electron redistribution were slowed during RET then the enhanced lifetime of FMN• might also enhance O2•– production (Birrell et al., 2009). As we suggest that superoxide generation by RET at complex I upon reperfusion is the major source of IR injury, it is of particular relevance that complex I RET is dramatically altered by a structural change known as the active/deactive transition (Gavrikova & Vinogradov, 1999a, 1999b). When complex I is not oxidizing NADH and pumping protons across the mitochondrial inner membrane, it gradually converts to a “deactive” state, which is characterized by a conformational transition (Dröse, Stepanova, & Galkin, 2016; Gavrikova & Vinogradov, 1999a, 1999b; Kotlyar & Vinogradov, 1990) (Fig. 4A). The physiological role of the active/deactive transition

525

Active

526

NAD+

Cys39

Deactive

NADH

SH SH Q QH2

Reperfusion

Ischemia

ND3

ND3 Cys39

Slow reactivation of complex I prevents ROS production upon reperfusion

(B) SX

Electrophile SNO

S-X ND3 SH AcylCoA

(A)

Temporarily inhibited complex I

(C)

SAc

H2 O 2 GSSG

H2S

SNO SOH

H2O2

SOn

SSG

SSH

FIG. 4 The complex I active/deactive transition during IR injury. (A) Schematic of complex I in the active and deactive states. During ischemia, the complex adopts the deactive conformation that leads to cysteine 39 (red; bovine nomenclature) on the ND3 subunit (yellow) becoming exposed to the solvent. Upon reperfusion, complex I is rapidly reactivated and then produces a burst of superoxide by RET. When exposed to the solvent, Cys 39 can be reversibly modified (indicated as S-X) and can then be reactivated by mitochondrial matrix reductants such as the glutathione or thioredoxin systems. This slows the reactivation of complex I upon reperfusion; consequently, by the time complex I is fully active, the succinate that drives RET has been oxidized, and there is less superoxide production upon reperfusion. (B) The inset shows the structure of the homologous cysteine residue in the structure of deactive complex I from Yarrowia lipolytica (Zickermann et al., 2015) in the deactive state showing the cysteine equivalent to Cys 39 in mammals. (C) The Cys 39 of the ND3 subunit of complex I that is exposed in the deactive complex I can react with a number of agents that modify the cysteine and thereby hold complex I in the deactive state. These modifications can be reversible (in red) such that complex I can be restored to full activity or irreversible modifications that render complex I permanently inactive (in green).

CHAPTER 26  Mitochondrial ROS production during IR injury

180°

­Induction of the mitochondrial permeability transition pore

is unclear, but in the context of IR injury, the most interesting aspect is that complex I readily undergoes deactivation during ischemia, with a half-life for deactivation of about 10–12 min (Galkin & Moncada, 2007; Gorenkova, Robinson, Grieve, & Galkin, 2013). Upon reperfusion, complex I is rapidly reactivated and can support superoxide production by RET (Dröse et  al., 2016; Gorenkova et  al., 2013). The active/deactive transition of complex I provides an appealing explanation for many therapeutic interventions that decrease IR injury. Deactive complex I exposes a critical cysteine residue (Cys39 in the ND3 subunit in mammals) that is occluded in active complex I (Chouchani et al., 2016; Galkin et al., 2008; Gavrikova & Vinogradov, 1999a, 1999b; Gorenkova et al., 2013). Covalent modification of Cys39 locks complex I in the deactive state (Fig. 4B). In addition, reversible modification of this cysteine residue by thiol reactive agents also temporarily locks complex I in the deactive state in vivo, thereby preventing ROS production by RET (Chouchani et al., 2010; Chouchani et  al., 2013; Chouchani et  al., 2016; Nadtochiy, Burwell, & Brookes, 2007; Prime et al., 2009) (Fig. 4C). The overall effect is that electron flow through complex I is temporarily prevented upon reperfusion, thereby blocking superoxide production by RET during the crucial first few minutes of reperfusion. As the modification is reversible, complex I returns to full activity a few minutes after reperfusion, by which time the succinate accumulated during ischemia will have been oxidized or released from the cell and returned to preischemic levels. This modification was demonstrated using the mitochondrion-targeted S-nitrosothiol compound, MitoSNO, and underlies the protective action of S-nitrosating agents such as MitoSNO against IR injury (Chouchani et al., 2013; Chouchani et al., 2016). It is likely that many other agents that protect against IR injury, such as hydrogen sulfide (Elrod et al., 2007; Karwi et al., 2017), act in a similar way to decrease ROS production upon reperfusion (Chouchani et al., 2016).

­Induction of the mitochondrial permeability transition pore Succinate oxidation upon reperfusion generates ROS at complex I by RET. The initial burst of ROS production upon reperfusion directly causes oxidative damage to mitochondria, and in conjunction with dysregulation of calcium levels, elevated ROS can also lead to induction of the mitochondrial permeability transition pore (MPTP) and on to cell death following reperfusion (Baines et  al., 2005; Chouchani et  al., 2016; Murphy & Steenbergen, 2011; Nakagawa et al., 2005; Schinzel et al., 2005). The cell death and organ dysfunction caused by the induction of the MPTP leads to the release of mitochondrial and cell contents, resulting in the activation of an inflammatory response that can further damage tissue and ultimately give rise to tissue scarring and remodeling (Chouchani et al., 2016; Lutz, Thurmel, & Heemann, 2010). Blocking MPTP induction is the next point to protect mitochondria during IR injury (Baines et al., 2005, Nakagawa et al., 2005, Schinzel et al., 2005). While the nature of the MPTP is still not definitively established, it is clear that the mitochondrial cistrans prolyl isomerase, cyclophilin D (CyD), is required for induction of the MPTP

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under pathological conditions (Baines et  al., 2005; Halestrap & Davidson, 1990; Nakagawa et al., 2005; Schinzel et al., 2005). The MPTP can be blocked by the CyD inhibitor, cyclosporin A (CsA) (Halestrap & Davidson, 1990; Tanveer et al., 1996). Infusing CsA in vivo, at reperfusion, resulted in reduced cardiac infarct size (Argaud et  al., 2005; Skyschally, Schulz, & Heusch, 2010), immediately suggesting a translatable drug treatment for IR injury in patients. When CsA was administered at the same time as PPCI in a Phase II trial of STEMI patients, it showed promising results (Piot et al., 2008). However, when extended to Phase III in the CIRCUS (Cung et al., 2015) and a larger Phase II CYCLE trial (Ottani et al., 2016), it was unsuccessful. The drug TRO40303, which binds to mitochondrial outer membrane translocator protein (TSPO) and is thereby thought to inhibit the MPTP, was also unsuccessful against STEMI in the MITOCARE study (Atar et al., 2015; Schaller et al., 2010). The mitochondrion-targeted peptide Bendavia (SS31) showed promising results against IR injury in animal studies (Cho et al., 2007), although its mechanism of action is unknown, but it too was unsuccessful when administered to STEMI patients during PPCI in the EMBRACE STEMI study (Gibson et al., 2016). Currently, despite the continued success of MPTP inhibition in small and larger animal models, it remains unclear as to why these are yet to succeed in clinical trials.

­Conclusion While it has been generally accepted for a long time that mitochondrial ROS upon reperfusion were the major driver for IR injury, this process had been tacitly assumed to be a random consequence of the reperfusion of ischemic tissue. However, recent work suggests that IR injury occurs as a result of specific processes and is not just a catastrophic breakdown of cell function (Chouchani et al., 2016; Pell, Chouchani, Murphy, Brookes, & Krieg, 2016). During ischemia, the mitochondrial metabolite succinate builds up; then upon reperfusion, the accumulated succinate is rapidly oxidized driving superoxide production at complex I by RET leading to IR injury (Chouchani et al., 2016). The unifying mechanism for mitochondrial ROS production during IR injury that we propose raises intriguing issues. One pertinent question is why mitochondria are set up to produce large amounts of superoxide by RET at complex I. It seems probable that complex I RET has a physiological role, perhaps as a way for mitochondria to rapidly communicate their status by a retrograde redox signal to the rest of the cell. The produced superoxide is dismutated to H2O2, which acts as a redox signal, both within the mitochondria and in the cytosol (Collins et al., 2012; Finkel, 2011; Holmstrom & Finkel, 2014; Janssen-Heininger et  al., 2008). This mode of signal transduction arises via the reversible oxidation of protein thiols that pass on the modification to effector proteins as a redox relay (Collins et al., 2012, Finkel, 2011, Holmstrom & Finkel, 2014, Janssen-Heininger et al., 2008). Most importantly, preventing mitochondrial damage during IR injury remains a promising clinical strategy with the hope that novel treatments focused on mitochondria could

­References

be utilized in a range of pathologies. The common mitochondrial pathway for IR injury suggests that many of the therapies under development can be applied to other clinical situations when IR injury arises, such as elective surgery, organ transplantation, acute trauma, or stroke.

­Funding Work in the author’s laboratory is supported by the UK Medical Research Council MC_U105663142 and by a Wellcome Trust Investigator award (110159/Z/15/Z) to MPM. The author (MPM) holds patents and has financial interests in the development of mitochondrion-targeted antioxidants and therapies.

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Stone, R. L., Zalkin, H., & Dixon, J. E. (1993). Expression, purification, and kinetic characterization of recombinant human adenylosuccinate lyase. The Journal of Biological Chemistry, 268, 19710–19716. St-Pierre, J., Buckingham, J. A., Roebuck, S. J., & Brand, M. D. (2002). Topology of superoxide production from different sites in the mitochondrial electron transport chain. The Journal of Biological Chemistry, 277, 44784–44790. Tannahill, G. M., Curtis, A. M., Adamik, J., Palsson-McDermott, E. M., Mcgettrick, A. F., Goel, G., et al. (2013). Succinate is an inflammatory signal that induces IL-1beta through HIF-1alpha. Nature, 496, 238–242. Tanveer, A., Virji, S., Andreeva, L., Nf, T., Hsuan, J. J., Ward, J. M., et al. (1996). Involvement of cyclophilin D in the activation of a mitochondrial pore by Ca2+ and oxidant stress. European Journal of Biochemistry, 238, 166–172. Valls-Lacalle, L., Barba, I., Miro-Casas, E., Alburquerque-Bejar, J. J., Ruiz-Meana, M., Fuertes-Agudo, M., et al. (2016). Succinate dehydrogenase inhibition with malonate during reperfusion reduces infarct size by preventing mitochondrial permeability transition. Cardiovascular Research, 109, 374–384. Valls-Lacalle, L., Barba, I., Miro-Casas, E., Ruiz-Meana, M., Rodriguez-Sinovas, A., & Garcia-Dorado, D. (2018). Selective inhibition of succinate dehydrogenase in reperfused myocardium with intracoronary malonate reduces infarct size. Scientific Reports, 8, 2442. Venable, P. W., Taylor, T. G., Sciuto, K. J., Zhao, J., Shibayama, J., Warren, M., et al. (2013). Detection of mitochondrial depolarization/recovery during ischemia—reperfusion using spectral properties of confocally recorded TMRM fluorescence. The Journal of Physiology, 591, 2781–2794. Wallace, D. C. (1999). Mitochondrial diseases in man and mouse. Science, 283, 1482–1488. White, H. D., Norris, R. M., Brown, M. A., Brandt, P. W., Whitlock, R. M., & Wild, C. J. (1987). Left ventricular end-systolic volume as the major determinant of survival after recovery from myocardial infarction. Circulation, 76, 44–51. Wilson, R. J., Drake, J. C., Cui, D., Lewellen, B. M., Fisher, C. C., Zhang, M., et al. (2018). Mitochondrial protein S-nitrosation protects against ischemia reperfusion-induced denervation at neuromuscular junction in skeletal muscle. Free Radical Biology & Medicine, 117, 180–190. Wong, H. S., Dighe, P. A., Mezera, V., Monternier, P. A., & Brand, M. D. (2017). Production of superoxide and hydrogen peroxide from specific mitochondrial sites under different bioenergetic conditions. The Journal of Biological Chemistry, 292, 16804–16809. Yellon, D. M., & Hausenloy, D. J. (2007). Myocardial reperfusion injury. The New England Journal of Medicine, 357, 1121–1135. Yousif, L. F., Stewart, K. M., & Kelley, S. O. (2009). Targeting mitochondria with organellespecific compounds: Strategies and applications. Chembiochem, 10, 1939–1950. Zaidat, O. O., Yoo, A. J., Khatri, P., Tomsick, T. A., Von Kummer, R., Saver, J. L., et al. (2013). Recommendations on angiographic revascularization grading standards for acute ischemic stroke: A consensus statement. Stroke, 44, 2650–2663. Zhang, L., Deng, S., Zhao, S., Ai, Y., Zhang, L., Pan, P., et al. (2016). Intra-peritoneal administration of mitochondrial DNA provokes acute lung injury and systemic inflammation via toll-like receptor 9. International Journal of Molecular Sciences, 17(9), 1425. Zhang, J., Wang, Y. T., Miller, J. H., Day, M. M., Munger, J. C., & Brookes, P. S. (2018). Accumulation of succinate in cardiac ischemia primarily occurs via canonical Krebs cycle activity. Cell Reports, 23, 2617–2628.

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Zhu, J., Vinothkumar, K. R., & Hirst, J. (2016). Structure of mammalian respiratory complex I. Nature, 536, 354–358. Zickermann, V., Wirth, C., Nasiri, H., Siegmund, K., Schwalbe, H., Hunte, C., et al. (2015). Mechanistic insight from the crystal structure of mitochondrial complex I. Science, 347, 44–49. Zweier, J. L., Flaherty, J. T., & Weisfeldt, M. L. (1987). Direct measurement of free radical generation following reperfusion of ischemic myocardium. Proceedings of the National Academy of Sciences of the United States of America, 84, 1404–1407.

­Further reading Swain, J. L., Hines, J. J., Sabina, R. L., & Holmes, E. W. (1982). Accelerated repletion of ATP and GTP pools in postischemic canine myocardium uisng a precursor of purine de novo synthesis. Circulation Research, 51, 102–105. Valko, M., Leibfritz, D., Moncol, J., Cronin, M. T. D., Mazur, M., & Telser, J. (2007). Free radicals and antioxidants in normal physiological functions and human disease. The International Journal of Biochemistry & Cell Biology, 39, 44–84.

CHAPTER

Redox signaling in cellular differentiation

27 Katrin Schröder

Institute for Cardiovascular Physiology, Medical Faculty of the Goethe-University, Frankfurt am Main, Germany

Abstract Differentiation is a process of transformation of precursor into a specialized cell. Within this process, the cell changes its gene expression pattern, which results in a switch of its metabolic profile, its shape, and its proliferative capacity. All of those processes are at least in part redox sensitive or redox regulated. Redox signaling itself depends on a controlled activity of different sources of reactive oxygen species (ROS). Such sources include organelles like mitochondria and enzymes. ROS may be produced as a side product, as in the case of NO synthases or xanthine oxidase or by professional ROS generators such as NADPH oxidases. Accordingly, the amount, localization, and kind of ROS that mediates the effects are different, if produced by distinct sources. In this chapter, the effect of distinct kinds of ROS and their appropriate sources on metabolism gene expression are discussed. ­Keywords: Reactive oxygen species, Differentiation, Metabolism, Epigenetic

­Introduction into cellular differentiation Cellular differentiation is the conversion of one cell type into another, most commonly into a more specialized one (Slack, 2007; Slack, 2013). Within the process of differentiation, cells may change their size, shape, membrane potential, metabolic activity, and responsiveness to signals. Those differences are based on specific modifications of gene expression in the course of differentiation. The number of cell types that can be generated from a stem or precursor cell is determined by its potency. A totipotent cell can differentiate into all cell types of the organism. In mammals, those include the zygote and subsequent blastomeric cells. A cell that can differentiate into all cell types of the adult organism is known as pluripotent. Such cells are embryonic stem cells (ESCs) in animals. Three transcription factors, Oct4, Sox2, and Nanog are highly expressed in undifferentiated embryonic stem cells and are necessary for the maintenance of their pluripotency (Blinka & Rao, 2017; Hou, Srivastava, & Jauch, 2017; She, Wei, Kang, & Wang, 2017). Cells Oxidative Stress. https://doi.org/10.1016/B978-0-12-818606-0.00027-4 © 2020 Elsevier Inc. All rights reserved.

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that can differentiate into multiple different cells are called multipotent. Usually, pluripotent stem cells specialize into multipotent progenitor cells, which then may give rise to functional cells. Oligopotent cells can differentiate into few closely related cell types, and eventually, unipotent cells can differentiate only into one cell type but still are capable of self-renewal (Schipanski, 2007). Terminal differentiation is the loss of self-renewal potential: The cells permanently leave the cell cycle. In vertebrates, “terminal differentiation” occurs, for example, in the nervous system, striated muscle, epidermis, and intestine. This terminal differentiation, however, must not be confused with no plasticity. A terminal differentiated cell still adapts, if needed, without changing its basic features. Potency is not restricted to be lost unidirectionally. In fact, potency can be increased in a process called dedifferentiation. In basal life forms such as worms and amphibians, dedifferentiation of partially or terminally differentiated cells into an earlier developmental stage is part of a regenerative process (Casimir, Gates, Patient, & Brockes, 1988; Stocum, 2004). Cells in cell culture can lose properties of differentiation they originally had, such as protein expression or their cell type-specific shape (Schnabel et  al., 2002). It might be worth mentioning that usually in cell culture only a partial dedifferentiation occurs, for example, endothelial cells: After isolation, those cells start to proliferate. Around passage 3–4, the cells lose their angiotensin II (Ang II) receptor. If further cultured, cells eventually become senescent and stop proliferation. In contrast, pluripotent cells (induced pluripotent stem cells [iPSCs]) can be created from adult fibroblasts by virally induced expression of four transcription factors Oct4, Sox2, c-Myc, and Klf4 called Yamanaka factors (Takahashi & Yamanaka, 2006). Those modified cells then exhibit embryonic stem cells like features and can be passaged on and on, as long as they do not differentiate (Lister et al., 2011). The stem cell character of those iPSCs is reflected by similar transcriptional activities of ESCs and iPSCs and by the fact that both are pluripotent and can give raise to whole organisms (Boland et al., 2009). In adult organisms, tasks like tissue repair and cell turnover are maintained by adult stem cells. Examples of adult stem cells are neuronal stem cells (NSCs) in the brain's subventricular zone, satellite cells lying beneath skeletal muscles’ basal lamina, intestinal stem cells in the deep base of gut crypts or hematopoietic (HSCs), and mesenchymal stem cells (MSCs) in the bone marrow or in adipose tissue. The main difference between embryonic and adult stem cells is that adult stem cells are considered multipotent but not pluripotent. They are able to differentiate in a lineagerestricted manner, only. A major determinant of the differentiation status of a cell is the level of intracellular reactive oxygen species (ROS). ROS mentioned in this chapter most often refer to hydrogen peroxide (H2O2) as the most stable ROS with the best potential to modulate target molecules, without destroying them. Besides H2O2, other ROS are superoxide anions or hydroxyl radicals. Depending on the level of ROS produced, especially if too high or too low, ROS may elicit stress reactions in adult cells. While too low ROS as stressors were widely ignored, the term oxidative stress was used for decades mainly to describe detrimental effects of overwhelming ROS formation. A prominent

­Introduction into cellular differentiation

example is the oxidative burst in leukocytes needed for host defense. In this process, ROS unroll a tremendous destructiveness. In contrast, the same ROS mediate eustress. Via reversible redox modification of target molecules, they improve cellular signal transduction and maintain homeostasis (Schröder, 2019a). Accordingly, it is clear that ROS can elicit both stress and eustress. In stem cells, similar effects can be observed. Balanced ROS formation maintains the stem character of the cell. Loss of ROS induces stem cell death, while increased ROS are associated with the loss of quiescence and increased proliferation, eventually leading to stem cell exhaustion (Tan & Suda, 2018). Besides mitochondria, the family of NADPH oxidase is a major source of ROS. Mitochondrial ROS formation is determined by their functionality, their number, the balance of fusion and fission, and general turnover of mitochondria. ROS formation by the NADPH oxidase family is determined by the characteristics of individual homologues of the family. Nox1 to Nox3 require cytosolic subunits for their activation and produce superoxide anions. Those subunits are p47phox and p67phox or NoxO1 and NoxA1, which enable acute transient or constitutive activity of the individual Noxes, respectively. Nox4 is constitutively active and produces H2O2. Eventually, Nox5, Duox1, and Duox2 are activated by increasing intracellular Ca2+ and produce either superoxide anions or H2O2 (Brandes, Weissmann, & Schröder, 2014).

­General targets of ROS in differentiation The molecular targets of ROS promoting differentiation of adult stem cells are not well defined yet. In general, ROS oxidize molecules such as lipids, proteins, or even nucleic acids. In proteins, usually, specific cysteine residues function as redox-dependent switches. Nevertheless, reversible oxidation and reduction of other amino acids can occur as well. Methionine residues, which also contain sulfur, provide an analogous redox-dependent system (Drazic & Winter, 2014). Redox-sensitive cysteines have regulatory functions in phosphatases (Shp2 and PTP1B), kinases (Jnk and p38), transcription factors (AP1 and FoxOs), and others. Most of the redox-sensitive proteins are only transiently affected by oxidation, meaning oxidation can be reversed by reductases, such as thioredoxin and glutaredoxin (Schröder, 2019b). If oxidation is not reversed, further exposure to ROS results in hyperoxidation of Cys−SOH to cysteine sulfinic acid (Cys−SO2H) followed by cysteine sulfonic (Cys−SO3H) acid (Finkel, 2012) and Met−SO to methionine sulfone (Met−SO2) (Drazic & Winter, 2014). High oxidation states are generally irreversible, resulting in permanent damage of the target protein. Often, ligand-stimulated ROS production depends on the activation of NADPH oxidases. In bone marrow mesenchymal stem cells, erythropoietin and hepatocyte growth factor (HGF) induce an acute ROS formation by Nox2 and subsequent transient inhibition of phosphatases, which is required for proper cytokine signaling (Schröder et al., 2011; Schröder, Wandzioch, Helmcke, & Brandes, 2009). The

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dephosphorylating activity of protein tyrosine phosphatases (PTPs) depends on a conserved reactive cysteine residue within the activation domain of the enzyme. Consequently, oxidation of this particular cysteine results in inactivation of the phosphatase (Denu & Tanner, 1998) and prolonged kinase or signal pathway activity. In addition, ROS can also directly affect kinases. The oxidation of a reactive cysteine residue in the cytoplasmic domain of the EGF receptor enhances its tyrosine kinase activity (Paulsen et al., 2012). Besides the PTPs, dual-specificity phosphatases, such as phosphatase and tensin homolog deleted on chromosome 10 (PTEN), are wellestablished redox targets (Kwon et al., 2004). Indeed, the NADPH oxidase Nox2 can promote neuronal stem cell self-renewal and neurogenesis via an increased PI3K/Akt activity due to PTEN oxidation (Le Belle et al., 2011). A direct evidence for ROS modified lipids on proliferation is currently not available. Rather indirect mechanisms have been proposed. Inhibition of the eicosanoid pathway, which oxidizes unsaturated metabolites, can delay differentiation and promote a pluripotent state of embryonic stem cells (Yanes et al., 2010). Oxidized lipids either can form new ROS via an alteration of the intracellular Ca2+ homeostasis or can directly modify signaling proteins by posttranslational modification of thiols. One famous example is the adduct formation with Keap1 (kelch-like erythroid cell-derived protein with CNC homology [ECH]-associated protein-1) with the cyclopentenone 15-deoxy-△12,14-prostaglandin J2. This modification results in the upregulation of cellular antioxidant proteins (Zmijewski et al., 2005). The Keap1/ Nrf2 (nuclear factor erythroid 2-related factor 2) system in fact is a major redox sensor and signal transduction system to keep a cell's redox balance. Nrf2 acts as a transcription factor for antioxidative enzymes such as SOD, catalase, and glutaredoxin peroxidase. Importantly, not Nrf2 itself is the target of oxidants, but instead, Keap1 is. Keap1 binds to both Nrf2 and the E3 ubiquitin ligase cullin 3, resulting in constitutive ubiquitylation and subsequent proteasomal degradation of Nrf2. Increased ROS level trigger the oxidation of several reactive cysteines in Keap1, which then releases Nrf2. Consequently, Nrf2 is not degraded anymore, and its level rises, which leads to its translocation and accumulation in the nucleus. In the nucleus, Nrf2 binds to antioxidant response elements, promoting transcription of the antioxidative enzymes mentioned earlier, as well as haem oxygenase-1, thereby augmenting the overall defense of the cell against ROS (Taguchi, Motohashi, & Yamamoto, 2011). DNA as a large macromolecule is highly susceptible to ROS-mediated damage of the DNA backbone (i.e., 2-deoxyribose) and DNA bases that can cause DNA strand breaks (Halliwell & Gutteridge, 2015). Guanine is most vulnerable to oxidation, and 2-deoxyguanosine in DNA is frequently oxidized to 8-oxo-2′-deoxyguanosine, which mimics a thymine, resulting in G-to-T transversion mutations or even doublestrand breaks, if not corrected before DNA replication (Neeley & Essigmann, 2006). DNA strand breaks and 8-oxo-2′-deoxyguanosine lesions can be rescued by the action of the transcription factor FoxO3 and subsequent expression of antioxidative enzymes as well as base excision and nucleotide excision repair genes in hematopoietic stem cells (Bigarella et al., 2017).

­ROS in epigenetic mechanisms of cellular differentiation

­ER stress in differentiation Overproduction of ROS interferes with protein folding and leads to the accumulation of misfolded proteins within the endoplasmic reticulum (ER). This results in ER stress and ER stress-associated ROS generation. ER stress, via Ca2+ release, stimulates mitochondrial ROS production, which in turn enhances the release of Ca2+, followed by a further increase in ROS formation that ultimately triggers apoptosis (Cao & Kaufman, 2014). The so-called unfolded protein response (UPR) is a collection of pathways to resolve protein misfolding and restore an efficient protein-folding environment. If in response to ER stress the UPR is activated, it induces apoptosis in HSCs, an effect coupled to the activation of protein kinase RNA-like endoplasmic reticulum kinase (PERK) (van Galen et al., 2014). ER stress and UPR are associated with impaired engraftment of human HSCs and long-term reconstitution ability of murine HSCs, which both were rescued by reducing ER stress (Miharada, Sigurdsson, & Karlsson, 2014; van Galen et al., 2014). Protein synthesis in HSCs is lower, when compared with lineage-committed progenitors or differentiated hematopoietic cells. Perturbation of HSC proteostasis by both increasing and decreasing protein synthesis is associated with a decline in long-term multilineage reconstitution ability (Signer, Magee, Salic, & Morrison, 2014).

­ROS in epigenetic mechanisms of cellular differentiation Differentiation most often is the result of a change in the cells vicinity, including cytokine or hormone composition, nutrition, or mechanical needs, all of which need to be transformed into a signal that will lead to the appropriate changes in gene expression. Each of the 3.72×1013 cells in an adult human has its own copy of the genome, except certain cell types, such as red blood cells, that lack nuclei in their fully differentiated state. Differentiated cells within one organism can have very different physical characteristics despite having the same genome. Importantly, cells vary in their ability to respond to external signals, depending on their set of expressed receptors or signal transduction molecules. This signal response equipment will change in the course of differentiation, providing a feed forward mechanism to reinforce the process of differentiation into a certain direction. Determination of cell type is tightly controlled based on processes that can be summarized as epigenetics. A major factor of inheritable control of gene expression is the control of the density of chromosomal DNA, packed as chromatin in a DNA/protein complex. Its fundamental unit, the nucleosome, is composed of two copies of the histone proteins H3, H4, H2A, and H2B creating a bead-like structure, with 146 bp of DNA wrapped around its surface. The linker histone, H1, binds the nucleosome at the entry and exit sites of the DNA wrapped around the nucleosome core particle, thus locking the nucleosomal particle in place (Tollervey & Lunyak, 2012). Nucleosomal packaging is dictated by modifications such as DNA and histone modifications that will be discussed later. Six nucleosomes per 11 nm are present in transcriptional inactive euchromatic chromatin and 12–15 nucleosomes per 11 nm in transcriptional active heterochromatin (Bassett, Cooper, Wu, & Travers, 2009).

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­DNA methylation DNA packaging and thereby regulation of gene expression can be controlled and inherited by the methylation state of DNA (Deaton & Bird, 2011). DNA methylation patterns are directed and preserved by the action of the DNA methyl transferase (DNMT) family, whereas the effects of DNA methylation are mediated by recruitment of methyl-CpG-binding domain (MBD) proteins (Tollervey & Lunyak, 2012). DNA methylation itself impedes the binding of transcription factors and thereby reduces the transcription of the affected genes. Additionally, methylated DNA bounds more efficiently to MBDs, which then recruit histone-modifying proteins to the locus. Such proteins are histone deacetylases or chromatin remodeling proteins that can determine the switch between eu- and heterochromatin (Tollervey & Lunyak, 2012). ROS can function as catalysts of DNA methylation. The DNA oxidation structure, 8-hydroxy-2'-deoxyguanosine, can induce DNA hypomethylation by inhibiting DNA methylation at nearby cytosine bases. 5-Hydroxy-methylcytosine (5hmC) achieves DNA demethylation and subsequently DNA hypomethylation. Moreover, ROS may induce site-specific hypermethylation via either upregulating expression of DNA methyltransferases (DNMTs) or formation of new DNMT-containing complexes (Wu & Ni, 2015). Suppression of Nanog has been identified as a necessary prerequisite for differentiation, regardless of the lineage cells differentiated into. In differentiated cells, the Oct4, Nanog, and Sox2 promoter regions are highly methylated and inactive, whereas in stem cells these promoters are unmethylated and active (Han & Yoon, 2012). Accordingly, their expression drops in the course of differentiation.

­Histone methylation Many studies have implicated a role for nucleosome positioning and histone modifications during differentiation (Zhang et al., 2016). In fact, besides DNA modifications, histone modifications are of major importance to keep a cell’s fate, as they allow an adequate recruitment of transcription factors. The polycomb group (PcG) proteins are a large and diverse family of proteins that epigenetically repress the transcription of key developmental genes by modifying histones. They form three broad groups of polycomb repressive complexes (PRCs) known as PRC1, PRC2, and polycomb repressive deubiquitinase, each of which modifies and/or remodels chromatin by distinct mechanisms. Polycomb repressive complex 1 (PRC1) complexes are E3 ubiquitin ligases that monoubiquitinate lysine 119 of histone H2A (H2AK119ub1). Polycomb repressive deubiquitinase opposes the action of PRC1 by deubiquitinating H2AK119. Polycomb repressive complexes 2 are methyltransferases that target histone H3 lysine 27 for mono-, di-, and trimethylation (H3K27me1, H3K27me2, and H3K27me3) (Chittock, Latwiel, Miller, & Müller, 2017). Both activities of PRC1 and PRC2 result in repression of differentiation and expression of development-promoting genes. Upon receiving differentiation signals, PcG proteins are recruited to promoters of pluripotency transcription factors, which results in suppression of their activity and subsequent reduction of stem gene expression.

­ROS in epigenetic mechanisms of cellular differentiation

Importantly, PcG-deficient embryonic stem cells can begin differentiation but cannot maintain the differentiated phenotype (Christophersen & Helin, 2010). Together with reduction of stem gene expression by the action of PcGs, differentiation and development-promoting genes are activated by trithorax group (TrxG) chromatin regulators (Schuettengruber, Chourrout, Vervoort, Leblanc, & Cavalli, 2007). TrxG protein complexes are recruited to transcriptionally active promoters, most likely by the transcription initiation apparatus, where they catalyze a histone trimethylation modification (H3K4me3) and promote transcription through histone acetylation. At promoters where activating transcription factors continuously recruit the transcription initiation apparatus, demethylation of H3K27me3 can occur, and PcG binding is lost (Guenther & Young, 2010). The PcG and TrxG system represents a three-state system. The silenced state is generated by PcG proteins and contains the PcG proteins themselves in addition to chromatin that is trimethylated on H3K27 and/or ubiquitinated on H2A (Han & Yoon, 2012). The opposing active state is generated by TrxG proteins and contains the TrxG proteins themselves in addition to chromatin that is methylated on histone H3K4 and H3K36, acetylated on H3K27, or both (Tollervey & Lunyak, 2012). In the intermediate neutral state, these residues are unmodified in the nucleosomes. The H3K27 residue represents a pivot point for the system, as it cannot be both methylated and acetylated on the same histone tail at the same time. Noncoding RNAs may be required to destabilize both active and silent states, and the same noncoding RNA can cause either recruitment or eviction of a protein that binds it depending on the rate of release of the RNA from chromatin (Ringrose, 2017).

­ROS in methylation-controlled differentiation Dioxygenases, such as Jumonji C (JmjC) domain-containing histone demethylases and ten-eleven translocation (TET) methylcytosine dioxygenases, remove histone and DNA methylation (e.g., H3K9me3, H3K27me3, H4K20me3, and DNA) and thereby contribute to the expression of genes mediating differentiation. Alternatively, ascorbate (vitamin C) appears to enhance reprogramming of somatic cells into iPSCs through H3K9me3 demethylation in response to BMP treatment (Chen et al., 2013). The epigenetic state of stem cells and the activity of the dioxygenases is critically altered by α-keto-gluterate (αKG) /Fe2+. αKG is an intermediate of the mitochondrial Krebs cycle and represents a precursor of succinate. Vitamin C is another cofactor of αKG/Fe2+-dependent dioxygenases, which cannot be generated by mammalian cells and therefore must be provided from an extrinsic source. In the presents of the antioxidant vitamin C, Tet1 deficiency enhances, while its overexpression impairs reprogramming via promotion of DNA 5-methyl-cytosine formation and thereby the expression of cadherin 1 and Epcam. Importantly, 5hmC levels at loci involved in pluripotency such as Pou5f1 (Oct4) can be increased with either Tet1 or vitamin C but not with both, suggesting that Tet1 and vitamin C do not function in synergy. In the absence of vitamin C, Tet1 promotes somatic cell reprogramming. In contrast, Tet2, independently of vitamin C, promotes dedifferentiation and reprograming (Chen et al., 2013). The mechanism of this difference remains elusive but potentially

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is a result of different features of the members of the Tet family. Unlike other members of the Tet family, Tet2 does not contain a DNA-binding domain. Instead, Tet2 chomatin recruitment is realized by the RNA-binding protein Paraspeckle component 1 (PSPC1) through transcriptionally active loci (Guallar et al., 2018). Inhibition of Tet2 affects hematopoietic differentiation and increases HSC self-renewal, and Tet genes are involved in ESC differentiation [92]. Despite this, pyruvate dehydrogenases and αKG dehydrogenase in the Krebs cycle are key enzymes, which are capable to contribute to ROS formation. Maintaining low levels of ROS corresponded to the quiescent state of stem cells in vivo and is a crucial feature of hematopoietic stem cell precursors (Jang & Sharkis, 2007).

­Metabolism and ROS in differentiation In mouse and human stem cells, metabolism and epigenetics are tightly connected. Essentially all known protein methyltransferases and demethylases utilize cofactors that are intermediate metabolites in core metabolic pathways. This inspired a tantalizing hypothesis, which proposed that changes in metabolism might induce adaptive responses through changes in the methylation of proteins, especially histones (Teperino, Schoonjans, & Auwerx, 2010).

­S-adenosylmethionine (SAM) and homocysteine Threonine and S-adenosylmethionine are required for maintenance of stemness and self-renewal of embryonic stem cells. A prolonged methionine deprivation results in cell apoptosis. One mechanism of how differentiation may be enhanced by depletion of S-adenosylmethionine is the reduction of the number of H3K4me3 marks, which results in a loss of Nanog expression and hinders the maintenance of the stem cell state (Shiraki et al., 2014). Homocysteine (Hcy) formed from methionine is the major pathway for Hcy biosynthesis in humans [13]. Folate intermediates can be used to force the remethylation of homocysteine, which results in the formation of methionine and S-adenosyl-methionine (SAM). SAM is the substrate for almost all protein methylations, including methylation of histones (Shyh-Chang et  al., 2013). The transfer of a methyl group from SAM to a substrate converts SAM into S-adenosyl-homocysteine (SAH), which is hydrolyzed to homocysteine and adenosine. This reaction is reversible, with SAH synthesis being favored over its hydrolysis [12]. Hcy is rapidly removed either by cystathionine β-synthase (CBS) and the transsulfuration pathway or by its remethylation to methionine. Remethylation can be folate-dependent (requiring the enzymatic activities of methionine synthase (MS) and 5,10-methylenetetrahydrofolate reductase [MTHFR]) or folate-independent (requiring the enzymatic activity of betaine-homocysteine methyltransferase [BHMT]) (Esse, Barroso, Tavares de Almeida, & Castro, 2019). Transsulfuration is the main route for methionine disposal, through which the sulfur atom is integrated into the cysteine molecule [14]. Transsulfuration occurs mainly in the liver

­Metabolism and ROS in differentiation

and ­kidney, and it begins with the condensation of Hcy with serine to form cystathionine via cystathionine β-synthase with pyridoxal phosphate (PLP) acting as a cofactor. The balance between transsulfuration and remethylation is regulated by SAM, which acts as an allosteric inhibitor of the methylene-tetrahydrofolate reductase reaction and as an activator of cystathionine β-synthase. Hydrogen peroxide increases the conversion of homocysteine to cystathionine (Mosharov, Cranford, & Banerjee, 2000). In contrast, methionine-adenosyl-transferase activity, and thereby the formation of SAM, is reduced in conditions of oxidative stress (Corrales et  al., 2002). Cystathionine is cleaved to cysteine and α-oxobutyrate by another PLP-requiring enzyme, cystathionine gamma-lyase. In addition to protein synthesis, cysteine is used for the synthesis of glutathione [15]. Glutathione is a major determinant of the intracellular redox balance. It is present in millimolar concentrations, either in reduced form or as oxidized glutathione disulfide (GSH/GSSG 50:1). Depletion of intracellular glutathione by buthionine sulfoximine treatment reduces HSC reconstitution ability in a dose-dependent manner without affecting progenitor cell differentiation (Ito et al., 2006), suggesting that glutathione is essential, selectively for maintaining the stem cell fate. Cysteine represents an extracellular redox buffer, with a ratio of reduced cysteine to oxidized cysteine disulfide in plasma of approximately 1:4 (Joseph & Loscalzo, 2013). Excess cysteine is oxidized to taurine or inorganic sulfates or is excreted in the urine (Selhub, 1999). Hyperhomocysteinemia of the plasma is associated with a higher risk of cardiovascular disease, but no developmental defects were observed in mice or men. It rather appears that hyperhomocysteinemia is associated with inflammation. If homocysteine is not metabolized, the level of glutathione drops and the antioxidative defense is reduced (Upchurch et al., 1997). Hcy can induce activity and expression of NADPH oxidases and reduce the expression of thioredoxin, SOD, and catalase. An intrinsic source of ROS, regulating the redox status of those enzymes is Nox4. Ablation of Nox4 in mice increases the remethylation of homocysteine, catalyzed by betaine-homocysteine-methyltransferase (folate independent) within the liver. As a consequence, Nox4-/- mice display significantly lowered plasma homocysteine, and the flux of homocysteine through the transsulfuration pathway is reduced, resulting in lower hepatic cysteine and glutathione levels (Murray et al., 2015). According to the assumption that ROS inhibit the stem cell status of a cell, a few studies show an inhibitory effect of hyperhomocysteinemia in NSCs and endothelial progenitors (Wang et al., 2016; Zhu et al., 2006).

­Glycolysis in stem cells Within a cell's life or within the course of differentiation, metabolism switches between glycolysis and oxidative phosphorylation. Interestingly, naïve murine PSCs use more oxidative phosphorylation (OxPhos) than murine epiblast stem cells (EpiSCs) and human embryonic stem cells (Davidson, Mason, & Pera, 2015). Mechanistically, this shift may rely on pluripotency factors such as LIF-induced Stat3, which can promote mitochondrial transcription, and Esrrb, which can promote transcription of

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mitochondrial OxPhos genes (Shyh-Chang & Ng, 2017). If a cell further differentiates, the metabolism switches back to oxidative phosphorylation rather than glycolysis, as shown, for example, for human mesenchymal stem cells in osteoblast differentiation. This switch goes along with a decrease of intracellular reactive oxygen species (ROS), most likely as a consequence of upregulation of antioxidant enzymes, such as manganese-dependent superoxide dismutase and catalase. Consequently, exogenous H2O2 and mitochondrial inhibitors prevent the differentiation of mesenchymal stem cells into osteoblasts (Chen, Shih, Kuo, Lee, & Wei, 2008). The high rate of glycolysis reminds on the Warburg effect seen in cancer cells. The Warburg effect is defined as a shift from oxidative phosphorylation to rapid aerobic glycolysis that goes along with a high rate of lactate production. It is likely that both cancer cells and pluripotent stem cells use the high energy yield for rapid proliferation and simultaneously reduce the involvement of mitochondria to prevent oxidative damage by accidentally formed ROS. The unwanted formation of ROS most often is a consequence of enzyme uncoupling (e.g., endothelial NO synthase), overactivation of NADPH oxidases, or mitochondrial dysfunction (Daiber et  al., 2017). Therefore, favoring glycolysis instate of oxidative phosphorylation has two advantages: A better ATP supply goes along with a lower risk of oxidative damage by overwhelming accidental ROS formation. It is worth noting that glycolysis differs between stem cells and cancer cells. The conversion of pyruvate into lactate ensures the rapid recycle of the rate-limiting NAD+ coenzyme in cancer cells. In contrast, in pluripotent stem cells, the cytosolic conversion of pyruvate into acetyl-CoA and not the lactate is important (Fig. 1). This reaction is catalyzed by a cytosolic ATP-citrate lyse and relies on the export of citrate out of the mitochondria. Loss of glycolysis in early differentiation downregulates acetyl-CoA and acetate production, causing loss of histone acetylation and associated loss of pluripotency (Moussaieff et al., 2015). Glycolysis is not the only way to elevate acetyl-CoA level in a cell. In fact, threonine catabolism provides a substantial fraction of both the cellular glycine and the acetyl-CoA needed for SAM synthesis. Accordingly, depletion of threonine from the culture medium or its catabolizing enzyme threonine dehydrogenase decreased accumulation of SAM and decreased H3K4me3, leading to slowed growth, and increased differentiation of embryonic stem cells (Shyh-Chang et al., 2013). Nicotinamide adenine dinucleotide (NAD) is a key electron carrier in the oxidation of hydrocarbon fuels. The normal intracellular ratio of NAD+/NADH is roughly 100:1, and high NAD+ levels indicate energetic stress (Kaelin & McKnight, 2013). One major sensor of NAD+ is the sirtuin family of histone deacetylases. Acetylation is an important posttranslational modification of proteins. It is catalyzed by acetyltransferases and deacetylases that add and remove acetyl groups to and from lysine residues. Sirtuins deacetylate several proteins, including histones, thereby regulating gene expression activity. In differentiation, sirtuins mediate their effects particularly via Notch signaling. Sirt1 promotes differentiation of neuronal precursor cells by repressing the Notch target Hes1 (Hisahara et al., 2008). Sirt1 also acts as an intrinsic negative modulator of Notch signaling in endothelial cells. Acetylation of the Notch1 intracellular domain (NICD) on conserved lysines controls the amplitude and

­Metabolism and ROS in differentiation

FIG. 1 The metabolic programming of stem cells. Mitochondrial reactions that are more enriched in naïve stem cells and cytosolic reactions that are more enriched in differentiating stem cells.

d­ uration of Notch responses. Suppression of Notch signaling impairs growth, sprouts elongation, and enhances Notch target gene expression in response to delta-like-4 (DLL4) stimulation, thereby promoting a nonsprouting, stalk cell-like phenotype. Consequently, inactivation of Sirt1 in zebrafish and mice causes reduced vascular branching and vessel density (Guarani et al., 2011). In response to oxidative stress, Sirt1 suppresses proliferation of murine neural progenitor cells and directs their differentiation toward the astroglial lineage at the expense of the neuronal lineage by blocking expression of the proneuronal transcription factor Mash1 (Prozorovski et al., 2008). In contrast, endogenous ROS are essential for the proliferation of embryonic neuronal stem/progenitor cells, and ROS scavenging can repress neurosphere formation (Yoneyama, Kawada, Gotoh, Shiba, & Ogita, 2010). It has been proposed that the high rate of glycolysis in pluripotent stem cells results from an immature mitochondrial network. This is characterized by low numbers of mitochondria and a reduced oxidative capacity (Bukowiecki, Adjaye, & Prigione, 2014). Accordingly, during cell reprogramming, mitochondria fission is an essential step. This means that mtDNA replication is decreased and existing mitochondria become fragmented and rejuvenate. Subsequently, their functionality as

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e­ nergy-producing organelles is reduced. In contrast, in the course of organ specific differentiation, mitochondria form a complex network by a process called fusion. Together with the tightening of the mitochondrial permeability transition pore and the subsequent increase in mitochondrial membrane potential, this results in a reduction of ROS formation (Prieto & Torres, 2017). Oxidative phosphorylation in mitochondria itself represents a differentiating force. At least establishment of mitochondrial oxidative metabolism over glycolysis is a prerequisite for the differentiation of embryonic stem cells into cardiomyocytes (Chung et al., 2007). Reversely, reprogramming fibroblasts into induced pluripotent stem cells is accompanied by an upregulated glycolysis (Panopoulos et al., 2012). Amino acid metabolism plays a major role for maintenance of pluripotency and for differentiation. As mentioned earlier, especially threonine appears to be important for murine pluripotent stem cells to maintain their stemness. Threonine is catabolized into glycine, which then serves as a source for the generation of folate, a reaction catabolized by glycine decarboxylase, which is highly expressed in pluripotent stem cells. Inhibition of this enzyme reduces the stem cell character of the cells, an effect linked to alterations of the metabolome and accumulation of advanced glycation end products (AGEs). In contrast, during reprogramming of somatic cells, accumulation of AGEs decreases gradually, and eventually, they disappear in iPSCs. In addition, ectopic expression of glycine decarboxylase or treatment with the AGE inhibitor LR90 promoted reprogramming (Kang et al., 2019).

­ROS in differentiation of embryonic stem cells A major and evolutionary highly conserved component of differentiation, especially in embryonal development, is the group of Wnt signaling pathways (Nusse, 2005). Next to embryonal development, Wnt is also an essential mediator and inducer of differentiation of mesenchymal stem cells. Wnts play a role in cardiac development and differentiation, angiogenesis, hepatogenesis, adipogenesis, bone turnover, neurogenesis, and aging (Visweswaran et al., 2015). Within Wnt signaling pathways, both are possible, either nearby cell-cell communication (paracrine) or same-cell communication (autocrine). In humans, 19 members of the Wnt family and 10 members of their receptors, Frizzled, are known (Rao & Kühl, 2010). The redox-sensitive canonical Wnt pathway leads to regulation of gene transcription, the noncanonical planar cell polarity pathway (also called the Wnt/jun N-terminal kinase [Jnk]) regulates the cytoskeleton, and the noncanonical Wnt/calcium pathway regulates intracellular calcium distribution. The canonical Wnt pathway involves the downstream target protein β-catenin, while the noncanonical pathways do not. Interestingly, nuclear translocation of β-catenin upregulates the expression of c-Myc and pluripotency related genes, such as Oct4, Sox2, and Nanog (He et al., 1998; Zhang et al., 2012). By that mechanism, the Wnt/β-catenin pathway contributes to stemness of cells or even dedifferentiation of somatic cells, which bears the danger of cancer formation. High levels of H2O2 inhibit Wnt/β-catenin signaling and favor, for e­ xample, ­osteogenesis,

­ROS in differentiation of embryonic stem cells

while low levels of H2O2 activate Wnt/β-catenin signaling to promote adipogenesis out of mesenchymal stem cells (Visweswaran et al., 2015). The canonical Wnt/ Jnk pathway involves activation of small GTPases of the Rho family, including rac, cdc42, and Rho as well as Jnk or Rho-kinase. Within the Wnt/calcium pathway, the Wnt-induced, heterotrimeric G-protein-dependent Ca2+ release (Ma & Wang, 2006) activates calcium-dependent enzymes like calcium/calmodulin-dependent kinase (CaMK)II, protein kinase C, or calcineurin. Subsequently, the Wnt/calcium pathway, via activation of CaMKII and calcineurin, activates histone deacetylases (HDACs) that regulate DNA packaging and the transcription factor nuclear factor of activated T cells (NFAT) (Backs, Song, Bezprozvannaya, Chang, & Olson, 2006; Saneyoshi, Kume, Amasaki, & Mikoshiba, 2002). Both Wnt signal pathways may interact via Ca2+, which accumulates in mitochondria and triggers ROS production. Low ROS delay the onset of the Wnt/β-catenin pathway activation (Rharass et al., 2014). Signaling by FoxO transcription factors is also mediated by ROS, with high ROS levels inducing phosphorylation and translocation into the nucleus where it is activated. In osteoblast differentiation, increased levels of H2O2 promote FoxOs transcriptional activity at the expense of Wnt signaling. H2O2 promotes the association of FoxOs with β-catenin, a step required for the stimulation of FoxO target genes expression that simultaneously inhibits Wnt signaling (Almeida, Han, MartinMillan, O'Brien, & Manolagas, 2007). Eventually, deletion of the redox-sensitive deacetylase Sirt1 in osteoprogenitor cells results in low cortical bone mass in mice, due to decreased bone formation resulting from increased β-catenin sequestration by FoxOs (Iyer et al., 2014). Growth factors, such as transforming growth factors (TGFs) and fibroblast growth factors (FGFs), represent another set of molecules crucial for differentiation. TGFβ family signals play critical roles in both the maintenance of the pluripotency of embryonic stem cells and iPSCs by inducing the expression of Nanog, Oct4, and Sox2 and in stem cell differentiation into various cell types (Itoh, Watabe, & Miyazono, 2014). Interestingly, although TGF-mediated signaling is important for maintenance of self-­renewal and pluripotency of stem cells across species, the mechanisms of action is divers. The undifferentiated state in cell culture is maintained, when cells are cultured on a layer of feeder cells such as mouse embryonic fibroblasts, which produce leukemia inhibitory factor (LIF). These feeders can be omitted by direct addition of LIF into the cell's culture medium (Williams et al., 1988). LIF transduces its signaling through dimerization of the cytokine receptors LIF-R and gp130 and activates the transcription factor STAT3 (Ying, Nichols, Chambers, & Smith, 2003). Other signaling factors that play a role in maintenance of stemness and/or differentiation are retinoic acid that can induce differentiation of human and mouse ESCs; Notch signaling, which is involved in proliferation and self-renewal of stem cells; and eventually Sonic hedgehog (Shh), which acts as a morphogen, that promotes embryonic stem cell differentiation and the self-renewal of somatic stem cells. Shh upregulates the production of BMI1, a component of the PcG complex that recognizes H3K27me3. Accordingly, as pointed out earlier, expression of differentiating genes is repressed, and the cells remain in the stem cell state (Lee, Sun, & Veltmaat, 2013).

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­ROS in differentiation of adult stem cells Adult stem cells reside in well-defined niches outside or beneath organs. Upon activation, they contribute to the long-term self-renewal capacity for tissue maintenance and repair. Relative to committed progenitors, ROS are maintained at low levels in HSCs and increase with differentiation (Holmström & Finkel, 2014). The low basal ROS levels preserve stem cell potential and an appropriate balance between stem cell quiescence, differentiation, and self-renewal. In contrast, reduction of ROS significantly reduces the regenerative potential of NSCs and HSCs (Le Belle et al., 2011).

­The niche of adult stem cells The vascular architecture of the bone marrow niche has an impact on ROS formation in HSCs. Less permeable arterial blood vessels maintain hematopoietic stem cells in a low ROS state, whereas the more permeable sinusoids promote activation of hematopoietic stem cells (Itkin et al., 2016). Besides the anatomical circumstances, the cellular neighborhood influences the function of hematopoietic stem cells as well. Bone marrow monocytes and macrophages with high expression of α-smooth muscle actin and the cyclooxygenase-2 reside adjacent to the stem cells and release prostaglandin E2. Via an Akt- and CXCL12-dependent mechanism, those cells prevent ROS formation and hematopoietic stem and progenitor cell exhaustion. Another interesting mechanism to keep ROS low in HSCs is the transfer of ROS to bone marrow stromal cells via connexin-43 (Taniguchi Ishikawa et al., 2012). Most stem cell niches have a strongly hypoxic environment in common, and 2%–5% O2 enhances the survival and proliferation of stem cells in vitro compared with atmospheric 21% O2 (Kwon et al., 2017). This implies a relatively high activity of hypoxia-induced factor (Hif). Next to hypoxia itself, ROS generated by mitochondrial complex III ensure full activation of Hif (Klimova & Chandel, 2008). Pyruvate dehydrogenase kinases PDK2 and PDK4, both of which are Hif1α targets, are required for hematopoietic stem cell self-renewal. Elevated PDK expression suppresses the influx of glycolytic metabolites into mitochondria, thereby promoting glycolysis, cell cycle quiescence, and stem cell capacity of murine hematopoietic stem cells (Takubo et al., 2013). In embryonic and neuronal stem cells, Hif1α activates Wnt/β-catenin signaling and thereby promotes proliferation and neurogenesis in the dentate gyrus of the hippocampus (Mazumdar et al., 2010). Nevertheless, the theory that hypoxia in a niche in general preserves the stem cell character of cells is questionable. Many hematopoietic stem cells closely interact with bone marrow microvessels and still exhibit a hypoxic profile, defined by strong retention of pimonidazole and expression of Hif1α (Nombela-Arrieta et al., 2013). In conclusion, the hypoxic state of at least hematopoietic stem cells is not solely the result of a hypoxic niche but may represent a cell-specific feature.

­ROS in differentiation of adult stem cells

­Differentiation of adult stem cells Murine hematopoietic stem cells and human mesenchymal stem cells during osteogenic differentiation utilize glycolysis instead of mitochondrial oxidative phosphorylation to meet their energy demands (Chen et al., 2008; Simsek et al., 2010). In contrast, mitochondrial oxidative phosphorylation appears to play a minor role for maintaining the stem cell character: mitochondrial fragmentation and presumably compromised oxidative phosphorylation had no effect on primary adult hematopoietic stem cell survival and self-renewal (Luchsinger, MJD, Corrigan, Mumau, & Snoeck, 2016). The relative independence of adult stem cells on oxidative phosphorylation has several advantages: In a hypoxic environment, mitochondrial function can be easily damaged and using anaerobic glycolysis minimizes the formation of ROS. Most adult stem cells are highly sensitive ROS. Elevated ROS level tier stem cells out of their quiescent status. In mesenchymal stem cells, ROS levels increase before the cells enter the S phase of the cell cycle, and antioxidants block the G1-S transition (Lyublinskaya et al., 2015). Activation of proliferation is followed by differentiation or apoptosis, a process that involves the transcription factor FoxO3 (Renault et al., 2009; Tothova et al., 2007). Aside from mitochondria, other sources of ROS exert specific signals that regulate differentiation and proliferation of adult stem cells. Such sources are the members of the NADPH oxidase family. Colon intestinal stem cells highly express Nox1 and its subunits NoxA1 and NoxO1 and accordingly produce ROS in a constitutive manner. In NoxO1 knock out mice, the radical formation of colon intestine stem cells not only is vanished but also results in a diminished differentiation, more proliferation, and less apoptosis in epithelial cells of colon crypts (Moll et  al., 2018). Accordingly, colon intestinal stem cells require a controlled formation of ROS for controlling their quiescent status. If ROS formation is reduced, the cells start to proliferate. As a result, they are more prone for exhaustion, DNA damage, and cancer formation. This in fact appears to be a general concept of how NADPH oxidase-derived ROS act in cells, if constitutively produced. The NADPH oxidase Nox4 is the prototype of a constitutive active source of H2O2 and is a major driving force for a cell to differentiate and stay differentiated. This is especially important for mesenchymal stem cells. The differentiation of osteoclasts (bone-degrading cells) out of bone marrow-derived murine and peripheral blood-­derived human mesenchymal stem cells strongly depends on the presents of Nox4 and Nox4-derived H2O2 formation. In the course of differentiation, not only expression of Nox4 is induced, but also its expression pushes the differentiation itself (Goettsch et  al., 2013). Accordingly, differentiation of human mesenchymal stem cells to adipocytes requires increased oxygen consumption and intracellular ROS generation from mitochondrial complex III and constitutive H2O2 formation by Nox4 (Schröder et al., 2009; Tormos et al., 2011). Nox4 therefore is a major control instance of differentiation. Its absence will activate quiescent cells to become fidget and restless, and deletion of Nox4 results at least in mice in a systemic hyperinflammation (Schröder et al., 2012; Schürmann et al., 2015), potentially based on reduced DNA damage repair capacity (Helfinger

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et al., 2017). This eventually is a bias toward more cancer development in situations of reduced ROS formation as in proinflammatory cancer models in mice (Helfinger et  al., 2017). Besides the constitutive alteration of ROS level, NADPH oxidases such as Nox2 contribute to acute transient ROS formation and signal transduction. Deletion of Nox2 causes reduced stem cell mobilization from the bone marrow in hypoxia and in response to erythropoietin. As a consequence, hypoxia-induced vascular repair is delayed in Nox2-deficient mice (Schröder, Wandzioch, et al., 2009). Together, ROS are essentially involved in maintain stem cells in a quiescent status, and simultaneously, they are required for an optimal activation and differentiation of adult stem cells.

­Interplay of ROS, Akt, and p38 in adult stem cells With age, viability and renewal and repair capacity of adult stem cells decline. This is due to accumulating DNA damage, chromatin perturbation, and reduction in telomere length. One possible reason for adult stem cells to become senescent is external stress, including ROS (Campisi & Di d'Adda Fagagna, 2007). ROS accumulation severely impairs long-term reconstitution potential and exhausts the stem cell pool. An effect attributed to p38 mitogen-activated protein kinase (MAPK)-mediated loss of quiescence and senescence induction (Ito et al., 2006). The receptor tyrosine kinase c-Kit (CD117) is a reliable marker of hematopoietic stem cells and is critical for their function (Thorén et al., 2008). c-Kit signaling determines HSC reconstitution potential by activating multiple downstream signaling pathways, including Akt and MAP-kinases. ROS modulate the c-Kit signaling cascade via oxidative modification of protein tyrosine phosphatases (PTPs) in hematopoietic stem cells and dual-specific phosphatases (DUSPs) in satellite cells (Chan et al., 2011; Yue et al., 2017). Both hematopoietic stem cell and satellite cell exhaustion, loss of quiescence, and hyperproliferation can result from inactivation of PTEN, a redox-responsive negative regulator of Akt (Yue et  al., 2017; Zhang et al., 2006). Akt itself is susceptible to H2O2-mediated oxidation at two conserved Cys residues (Cys-297 and Cys-311). Exposure of Akt to H2O2 results in disulfide bond formation between those two cysteines, recruitment of protein phosphatase 2A (PP2A), Akt dephosphorylation, and subsequently suppression of Akt activity (Murata et al., 2003). Inhibition of Akt activity appears to be crucial for stem cell fate of hematopoietic stem cells, as ectopic expression of constitutively active Akt induces hyperproliferation, apoptosis, and eventual exhaustion of the stem cell compartment (Kharas et al., 2010). One possible explanation for this phenomenon is the ability of Akt to inhibit FoxO3 (Brunet et al., 1999). The transcription factor FoxO3 forces the expression of antioxidant genes (Kops et al., 2002). Accordingly, overexpression of Akt will increase cellular ROS level, which eventually results in loss of stem cell quiescence (Juntilla et al., 2010). Likewise, activation of p38 results in loss of quiescence and eventual exhaustion of the HSC pool as consequence of elevated intracellular ROS levels (Jung et al., 2016). In human satellite cells, p38 activation is associated with differentiation, while p38 inhibition forces

­Concluding remarks

satellite cell expansion (Charville et al., 2015). Interestingly, ­asymmetric division of activated murine satellite cells involves asymmetric inheritance of activated p38α/β in daughter cells. Daughter cells containing activated p38α/β differentiate, whereas those without activated p38α/β stay quiescent and retain their self-renewal capability (Troy et  al., 2012). Consequently, ROS via controlling the activity of Akt and p38 control the stem cell status of HSCs.

­Transdifferentiation: Epigenetic reprogramming? Transdifferentiation is defined as the process of conversion a somatic cell of one type into another without full dedifferentiation. Especially for the transdifferentiation of fibroblasts into endothelial cells, innate immunity plays a pivotal role. Additionally, transdifferentiation strongly depends on metabolic switches as seen in stem cells: The glycolytic switch begins immediately after activation of innate immunity in fibroblasts. Inhibiting glycolysis then impairs, whereas facilitating glycolysis enhances the transdifferentiation of fibroblasts into endothelial cells. Importantly, the glycolytic switch seen here is similar to that observed in pluripotency (see earlier). Activation of innate immunity with the toll-like receptor 3 agonist poly I:C increases the expression of the mitochondrial citrate transporter Slc25A1 and the nuclear ATPcitrate lyase. This is associated with intracellular accumulation of citrate, which represents the precursor for acetyl-CoA. Eventually, these metabolic changes result in an increased histone acetylation necessary for transdifferentiation (Lai, Reineke, Hamilton, & Cooke, 2019). Importantly, long time before the discovery of the stem cell-specific acetyl-CoA producing glycolysis, the export of citrate from mitochondria into the cytosol was discovered to be essential for the differentiation of 3T3 fibroblasts into adipocytes (Kajimoto, Terada, Baba, & Shinohara, 2005).

­Concluding remarks ROS play divers roles in stem cells (Fig. 2). Oxidative eustress is a major component for cellular signaling, differentiation, and homeostasis. Low ROS is needed to maintain stemness, while increased levels of ROS promote differentiation. Mechanisms that maintain low ROS formation in stem cells include metabolic favor of glycolysis. In the course of differentiation, mitochondrial oxidative phosphorylation becomes more prominent. Together with an increased expression of ROS sources such as NADPH oxidases and a decrease in expression of antioxidative enzymes, the resulting increase in ROS is a necessary precaution for asymmetric cell division and proper differentiation. Oxidative stress, meaning too high or too low formation of ROS, is detrimental for cells in general and stem cells as well. Accordingly, ROS formation is no unavoidable side effect of metabolic processes, but rather represents a component of a sensible and extremely fine-tuned network of signaling cascades.

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FIG. 2 Relationship between ROS and stem cell potential. Low basal levels of ROS preserve stem cell quiescence. A lower ROS level is associated with impaired stem cell function, exit from quiescence, and regenerative potential. Intermediate ROS levels lead to loss of quiescence and induction of senescence, resulting in stem cell exhaustion and impaired regenerative potential. Further accumulation of ROS to high levels ultimately results in cell death.

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Redox-regulated brain development

28

Carsten Berndta, Christina Wilmsa, Marion Thauvinb, Sophie Vrizb a

Department of Neurology, Medical Faculty, Heinrich-Heine Universität, Düsseldorf, Germany b Center for Interdisciplinary Research in Biology (CIRB) Collège de France, Paris, France

Abstract The brain is one of the best examples to describe oxidative eu- and distress. Whereas the vast majority of neurological deficits are linked to oxidative distress, oxidative thiol modification of specific target proteins is a prerequisite for proper brain development. In this book chapter, we summarize the recent knowledge regarding the impact of the regulation of the thiol redox state of several proteins via H2O2 and oxidoreductases of the thioredoxin family toward the establishment of a functioning neuronal network. A variety of different steps during brain development are redox regulated such as proliferation and differentiation of stem cells, neuronal pruning, and axonal guidance. ­Keywords: Hydrogen peroxide, Glutaredoxin, Neurons, Central nervous system, NADPH oxidase, Histone deacetylase, Promyelocytic leukemia nuclear bodies

In the research community, especially in medical research, the term oxidative stress is still linked to oxidative damage and diseases. Recently, oxidative stress was divided into oxidative distress (pathological situations) and oxidative eustress (physiological conditions) (Sies, Berndt, & Jones, 2017). Whereas many—if not all—diseases of the central nervous system are linked to oxidative distress, oxidative eustress plays a central role during brain development and regeneration.

­Oxidative eustress and distress in the brain The brain is highly dependent on oxygen. It contributes with approximately 2% to the human body weight but consumes 20% of the oxygen taken up (Clarke & Sokoloff, 1999). Ischemic stroke, the lack of oxygen, for 30 min leads to the loss of 1.9 million neurons—every minute (Saver, 2006). The brain is quite susceptible to oxidative distress. However, this susceptibility is not explained by the simple assumption that Oxidative Stress. https://doi.org/10.1016/B978-0-12-818606-0.00028-6 © 2020 Elsevier Inc. All rights reserved.

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the high consumption of oxygen leads to high amounts of reactive oxygen species (ROS). ROS are also formed during specific processes, for example, neurotransmitter metabolism. Deamination of amine-based neurotransmitters (dopamine, tyramine, tryptamine, and noradrenaline) via monoamine oxidases produces H2O2 during the catalytic cycle (Edmondson, 2014). Tyramine deamination, for example, leads to the formation of 1.6-nmol H2O2/min/mg protein in neuronal mitochondria (Hauptmann, Grimsby, Shih, et al., 1996). Some neurotransmitters (dopamine, serotonin, and adrenaline) are also able to autoxidize. 6-Hydroxydopamine-dependent reactions lead to increased amounts of H2O2, O2−•, and OH• (Cobley, Fiorello, & Bailey, 2018). Two other molecules important for signal transduction can disturb redox homeostasis, calcium (Görlach, Bertram, Hudecova, et al., 2015), and glutamate (Cobley et al., 2018). Furthermore, neurons provide only a minor endogenous antioxidative capacity. They contain just half of the glutathione (GSH) concentration than other cell types that might affect activity of GSH-dependent enzymes, such as glutathione peroxidase 4 (GPx4). Together with higher amounts of iron (Halliwell, 1992) lowered GPx4 activity could explain the susceptibility of neurons against ferroptosis (Gascón, Murenu, Masserdotti, et al., 2016). All these reasons synergistically contribute to the high vulnerability of the brain to oxidative distress. Therefore, many neurological deficits are linked to oxidative damage: neurodegeneration, neuroinflammation, and psychiatric disorders. Oxidative distress is a common feature in all neurodegenerative diseases. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting motor neurons. In 20% of the patients, ALS is induced by mutations of superoxide dismutase 1 (SOD1). Several mutations in the respective gene have been described, all leading to a further diminished enzymatic antioxidant capacity within neurons. Another neurodegenerative disease partly depending on mutations is Parkinson’s disease. Here, several different mutated proteins, for example, α-synuclein, DJ-1, or PINK, lead to oxidative distress and to the loss of the dopaminergic neurons. Mitochondrial dysfunction is the main underlying mechanism for epileptic seizures. Recently, loss of parvalbumin positive neurons upon ferroptosis has been described to induce epilepsy (Ingold, Berndt, Schmitt, et al., 2018). Neuroinflammatory diseases like multiple sclerosis are induced by oxidative/nitrosative damage of the oligodendrocytes, the myelinating cells of the CNS, by nitric oxide released by activated microglia, the immune cells of the CNS (Trapp, 2004). Oligodendrocytes are especially vulnerable to peroxynitrite formed by the reaction between nitric oxide and superoxide (Jack, Antel, Brück, et al., 2007) and are protected against inflammatory damage when the formation of peroxynitrite is inhibited (Lepka, Volbracht, Bill, et al., 2017). Up to 50% of multiple sclerosis patients display depressive symptoms. Of note, major depressive disorder and multiple sclerosis display many overlapping features, including chronic oxidative and nitrosative stress (Morris, Puri, Walder, et al., 2018). In line, schizophrenia is also connected to redox imbalance (Maas, Vallès, & Martens, 2017). Next to damages occurring in adult and aging brains, developmental failures are connected to oxidative damage as well. Complications such as retinopathy of prematurity, punctate white matter lesions, necrotizing enterocolitis, ­intraventricular

­H2O2 signaling during development of the nervous system

h­emorrhage, or periventricular leukomalacia are related to oxidative distress (Ozsurekci & Aykac, 2016). Nevertheless, as described later, oxidative eustress is an essential trigger of processes regulating proper brain development.

­Brain development The fundamental process of brain development appears similar in all vertebrates. During early embryonic development, the neural plate gives rise to a neural tube, which bulges into the three primary brain vesicles along the anterior–posterior axes: the forebrain, the midbrain, and the hindbrain vesicle. These three vesicles develop into five secondary vesicles that eventually give rise to all brain derivatives in the adult (Fitzgerald, Gruener, & Mtui, 2012). The wall of the neural tube consists of rapidly dividing neuronal stem cells, which differentiate into neurons, oligodendrocytes, and astrocytes. These cells migrate to different parts of the developing brain and form local circuits in an activity-independent, genetically encoded process, while some of them are eliminated by tightly regulated cell death (Bear, Connors, & Paradiso, 2015; Gilbert, 2014). Once these local circuits have formed, sensory experience will constantly refine nascent neuronal circuits via neuronal activity and synapse formation/degradation, these processes being influenced by the environment at various levels (intracellular metabolism, interior milieu, or whole organism surroundings). All processes contributing to brain development, from neural stem cell proliferation, survival, and differentiation; to neuronal polarization, axonal outgrowth, synapse formation, and stabilization; up to myelin production and glia–neuron interactions were shown to be redox regulated. Here, we focus on the role of H2O2 and oxidoreductases of the thioredoxin family and its impact on different aspects of brain development.

­H2O2 signaling during development of the nervous system Development of any multicellular organism depends on complex interactions of fundamental cellular processes like proliferation, differentiation, migration, and apoptosis. Cellular redox status plays an essential role in all these processes and thereby in embryonic development (Covarrubias, Hernández-García, Schnabel, et al., 2008; Dennery, 2010). Indeed, the redox balance regulates differentiation of neural progenitors, death of postmitotic neurons during development, and neuronal function. It is thus important to better understand how redox signals are produced and interpreted in the developing nervous system, which should give clues to better means for preserving healthy state or shielding from dysfunction.

­H2O2 production in physiological situations H2O2 is believed to be the major second messenger acting in redox signaling pathways (see also Toledano’s chapter in this book). The main sources of H2O2 are the mitochondrial respiratory chain and NADPH oxidases (NOXs) (Holmström & Finkel, 2014),

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and NOX complexes are the main producers of O2−• and subsequently H2O2 in the central nervous system (Rastogi, Geng, Li, et al., 2016). NOXs are transmembrane proteins that use cytosolic NADPH as an electron donor and belong to multicomponent complexes that generate extracellularly either O2−• (NOX 1, 2, 3, and 5) or H2O2 (NOX 4, DUOX 1, and DUOX 2) upon appropriate stimulation, for example, by growth factors and cytokines (Bedard & Krause, 2007; Brandes, Weissmann, & Schröder, 2014). Even when the primary product of NOX activity is O2−•, it is largely and immediately transformed into H2O2 by a SOD physically associated with NOX (see Petersen’s chapter in this book), or it dismutates spontaneously at low pH levels. H2O2 has access to the cytosol by crossing the plasma membrane via aquaporin channels (Bertolotti, Farinelli, Galli, et al., 2016; Bienert & Chaumont, 2014; Miller, Dickinson, & Chang, 2010). Some NOXs (e.g., NOX2) have a very broad distribution, whereas others (e.g., NOX3, DUOX 1, and DUOX 2) have a more limited distribution, but members of the NOX family are expressed in most parts (cerebellum, hippocampus, and cortex) and cell types (neurons, astrocytes, and microglia) of the central nervous system (Bedard & Krause, 2007; Katsuyama, 2010; Weaver, Leung, & Suter, 2016; Wilson, Muñoz-Palma, & González-Billault, 2018). Although it was described that NOX2 participates in neurogenesis in the subventricular zone (Le Belle, Orozco, Paucar, et al., 2011), we still lack a detailed picture of the respective spatiotemporal patterns of different NOX activities during development in vivo. However, in different cell models of neuronal differentiation, NOX activity is associated with induction of differentiation: PC12 cells (Suzukawa, Miura, Mitsushita, et al., 2000), SH-SY5Y cells (Nitti, Furfaro, Cevasco, et al., 2010), P19 cells (Kennedy, Ostrakhovitch, Sandiford, et al., 2010), and primary cultures of cerebellar granule neurons (Olguín-Albuerne & Morán, 2015). Moreover, NOX activity is controlled by several regulators of brain development, for example, cytokines, neurotrophins, fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF) (Rhee, 2006; Suzukawa et al., 2000). NOX2 activity, the main NOX expressed in neurons, depends also on membrane recruitment of cytoplasmic subunits, including Rac (Rastogi et al., 2016; Wilson, Terman, González-Billault, et al., 2016b). It is worth mentioning that, in the context of regeneration at least, NOX2 complexes may even be transferred between cells via exosomes (Hervera, De Virgiliis, Palmisano, et al., 2018). It is now clear that physiological H2O2 production is abundant, very dynamic, and of utmost importance in the development of the nervous system (Gauron, Meda, Dupont, et  al., 2016; Meda, Rampon, Dupont, et  al., 2018; Rampon, Volovitch, Joliot, et al., 2018; Weaver, Terzi, Roeder, et al., 2018; Wilson et al., 2018; Wilson & González-Billault, 2015).

­Redox regulation of neurogenesis It is now well established that stem cells and progenitor cells show higher levels of H2O2 than differentiated cells (Bigarella, Liang, & Ghaffari, 2014; HernándezGarcía, Wood, Castro-Obregón, et al., 2010; Rampon et al., 2018; Timme-Laragy,

­H2O2 signaling during development of the nervous system

Hahn, Hansen, et al., 2018; Yeo, Lyssiotis, Zhang, et al., 2013) and during embryonic development cell commitment goes along with a decrease in H2O2 levels (Fig. 1A) (Gauron et al., 2016). On the contrary, cell dedifferentiation during adult regeneration is possible thanks to a transient H2O2 level enhancement (Rampon et al., 2018; Zhou, Meng, Li, et al., 2016). These processes are all dependent on chromatin function. Redox-sensitive transcription factors were first identified in the context of oxidative distress (linked to inflammation, cancer, or hypoxia), the best examples being NRF2, NFkB, AP1, and HIF1, and they were subsequently shown to also play a role in physiological situations under the control of redox signaling (Marinho, Real, Cyrne, et al., 2014; Prozorovski, Schneider, Berndt, et al., 2015; Wilson et al., 2018; Wilson & González-Billault, 2015). Transcription factors dedicated to stemness have also been shown to be redox regulated as Oct proteins and more precisely Oct 4 (Covarrubias et al., 2008). In addition, oxidative eustress can impact gene expression by modulating various aspects of the epigenetic machinery and nuclear architecture (Fig. 1B).

FIG. 1 H2O2 levels during retinal development and neurogenesis. (A) H2O2 levels change in relation to retinal development revealed by the reporter line Tg(ubi:HyPer). The H2O2 levels are inferred from the YFP500/YFP420 excitation ratio of HyPer. HyPer imaging in the brain at 24 hpf shows homogeneous high levels of H2O2 throughout the proliferative epithelium. At 32 hpf, optical section of a Tg(ubi:HyPer) retina shows a lowering in H2O2 content in the central part of the tissue and remains high in the ciliary marginal zone (CMZ). (B) H2O2 level modifications during neurogenesis lead to different nuclear responses according to the range of modification. Neural cell differentiation is accompanied by several features developed in the text. (A) Adapted from Albadri et al. (2019).

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Three main protein groups control major epigenetic mechanisms targeting DNA, histones, and nucleosome positioning: DNA methyl transferases (DNMT) and demethylases, histone modifiers (histone acetyl transferases [HAT], histone methyl transferases [HMT], histone deacetylases [HDAC], histone demethylases [HDM]), and chromatin remodelers (SWI/SFN, ISWI, CHRD, INO80, etc.), often influenced by noncoding RNA (Goldberg, Allis, & Bernstein, 2007; Han & Chang, 2015; Saxena & Carninci, 2011; Tessarz & Kouzarides, 2014). The first link between ROS and epigenetic regulation came from the cancer research field. It was demonstrated that an enhancement of ROS levels modified the DNA methylation marks with an hypermethylation of tumor suppressor gene promoters and a global DNA hypomethylation (Chen, Wang, & Shen, 2012) as well as histone modification via HDAC regulation (Calonghi, Cappadone, Pagnotta, et al., 2005). In mammals, 5-hydroxymethylcytosine can be obtained by 5-methylcytosine oxidation and is often an intermediate form of cytosine demethylation (Lamadema, Burr, & Brewer, 2019). Global 5hmC levels decrease during embryonic stem cells (ESCs) differentiation toward neuroectoderm fate, while enrichment of 5hmC at the gene body of transcriptionally active genes is identified in neural progenitor cells (NPCs) (Kim, Park, et al., 2014). The reaction is catalyzed by methylcytosine oxidase teneleven translocation (TET) proteins, which are important for neuroectoderm specification (Wu, Li, & Xie, 2018). Overall, the transient oxidation of 5-­methylcytosine opens the door for local and dynamic chromatin opening involved in both cellular reprogramming and homeostatic modulation of cell function. Histone acetylation, which is catalyzed by HATs, increases accessibility to transcription factor binding, while histone deacetylation catalyzed by HDACs generally represses gene transcription by promoting DNA winding and thereby limiting access to transcription factors. Both enzyme types are subject to redox regulation during neuronal cell differentiation (Jänsch, Meyners, Muth, et al., 2019; Parolin, Calonghi, Presta, et al., 2012). HDAC1 is directly inhibited by 9-hydroxystearic acid (9-HSA), which relies on lipid peroxidation and is dependent on the redox state of the cell (Parolin et al., 2012). It has been demonstrated that lowering of H2O2 observed during retinal progenitor cell (RGC) differentiation is responsible for HDAC1 regulation via 9-HSA and allows expression of neuronal differentiation genes in vivo (Albadri et al., 2019). Both lowered H2O2 levels by catalase overexpression and modulation of 9-HSA concentration by direct injection in the retina impaired HDAC1 activity and the process of RGC differentiation in zebrafish (Albadri et al., 2019). In addition to the direct regulation of genes involved in differentiation, remodeling of the chromatin is involved in retrotransposon activation. Among transposons, focus has been put on long interspersed element 1 (LINE-1, L1). L1 transposition is a molecular copy-and-paste process during which an mRNA template is reverse transcribed and integrated into the host genome, hence duplicating the donor DNA sequence from which the RNA was transcribed (Boeke, Garfinkel, Styles, et  al., 1985). This phenomenon depends on the regulation of the L1 promoter, which is inhibited by methylation of its CpG islands (Bourc’his & Bestor, 2004; Coufal, Garcia-Perez, Peng, et al., 2009; Thayer, Singer, & Fanning, 1993). New insertion

­H2O2 signaling during development of the nervous system

by ­retrotransposition can impact the expression of nearby genes by the insertion of transcription factors binding sites or by generating new splice sites, cryptic promoters, or adenylation signals, all of which being susceptible to modify gene expression. The high H2O2 levels found in neural progenitors activate L1 element, which spread into the genome (Blaudin de Thé, Rekaik, Peze-Heidsieck, et al., 2018). One of the most exciting results in neurobiology in the last decade is the demonstration that L1 retrotransposition in the embryo and during adult neurogenesis leads to somatic genome mosaicism in neurons (Faulkner & Garcia-Perez, 2017). The significance of this mosaicism is not yet fully understood. Many L1 insertions are likely to be deleterious for the cell, but L1 transposition, coupled with Darwinian somatic selection, may generate a selective advantage for the surviving neurons (Bodea, McKelvey, & Faulkner, 2018; Singer, McConnell, Marchetto, et  al., 2010), further enhance the diversity among neurons, and create new properties in line with environmental changes. This has been recently illustrated by L1 transposition in selective environmental changes as in the adaptation of fish to cold (Chen, Yu, Chu, et al., 2017), the response to maternal care in mouse pups (Bedrosian, Quayle, Novaresi, et al., 2018), or the response to opioid receptor stimulation in neural human neuroblasts (Trivedi, Shah, Hodgson, et al., 2014). Promyelocytic leukemia nuclear bodies (PML NBs) correspond to spherical membraneless domains that assemble in the nucleus upon H2O2 increase (LallemandBreitenbach & de Thé, 2018). First identified through their disorganization in acute promyelocytic leukemia (APL), they are mostly composed of PML proteins and involved in posttranslational modifications after oxidative stress and during embryonic development, especially during neurogenesis. Indeed, PML NBs are abundant in stem cells during embryonic development, and their number decreases upon differentiation (Niwa-Kawakita, Wu, de Thé, et  al., 2018). The role of PML NBs in p53 and PRB activation is well documented (Ahmed, Wan, Mitxitorena, et al., 2017; Salomoni, Ferguson, Wyllie, et al., 2008), and emphasis is given to the growing list of transcription factors or chromatin modifiers that can be modified in PML NBs (Lallemand-Breitenbach & de Thé, 2018). These nuclear bodies appear to be factories for protein modification upon environmental changes or through cell lineage commitment (Korb & Finkbeiner, 2013). PML contributes to ESCs self-renewal and maintenance by controlling cell-cycle progression and sustaining the expression of crucial pluripotency factors (Korb & Finkbeiner, 2013). Moreover, NPCs failed to differentiate in PML knockout mice ending to a small brain size phenotype (Regad, Bellodi, Nicotera, et al., 2009).

­Redox regulation of postmitotic neuronal development Physiological levels of H2O2 have been shown to be involved in all steps of postmitotic development of neurons, from polarization to maturation (Wilson et  al., 2018; Wilson & González-Billault, 2015). Polarization is a stereotyped multistep process leading to the establishment of somatodendritic and axonal compartments. Maturation comes with axon growth cone navigation to reach their targets, followed

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by the establishment and refinement of connections. Pharmacological treatments, genetic ablation or genetic overexpression of NOX subunits, have shown that a correct balance of H2O2 levels is necessary for the establishment of neuronal polarity, neurite growth, or axon specification, in rodent hippocampal neurons, cerebellar granule cells, and aplysia bag neurons (Munnamalai, Weaver, Weisheit, et al., 2014; OlguínAlbuerne & Morán, 2015; Wilson & González-Billault, 2015; Wilson, Muñoz-Palma, Henríquez, et al., 2016a). Function of neurons depends on their connectivity via synapses. This connectivity is established during development by a process called neuronal pruning, the elimination of unwanted and unused connections. A very recent hypothesis links formation of mitochondrial O2−• and H2O2 to neuronal pruning. This hypothesis is based on the observation that O2−• and H2O2 block synaptic activity and that these inactive synapses are eliminated (Sidlauskaite, Gibson, Megson, et al., 2018). Within the large number of potential targets for H2O2 signaling during postmitotic development of neurons, the most obvious and best studied examples are part of the cytoskeleton. It is well established that microfilament and microtubule dynamics is regulated by oxidative species and redox enzymes based on oxidation at specific actin and tubulin residues highly susceptible to oxidation leading to modified properties (Gellert, Hanschmann, Lepka, et al., 2015; Wilson & González-Billault, 2015; Wilson, Terman, et al., 2016b). Moreover, a large number of regulatory partners of actin and tubulin are themselves subject to redox regulation. Direct oxidation of actin can be achieved in neuronal cells by flavin-containing monooxygenases of the molecule interacting with CasL (MICAL) family (first identified as binding partners of the transmembrane guidance receptor plexin) leading to actin filament severing and growth cone collapse. The regulatory significance of these findings is highlighted by the reversibility of actin oxidation by MICAL (via the activity of deoxidizing enzymes of the SelR type). But MICAL proteins can also modify actin filament dynamics indirectly by promoting cross talk between them and intermediate filaments via the interaction with proteins of the Cas family. MICAL1 also interferes with the addressing of the Par3/Par6/aPKC complex in the establishment of neuronal polarity by inhibiting kinases of the NDR family. Indirect modification of microtubule is also achieved by redox targeting of microtubule-associated proteins, notably MAP1B, MAP2, Tau, and collapsin response mediator protein 2 (CRMP2) (see the following paragraph 4 for detailed analysis of CRMP2 regulation).

­Enzymatic regulation of oxidative eustress in the brain Specific redox signaling events are mediated by reversible oxidative modifications of protein thiol groups (Jones, 2006). Key player in the control of the redox state of these thiol groups are the oxidoreductases thioredoxin (Trx, Fig. 2A) (Arnér & Holmgren, 2000) and glutaredoxin (Grx, Fig. 2B) (Lillig & Berndt, 2013). Mammals encode two Trxs, one in the cytosol (Trx1) and one in the mitochondria (Trx2), and two glutaredoxins functioning as oxidoreductases, again located in both cytosol

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Activities of oxidoreductases of the thioredoxin family affect axonal outgrowth. (A) Thioredoxins (Trx) reduce persulfidated (1) and nitrosylated (2) cysteine residues, as well as disulfides (3). Oxidized Trx is reduced by thioredoxin reductase (TrxR). (B) Glutaredoxins (Grx) reduce disulfides (1) and glutathionylated cysteine residues (2). Oxidized Grx is reduced by glutathione (GSH). (C) 2-Cysperoxiredoxins (Prx) reduce peroxides (here hydrogen peroxide, H2O2). Formation of a sulfenic acid at the peroxidatic cysteine (Sp) can lead to the formation of an inter- or intramolecular disulfide with the resolving cysteine (Sr), which is reduced by Trx (1). The sulfenic acid at Sp in 1-Cys-Prxs is reduced by GSH. Sulfenic acids can be further oxidized to sulfinic (2) and sulfonic acid (3). Sulfinic acid is recovered by sulfiredoxin (Srx). (D) Collapsin response mediator protein 2 (CRMP2) is oxidized via active semaphorin 3a signaling leading to growth cone collapse. Both Trx1 and Grx2 are able to reduce the disulfide connecting cysteines 504. This induces conformational changes allowing phosphorylation of CRMP2 and axonal outgrowth.

­Enzymatic regulation of oxidative eustress in the brain

FIG. 2

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(Grx1 and Grx2) and mitochondria (Grx2) (Hanschmann, Godoy, Berndt, et  al., 2013). Moreover, several peroxiredoxins (Prxs) are present in mammalian cells (Fig. 2C) (Hanschmann et al., 2013). Transcription of Grx1 and mitochondrial and cytosolic Grx2 has been demonstrated in early embryogenesis in mice (Jurado, Prieto-Alamo, Madrid-Rísquez, et  al., 2003), and Grx2 is already expressed ubiquitously throughout the first 24 h of zebrafish development, when all major organs, including the brain, are formed (Kimmel, Ballard, Kimmel, et  al., 1995). Bräutigam et  al. demonstrated that embryonic brain development depends on the enzymatic activity of Grx2 (Bräutigam, Schütte, Godoy, et al., 2011). Zebrafish with silenced expression of Grx2 lost virtually all types of neurons by apoptotic cell death and the ability to develop an axonal scaffold, which severely impaired embryo’s movements. Axon growth and guidance depend strongly on dynamic modifications of actin and tubulin, the essential components of the cytoskeleton, which are regulated by the GSH/Grx system (Gellert et al., 2015). For the establishment of an axonal scaffold, one of the most important signaling pathways is the semaphorin pathway (Fiore & Püschel, 2003). When axon steering is required, semaphorin 3A (Sema3A) binds to the receptor pair ­neuropilin-1/ plexin3A. Subsequently, plexin3A conveys the signal via MICAL to CRMP2, which induces growth cone collapse (Zhou, Gunput, & Pasterkamp, 2008). CRMP2 is essential during brain development (Charrier, Reibel, Rogemond, et al., 2003), highly conserved across vertebrate species (Schweitzer, Becker, Schachner, et  al., 2005), and controls axonal branching, guidance, and number of neuritis (Ip, Fu, & Ip, 2014). In the developing zebrafish embryo, CRMP2 is expressed in the major neural clusters, especially when the formation of the neuronal network takes place (Christie, Starovic-Subota, & Childs, 2006). Grx2 controls axonal outgrowth via thiol redox regulation of CRMP2 as demonstrated in zebrafish and in a human cellular model for neuronal differentiation (Fig. 2D) (Bräutigam et al., 2011). In mammals, Grx2 reduces a disulfide between cysteines 504, which stabilizes a CRMP2 tetramer (Möller, Gellert, Langel, et  al., 2017). Reduction of this disulfide leads to structural changes allowing phosphorylation and subsequently axonal outgrowth (Gellert, Venz, Mitlöhner, et al., 2013; Möller et al., 2017; Schmidt & Strittmatter, 2007). Another redox system in cells involved in the control of cellular redox balance is the Trx system, comprising of cytosolic (Trx1) and mitochondrial (Trx2) thioredoxins and the selenoenzymes thioredoxin reductases (TrxR) 1 and 2 (Arnér & Holmgren, 2000). Histopathological analysis of TrxR1 null brain showed striking cerebellar hypoplasia of lobules I–VI (Soerensen, Jakupoglu, Beck, et  al., 2008). Proper foliation requires a complex series of developmental steps, which are well coordinated and intricately connected (Sotelo, 2004), including proliferation or death of granule cells (Wahlsten & Andison, 1991), migration along Bergmann glial fibers (Ackerman, Kozak, Przyborski, et  al., 1997), and outgrowth of PC dendrites (Soerensen et al., 2008). Since numbers of mitotic granule cell precursors are reduced but increased cell death is not observed, the major defect underlying cerebellar hypoplasia induced by TrxR1 knockout might result from reduced proliferation of granule cells, the driving force of cerebellar growth and foliation (Soerensen et al., 2008).

­Enzymatic regulation of oxidative eustress in the brain

In the rat brain, regions with high energy demands and high activity that involves ­redox-reactive metabolites including the substantia nigra and the subthalamic nucleus showed high levels of Trx1 mRNA (Silva-Adaya, Gonsebatt, & Guevara, 2014). While the C1 area of the hippocampal formation shows very low expression, the CA2/CA3 area and the dental gyrus of the hippocampus showed stronger expression (Lippoldt, Padilla, Gerst, et al., 1995). Godoy et al. reported immunoreactivity to Trx1 in the Purkinje cell layer of the rat, the motor neurons of the spinal cord, the ependymal cell layer, and the cells of the choroid plexus. TrxR1 was abundantly expressed in the glial cells of the cerebellar white matter, in contrast to Trx1. While TrxR2 expression was detected in the cell bodies of neurons in the Purkinje and molecular cell layers in the cerebellum, Trx2 was present in the axonal fibers of the cerebral cortex, striatum, cerebellar white matter, and spinal cord (Godoy, Funke, Ackermann, et  al., 2011). Due to posttranscriptional regulations, localization of mRNAs and proteins of Trxs might differ in some regions such as the hippocampus (Silva-Adaya et al., 2014). Furthermore, it was shown that in the rat Trx1 and Trx2 expression occurs predominantly in brain neurons (Lippoldt et al., 1995; Rybnikova, Damdimopoulos, Gustafsson, et al., 2000), whereas TrxRs protein levels are higher in glial cells than in neurons (Rozell, Hansson, Luthman, et  al., 1985; Rubartelli, Bajetto, Allavena, et al., 1992). Knockout of either Trx1 or Trx2 in mice leads to early embryonic lethality (Silva-Adaya et al., 2014). Moreover, deficiency in Trx2 showed in in vivo and in vitro studies increased cellular ROS, apoptosis, exencephaly, and early embryonic lethality (Nonn, Williams, Erickson, et  al., 2003; Tanaka, Hosoi, Yamaguchi-Iwai, et al., 2002), demonstrating that both Trx isoforms have essential roles in neuronal differentiation, proliferation, and survival. Trx1 promotes the action of the nerve growth factor (NGF), a neurotrophic factor essential for the development and promotion of survival and function of the CNS (Huang & Reichardt, 2001). On the other hand, NGF induces transcription of Trx1 via cyclic AMP-responsive element (CREB). Another important signaling pathway regulated by Trx1 is the PTEN/ P13K/AKT pathway. Via phosphatase and tensin homolog (PTEN) inactivation, Trx1 activates protein kinase B AKT (Meuillet, Mahadevan, Berggren, et al., 2004), a mediator of proliferation and survival of NPCs (Groszer, Erickson, Scripture-Adams, et al., 2001). Finally, Trx1 is also able to reduce oxidized CRMP2 to promote axonal outgrowth (Fig. 1D) (Morinaka, Yamada, Itofusa, et al., 2011). In terms of regeneration, Trx1 administration to mice promotes neurogenesis after stroke (Tian, Nie, Zhang, et al., 2014; Zhou, Liu, Ying, et al., 2013). The third redox system playing a role in the cellular redox balance during brain development is the Prx system. Mammals express six isoforms of Prxs (Prxs 1–6), which reduce different types of peroxides (Trujillo, Ferrer-Sueta, Thomson, et al., 2007). As ubiquitous enzymes, Prxs have a particularly wide distribution in subcellular compartments. All isoforms are present in the cytosol and have been observed in the nucleus in certain conditions, except for Prx3 and Prx4 (Oberley, Verwiebe, Zhong, et al., 2001). Prx3 is addressed to mitochondria, while Prx5 is targeted to peroxisomes and mitochondria. Prx4 is the only isoform to be present in the endoplasmic reticulum and to be secreted (Rhee, Chae, & Kim, 2005). In contrast to their similar

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distribution, each Prx has a unique pattern of developmental expression. While Prx1 expression increases during late gestation with highest expression at postnatal day 1, Prx2 expression remains largely unchanged. In contrast, Prx6 expression continually increases during development (Shim, Kim, Kim, et al., 2012). Goemaere et al. used immunohistochemistry to map basal expression of Prxs throughout C57BL/6 mouse brain. They showed the neuronal localization of Prxs 2–5 and the glial expression of Prx1, Prx4, and Prx6. Prxs 2–5 are widely detected in the different neuronal populations and especially well expressed in the olfactory bulb, in the cerebral cortex, in pons nuclei, in the red nucleus, in all cranial nerve nuclei, in the cerebellum, and in motor neurons of the spinal cord (Goemaere & Knoops, 2012). Interestingly, Prx expression is abundant in regions that require high energy demands. A similar expression pattern was also observed previously in the rat brain for Trx1, a physiological reductant for Prxs 1–5 (Lippoldt et al., 1995). Prx1-deficient mice display a lack of postmitotic motor neurons, most likely via regulation of the thiol redox state of the proneurogenic factor glycerophosphodiester phosphodiesterase 2 (GDE2) (Yan, Sabharwal, Rao, et al., 2009). Not surprisingly, all three mentioned enzymatic systems regulating redox signaling are involved in a variety of neurological diseases (summarized, for example, in (Hanschmann et  al., 2013), including developmental failures such as perinatal asphyxia (Romero, Hanschmann, Gellert, et al., 2015).

­Conclusion Brain development is a highly complex process regulated by the interaction of many different pathways and signals. One of these signals is oxidative thiol modifications. Here, we summarized the recent knowledge regarding the impact of the regulation of the thiol redox state of several proteins via H2O2 and oxidoreductases of the thioredoxin family toward the establishment of a functioning neuronal network. A variety of different steps during brain development are redox regulated such as proliferation and differentiation of stem cells, neuronal pruning, and axonal guidance.

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CHAPTER

Eustress, distress, and oxidative stress: Promising pathways for mind-body medicine

29

Kirstin Aschbachera,b, Ashley E. Masonb,c a

Department of Medicine, Division of Cardiology, UCSF, San Francisco, CA, United States b Center for Health and Community, Department of Psychiatry, UCSF, San Francisco, CA, United States cOsher Center for Integrative Medicine, University of California San Francisco (UCSF), San Francisco, CA, United States

Abstract When is psychological stress “good” or “bad” for our health? There is no straightforward answer. In this chapter, we first address this question with a review of the best principles for the measurement of psychological distress and stressor exposures in human psychobiological studies. Next, we review the associations between psychological distress and stress-arousal biomarkers with markers of oxidative stress and antioxidant response. Finally, we review promising future directions for translational discovery along three interdisciplinary areas with the potential to transform clinical care: (1) cellular aging, (2) treatment of depression and anxiety, and (3) obesity and cardiometabolic disorders. ­Keywords: Oxidative stress, Eustress, Stress measurement, Psychoneuroimmunology, Psychoneuroendocrinology, Psychological stress, Depression, Treatment response, Obesity, Cellular aging

­Introduction “Stress is the salt of life; few people would like to live an existence of no runs, no hits, no errors” Selye (1976a).

There is intense interest in understanding exactly when psychological stress is “good” or “bad” for our health, yet there is no straightforward answer. The experience of psychological stress initiates a wide-ranging cascade of physiological responses affecting nearly every system in the body, and these responses can be adaptive, maladaptive, or both. However, the term “stress” is ambiguous and lacks ­scientific rigor. Oxidative Stress. https://doi.org/10.1016/B978-0-12-818606-0.00029-8 © 2020 Elsevier Inc. All rights reserved.

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Herein, we describe a range of established measurement tools and models, which focus on the rigorous measurement of psychological distress, as distinct from positive psychological states, stress exposures, and clinical diagnoses. To understand when psychological distress may be associated with poor health, we focus on the concept of “oxidative stress,” now understood to play a role in myriad chronic disease states. Oxidative stress, introduced by Sies (1985), denotes a state of physiological imbalance involving high levels of oxidants and comparatively low antioxidants. He defined “eustress” as low-level physiological oxidative stress, which is essential for healthy redox signaling (or signal transduction by electron transfer). In contrast, Sies defined “distress” as supraphysiological oxidative stress, which causes damage to biomolecules and thereby promotes disease (Sies, 2019). With these definitions clarified, we propose a framework to understand the mindbody mechanisms and pathways that underlie associations between psychological distress and oxidative stress.

­General theories of psychological stress and health ­The origin of stress: A brief history Hans Selye is credited with proposing the first general theory of stress (Selye, 1936), called the “general adaptation syndrome.” This theory described stress as a nonspecific response to acute demands, meaning that all stressors, both psychological and physical, would provoke a similar neuroendocrine response. Fundamental to Selye’s theory was the notion that the stress response evolves over time, from acute alarm, to resistance, to exhaustion (Selye, 1976b). “Chronic stress” is defined as exposure to prolonged periods of intense psychological distress or frequent acute demands over months to years, leading to vital “exhaustion” or breakdown of physiological stress response systems. Selye described the physiological outcomes of chronic stress exposure as adrenal hyperactivity, involution of lymphatic organs, and gastrointestinal disturbances (Selye, 1956). In the 1980s, Lazarus and Folkman published a broad definition stating that psychological stress occurs when we appraise the demands of a situation as taxing or exceeding our ability to cope (Lazarus & Folkman, 1984). This definition permeated subsequent research at the intersection of stress, adaptation, and health. Today, the wording used on gold-standard self-report instruments for assessing chronic psychological stress reflects these historical and conceptual influences (Cohen & Janicki-Deverts, 2012).

­Evidence for stressor specificity Evidence over several decades now contradicts the notion that the stress response is nonspecific. One meta-analysis of 208 studies of acute cortisol reactivity to laboratory stress tasks found that tasks characterized by social evaluative threat and uncontrollability evoked the greatest cortisol reactivity and slowest recovery (Dickerson & Kemeny, 2004). This analysis investigated a variety of stressor paradigms, such as

­General theories of psychological stress and health

cognitive performance, noise, public speaking, and emotion induction. Stress task paradigms that combined tests of cognitive performance with public speaking and social evaluative feedback (e.g., negative judgments that challenge self-esteem and social status) evoked the strongest cortisol responses. An individual’s motivation to perform well also determined greater cortisol reactivity, presumably because it heightens the stakes for failure (Dickerson & Kemeny, 2004). In sum, not all stressors recruit comparable physiological responses, and social evaluative threat is particularly potent.

­The presence of threat versus the absence of safety More recently, the generalized unsafety theory of stress (GUTS) (Brosschot, Verkuil, & Thayer, 2018) has arisen to address certain gaps in the existing models of acute and chronic stress. These models fail to explain why circumstances like loneliness, low socioeconomic status, or “technology overload” are associated with heightened stress-arousal and disease risk (Adler & Snibbe, 2003; Riedl, 2012; Wilson et al., 2007). Current understandings of the neurocircuitry underlying stress responses provide an explanatory framework. Brain regions activated during acute stress exposures and perceived threat, such as the amygdala, are subject to tonic inhibition by the prefrontal cortex (Arnsten, 2015). Inhibitory control is a finite resource eroded by cognitive load (Murphy, Groeger, & Greene, 2016). Hence, the authors of GUTS argue that the stress response need not be provoked by acute exposures, but is always “on,” restrained by the “brake” of prefrontal inhibition (Brosschot et al., 2018). A lapse in prefrontal oversight could unleash the effects of suppressed amygdalar action. Thus, external stressors are not needed to generate a prolonged stress response—unconscious perseverative worries and perceived lack of safety are sufficient. Indeed, rats exposed to chronic isolation stress (21 days) exhibit oxidative stress in the prefrontal cortex and depressive behaviors, despite no changes in corticosterone (Zlatković et al., 2014).

­Distinguishing transdiagnostic distress from diagnosis Both exposures to stressful life events and the presence of significant psychological distress are central features of the diagnostic criteria for many psychiatric diagnoses, such as major depressive disorder (MDD), generalized anxiety disorder (GAD), and posttraumatic stress disorder (PTSD). There are several psychometrically validated scales that assess the symptoms of each of these disorders. Such symptoms might also be thought of as falling under an umbrella construct of transdiagnostic psychological distress (Harvey & Watkins, 2004), a term that refers to common functional processes, which span several disorders and serve as useful treatment targets (e.g., experiential avoidance spans various anxiety disorders) (Harvey & Watkins, 2004). As a note of caution, it is important not to conflate symptom scores with true diagnoses. Diagnoses, as defined by the Diagnostic and Statistical Manual (DSM) of Mental Disorders (American Psychiatric Association, 2013), are made based on meeting

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c­ riteria for a certain number of symptoms with adequate severity within a given time frame, where the presentation is not better explained by various rule outs. Lastly, in cardiovascular and metabolic health, subclinical levels of distress symptoms are generally associated with changes in biomarkers in a linear rather than categorical way (Carney, Freedland, & Jaffe, 2001).

­RDoC and neurocircuitry based frameworks The value of DSM-based definitions in biological research has been increasingly questioned over the past decade as the National Institute of Mental Health (NIMH) launched a paradigm shift known as the Research Domain Criteria or the “RDoC” initiative (Insel, 2014). DSM categories are entirely based on signs and symptoms, whereas RDoC posits that symptoms arise from specific neurocircuits (Morris & Cuthbert, 2012). Much research has attempted to identify a distinct biomarker “fingerprint” associated with specific DSM diagnoses. Yet, this is arguably analogous to expecting consistent digestive effects from a vegetable soup, in which the specific ingredients differ from one batch to the next. Nonetheless, the modern healthcare system revolves around ascribing categorical diagnoses and reimbursing treatments accordingly. Therefore, one goal of this chapter is to outline how future researchers might translate the existing literature into a more circuit- or pathway-inspired framework.

­Eustress: Definition and discordance ­Disentangling exposures and experiences from impact Researchers have yet to reach a consensus on a definition of eustress. Eustress should be defined based on (1) the positive or negative valence of an individual’s emotional experience, (2) a biomarker of the stress response (e.g., neuroendocrine or autonomic), or (3) an oxidative stress biomarker end point? Hans Selye distinguished eustress as “agreeable or healthy” and distress as “disagreeable or pathogenic” (Selye, 1976a), leaving some room for confusion about whether eustress refers to the subjective psychological experience itself or the biological impact of that experience. In contrast, Helmut Sies uses the latter to define eustress (Sies, Berndt, & Jones, 2017). As to the question of emotion valence, Hans Selye opines, “A painful blow and a passionate kiss can be equally stressful” (Selye, 1976a). However, recent research suggests that arousal accompanied by positive emotions, confidence, or a sense of mastery (“a challenge state”) evokes a qualitatively different and more salutary acute cardiovascular reactivity response than that evoked by distress (“a threat state”) (Jamieson, Mendes, & Nock, 2013). It follows that “eustress” responses may arise from both positive and negative emotions and may be quantified across varying domains of measurement (psychological, neurocognitive, or peripheral biomarkers such as cortisol) (Shojaie, Ghanbari, & Shojaie, 2017). Nonetheless, herein, we focus on psychological distress, which has generated the vast majority of the evidence in relation to oxidative stress.

­The assessment of psychological distress

­Two paths to eustress: Hormesis versus stress buffering Eustress can be achieved through different paths, one of which is known as hormesis, and another, stress buffering. Hormesis is well-defined within toxicology as a biphasic response to a “stressor” or environmental factor, such that low to moderate doses produce optimal effects (eustress), and high doses produce toxic or inhibitory effects (Mattson, 2008). Physical activity is a good example of a behavioral exposure that can induce hormesis or a eustress response. Mice bred to engage in insufficient physical activity develop mitochondrial dysfunction and cardiometabolic disease, representing the fact that the lack of sufficient (physiological) stress can have adverse health effects (Wisløff et al., 2005). In contrast, by increasing the stress of physical activity to moderate levels, a eustress effect occurs, marked by improved oxidative stress levels. Over time, these episodic demands, following by a period of restoration, increase mitochondrial resilience, thereby improving the body’s ability to maintain a healthy oxidative milieu during everyday cellular metabolism. In contrast, physical activity that is excessive and prolonged, such as extreme marathon running, will exceed the eustress zone, leading to accumulated oxidative damage (Mrakic-Sposta et al., 2015). Eustress is sometimes conflated with “stress-buffering” effects, rather than recognized as a potential downstream outcome. For example, one study defined eustress as “a positive phenomenon that ameliorates the biological effects of distress” (Berk, Felten, Tan, Bittman, & Westengard, 2001). However, factors that ameliorate or inhibit the negative impact of stress should be considered stress buffers. For example, social support and a sense of mastery or self-efficacy are examples of factors that “buffer,” lessen, or protect against the adverse physiological effects of acute or chronic psychological distress (Hostinar, Sullivan, & Gunnar, 2014). Psychological strategies for emotion regulation, such as reappraising a stressor in a more positive manner, can activate prefrontal cortex-mediated inhibition of amygdalar threat responses (Banks, Eddy, Angstadt, Nathan, & Phan, 2007), thereby “buffering” the outflow of neuroendocrine or autonomic activation. Hence, it is conceivable that stress buffering might move a body from oxidative distress toward eustress, but it would achieve this end, not by activating the stress response (as hormesis does), but by dampening it. However, to our knowledge, only one published study has linked acute stress reactivity with oxidative damage markers (Aschbacher et  al., 2013). Hence, the idea that interventions to enhance stress buffering might conceivably improve the oxidative milieu in at-risk populations remains purely speculative.

­The assessment of psychological distress ­Standardized tools and tasks Behavioral and neuroscientists have developed a host of well-studied, standardized questionnaires, and computer-based tasks exist to assess the subjective experience of psychological distress. These tools range from one-time self-report measures, to

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acute experimental stress tasks performed in a standardized laboratory environment, to computer-based neurocognitive assessments of emotion regulation processes.

­The validity of self-reported psychological distress

A primary method for assessing distress has been the use of one-time, subjective, self-report questionnaires. Contrary to the popular misconception among nonsocial scientists that self-reports lack validity, the scientific evidence does not support this view. The most commonly used questionnaires assessing psychological and psychiatric constructs typically go through an extensive and deeply rigorous process of psychometric validation (Kim, 2009). It is true that subjective responses are subject to known biases, due to factors such as social desirability, memory distortions, lack of insight or conscious awareness of one’s mental state, and methodological considerations (e.g., those related to the structure of the response scale). Nonetheless, thoughtful frameworks exist to understand and address the implications of these biases (Podsakoff, 2003). It is not that self-reports are unreliable or invalid but that these questionnaires are optimized relative to a specific mental health outcome. Hence, psychosocial measures are well validated to maximize their predictive validity for DSM-defined mental health disorders, which in turn tend to be inconsistent predictors of biology (Insel, 2014). As interdisciplinary scientists, we advocate for triangulation of measures using different methods. For example, combining subjective reports with experimental perturbations, neurocognitive performance tasks, biomarkers of stress arousal or performance, and behavioral observations permits researchers to capture the complexity of mind-body pathways and minimize measurement “noise” (Mason et al., 2019).

­Self-report measures of perceived stress

Several different measures of perceived psychological stress symptoms have been well validated in large samples and are predictive biological end points, such as the Perceived Stress Scale (PSS) (Cohen & Janicki-Deverts, 2012). The effort-reward imbalance (ERI) model (Siegrist & Li, 2016) is often used to assess work stress in relation to health. The PSS includes items about difficulty coping, lack of control, and anxiety (“feeling nervous”), whereas the ERI scale focuses on the imbalance between demands and rewards. Meta-analytic evidence shows that ERI was more predictive of visceral adiposity than the PSS and that associations also differed by gender (Tenk et  al., 2018), thereby indicating that certain measures may be more closely aligned than others with the underlying neurocircuitry relevant to a given pathophysiological condition.

­Self-report measures of depressive symptoms

A number of validated measures of depressive symptoms have been used in relation to health outcomes, such as the Center for Epidemiological Studies-Depression (CES-D) scale (Devins et al., 1988), the Patient Health Questionnaire (PHQ) (Kroenke, Spitzer, & Williams, 2001), and the Beck Depression Inventory (BDI) (Beck, Steer, & Carbin, 1988), with some exhibiting greater sensitivity and s­ pecificity ­characteristics

­The assessment of psychological distress

than others in relation to clinical cohorts (Löwe et al., 2004). It is worth highlighting that depression is a highly heterogeneous diagnosis, and therefore, two patients with the same diagnosis can present with very different symptoms (e.g., elevated versus decreased appetite). Some questionnaires, such as the PHQ, which ask about “poor appetite or overeating,” do not distinguish between increased versus decreased appetite. In contrast, animal models of depression and oxidative stress have used sucrose preference as an indicator of depressive symptoms (Zlatković et al., 2014), which would align best with increased appetite. As per the RDoC approach (Insel, 2014), future research should examine whether certain dimensions of depression (e.g., low energy, anhedonia, and increased appetite), track better with markers of oxidative stress than the full, heterogeneous symptom totals.

­Self-report measures of anxious symptoms

The symptoms of anxiety (e.g., restlessness, fatigue, and irritability) are assessed through validated scales (Julian, 2011; Kraemer, Gullion, Rush, Frank, & Kupfer, 1994). It is noteworthy that depressive and anxious symptoms are highly comorbid with one another (Kaufman & Charney, 2000) and with perceived stress symptoms. Approximately 50%–60% of individuals who meet criteria for MDD also meet criteria for an anxiety disorder during their lifetime (e.g., generalized anxiety, panic, social phobia, and posttraumatic stress disorder [PTSD]) (Kaufman & Charney, 2000). In 2013, PTSD was reclassified from an anxiety disorder to a new category of trauma and stressor-related disorders in the DSM-V (American Psychiatric Association, 2013). Depression, anxiety, and PTSD are all associated with markers of oxidative stress in myriad studies, as discussed later (Hovatta, Juhila, & Donner, 2010; Lopresti, Maker, Hood, & Drummond, 2014; Maurya et al., 2016; Miller & Sadeh, 2014).

­Acute laboratory psychological stress tasks

The most widely used tool to study acute psychobiological stress responses in the lab is the Trier social stress test (TSST) (Kirschbaum, Pirke, & Hellhammer, 1993; Kudielka, Hellhammer, Kirschbaum, Harmon-Jones, & Winkielman, 2007). This laboratory-based protocol involves inducing moderate psychological stress using a social evaluation paradigm. Specifically, an individual plans, prepares, and anticipates performing a speech about a personally relevant topic and delivers this speech to an evaluative audience. This audience provides no positive feedback after this speech (or may provide negative feedback in some versions) and then asks the individual to perform math tasks. The TSST reveals differences in physiological stress responses between healthy normal and clinical populations (Burke, Davis, Otte, & Mohr, 2005; Takahashi et  al., 2005). Subjective stress appraisals assessed before and after the TSST may represent important and modifiable determinants of stress response type (i.e., eustress vs. distress) (Mcewen, 1998). The TSST offers an extremely well-validated paradigm to induce increases in physiological markers of many systems, including neuroendocrine hormones (e.g., cortisol) (Dickerson & Kemeny, 2004; Kudielka et al., 2007), cardiovascular reactivity and catecholamines

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(Aschbacher et al., 2008; Mendes, Blascovich, Hunter, Lickel, & Jost, 2007), inflammatory cytokines (Steptoe, Hamer, & Chida, 2007), coagulation/hemostasis (Von Känel & Dimsdale, 2003), markers of proatherosclerotic leukocyte trafficking into the vasculature (Aschbacher et al., 2008), and increases in heart rate (Kudielka et al., 2007). Additionally, one study reported that TSST-induced cortisol reactivity was associated with markers of oxidative damage among chronically stressed individuals (Aschbacher et  al., 2013). Gender, age, genetics, and other factors can impact stress responses to the TSST, underscoring the complexity of physiological stress responses (Kudielka et al., 2007).

­Neurocognitive tasks

Many of the drivers of the stress response may be phylogenetically old and therefore potentially unconscious. Hence, neurocognitive testing may capture these unconscious drivers of the stress response better than self-reports. Neurocognitive tests relevant to emotion regulation (Smaga et  al., 2015) are typically administered as brief tasks on a computer, potentially concurrent with functional neuroimaging or physiological assessments. Such tests can tap into the cognitive basis of threat and provide insights into the underlying neurocircuitry. Threat neurocircuitry regulates the outflow of stress-responsive hormones and nervous system efferent signaling to peripheral target tissues. Hence, it represents an important neural mechanism linking anxiety or trauma exposure with accelerated risk of biological aging (O’Donovan, Slavich, Epel, & Neylan, 2013).

­ aturalistic exposures to stressful life events: Methods and N assessment Two people faced with the same stressful event—a horrific natural disaster, a child’s chronic illness, or the experience of war and combat—will each tell a unique story. Do people who narrate these stories in a more “resilient” fashion have better health outcomes, or do major life events simply take a toll regardless of how we perceive them? It is tempting to speculate that “objective” measures (i.e., exposures) will be superior to “subjective” measures (i.e., self-reports) or that, to the contrary, it is only one’s subjective experience that matters. Such questions call for methods to contrast the subjective and objective aspects of human experience. In the subsequent section, we describe frequently used methods to assess naturistically occurring stress exposures such as chronic caregiver stress, early adversity, and the stressful events of everyday life. Finally, we review evidence that the mere act of anticipating a stressful event can produce a robust psychological stress response with relevance to oxidative stress.

­Assessing objective stress exposures

Measures such as the Life Events and Difficulties Schedule (LEDS) (Brown & Harris, 2012), the Stress and Adversity Inventory (STRAIN) (Slavich & Shields, 2018), and the Life Stressor Checklist (LSC) (Ungerer, Deter, Fikentscher, & Konzag, 2010)

­The assessment of psychological distress

attempt to quantify life stress exposures. Caregiving, defined as caring for a loved one (e.g., a spouse, parent, or child) with a chronic illness or disability (Pinquart & Sörensen, 2007), is a well-established human model of chronic stress and its adverse effects on health. As humans cannot be ethically randomly assigned to conditions of chronic or traumatic stress, such designs are quasi-experimental and require careful statistical handling of potential confounds. The dose-response exposure to chronic caregiver stress can also be objectively quantified as the duration and the number of hours of care provided per day. Intensity can be objectively assessed through illness severity of the care recipient (Aschbacher et  al., 2006). Both objective (caregiver status) and subjective definitions (self-reported distress) have predicted oxidative stress markers (Aschbacher et al., 2013) and mitochondrial respiratory chain activity (Picard et al., 2018). However, meta-analytic studies of caregiving suggest that subjective measures of distress and care-recipient problem behaviors are somewhat better predictors than objective measures (e.g., being a caregiver, the duration and intensity of caregiving) (Pinquart & Sörensen, 2007).

­Early adversity

Stress in early life, assessed by retrospective self-reports in humans or by experimental manipulation in animals, is associated with alterations in mitochondrial DNA copy number (Tyrka et al., 2016), brain oxidative stress (Schiavone, Colaianna, & Curtis, 2015), and telomere shortening (Price, Kao, Burgers, Carpenter, & Tyrka, 2013). Life history theory posits that biological systems are “tuned” through environmental exposures during sensitive windows to better survive the particular stressors in the environment. Interestingly, this anthropologic theory parallels systems engineering principles of robustness (Kitano, 2007). Sensitive periods are thought to reshape neuroimmune circuits, leading to heightened acute stress reactivity (arguably adaptive in the short term) at the cost of greater physiological wear and tear in the long term (Miller & Raison, 2015). The evidence also suggests a reallocation of resources from adaptive to innate immunity (and a proinflammatory milieu), acceleration of the onset of fertility, and a shorted life span (Miller & Raison, 2015). There are, however, challenges in evaluating oxidative stress as a crucial mechanism by which exposure to psychological stressors may shift the eustress optima and accelerate biological aging, as reviewed elsewhere (Shalev & Belsky, 2016). In sum, the timing of stress exposures in relation to sensitive windows of development may influence the nature and magnitude of the biological impact.

­Daily stressful exposures

Many studies report that daily stressor exposures (e.g., a work deadline or argument with one’s spouse) impact health (Almeida, Mogle, Sliwinski, Piazza, & Charles, 2012), though little is known about their impact on oxidative stress. Daily diary methodologies can be used to assess both daily stressor exposures and an individual’s subsequent positive or negative affective state. One study assessed the association of leukocyte mitochondrial function with daily affective responses (rather than stress exposures), using a maternal caregiving model of chronic stress (Breen et al., 2017;

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Picard et  al., 2018). That study reported a prospective association of positive affect with next-day mitochondrial function (a major determinant of oxidative stress). However, it did not specifically include data on whether or not the simple exposure to daily stressors was related to mitochondrial function.

­The role of anticipation

Just thinking about a stressful event can activate neurocircuits and physiological stress responses (Davidson, Marshall, Tomarken, & Henriques, 2000). Hence, assessing states of anticipatory threat or reward can be informative. Such assessments somewhat dissociate the psychological processes from the metabolic requirements needed to perform during a stressor, which we might consider to be an important aspect of a “eustress” response. The TSST (reviewed earlier) has a built in period of anticipation (Kirschbaum et al., 1993). One study found that the anticipatory cortisol reactivity to the TSST, but not the actual peak level of cortisol during the stress task, best predicted oxidative damage markers to DNA and lipids among chronically stressed women (Aschbacher et  al., 2013). Furthermore, positive emotions might be most likely to predict oxidative distress when there is a violation of positive expectations. A significant proportion of the brain’s resting state appears to be dedicated to a “prediction model” of expectation and anticipation (Bubic, Von Cramon, & Schubotz, 2010). Hence, the violation of expectations can strongly activate acute cardiovascular (Mendes et al., 2007) and inflammatory stress reactivity (Aschbacher et al., 2012).

­Stress system biomarkers Many of the effects of psychological distress on downstream target tissues (including the brain itself) are mediated by catecholamines, such as norepinephrine and epinephrine, or neuroendocrine hormones, such as cortisol. Their effects can transition from eustress into distress, dependent on the cellular context, frequency and duration, and interactions with other factors, as described elsewhere (Arango-Lievano & Jeanneteau, 2016).

­Cortisol: Functions and measurement Cortisol is one of the most widely studied biomarkers of stress arousal; however, its role as a transducer of psychological distress is complex. Exposure to psychological stressors can activate the hypothalamic-pituitaryadrenal (HPA) axis, leading to production of the glucocorticoid hormone cortisol by the adrenal cortex (or corticosterone in rodents). However, cortisol does not always elevate in response to acute psychological stress exposures (Julian, 2011). Cortisol has wide-ranging physiological effects, such as increasing glucose neogenesis (Khani & Tayek, 2001) and modulating inflammation (Arango-Lievano & Jeanneteau, 2016; Dandona et al., 1999). Cortisol also crosses the blood-brain barrier and impacts myriad brain functions, ­including

­Stress system biomarkers

emotional memory formation, habit formation, fear learning, and neuroplasticity (Arango-Lievano & Jeanneteau, 2016; Mcewen, 1998). Nonetheless, reviews of cortisol in relation to specific health outcomes often reveal inconsistent results (Miller, Chen, & Zhou, 2007; Nyberg et  al., 2012; Steptoe & Serwinski, 2016), and one contributor may be measurement heterogeneity. In humans, cortisol is measured in a variety of sample types (saliva, blood, urine, and hair) using a variety of protocols and models. These include acute laboratory stress reactivity (Dickerson & Kemeny, 2004), the circadian awakening response (Steptoe & Serwinski, 2016), time series or dynamic systems analysis (understanding the HPA axis as a feedback-regulated loop) (Aschbacher et al., 2012), and hair sampling (reflecting longer-term exposures) (Staufenbiel, Penninx, Spijker, Elzinga, & Van Rossum, 2013). Collectively, this evidence suggests that the dynamics of cortisol are as or more important in predicting health outcomes, than an average level or simple diurnal slope.

­Cortisol: No straightforward interpretation Psychological disorders characterized by dysregulated stress responding are often associated with alterations in cortisol, but the directionality is inconsistent. For example, cortisol generally appears to be elevated in depression and lowered in anxiety or PTSD (Staufenbiel et al., 2013). Moreover, this finding appears to conflict with other aspects of the literature. First, depression and PTSD exhibit high levels of comorbidity in the population (Kaufman & Charney, 2000). Second, both depression and PTSD are associated with elevated markers of oxidative/nitrosative stress and proinflammmatory cytokines (Hovatta et al., 2010; Lopresti et al., 2014; Passos et al., 2015). Hence, this apparent contradiction is difficult to reconcile with the assertion that cortisol explains or mediates the effects of psychological distress on oxidative stress and related inflammatory biomarkers. As reviewed elsewhere (Arango-Lievano & Jeanneteau, 2016), cortisol’s ultimate effects result from a complex cascade of interactions along cell signaling pathways. Hence, many researchers have turned to studying psychological distress in relation to glucocorticoid receptor sensitivity, transcriptional or epigenetic alterations, and functional readouts (Aschbacher et al., 2016; Cole, 2013; Somvanshi et al., 2018; Turecki & Meaney, 2016).

­Cortisol: From eustress to distress Evidence from humans and animals suggests that glucocorticoids can have both beneficial and adverse effects on the oxidative milieu (Aschbacher et  al., 2013; Caro et al., 2007; Dandona et al., 1999; Joergensen et al., 2011; Sanner, Meder, Zidek, & Tepel, 2002). Evidence suggests that synthetic glucocorticoids, such as dexamethasone, decrease ROS generation in leukocytes and platelets, thereby suppressing inflammation (Dandona et al., 1999; Sanner et al., 2002). However, a few human studies have reported that higher endogenous cortisol is associated with increases in markers of oxidative damage (Aschbacher et  al., 2013; Joergensen et al., 2011).

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­The autonomic nervous system and oxidative stress Stress is often said to evoke a “fight or flight” response, intended to mobilize the body to action. This response is mediated by activation of the sympathetic arm of the autonomic nervous system, resulting in secretion of the hormones (and neurotransmitters) epinephrine and norepinephrine. Moreover, some evidence suggests that ROS participate in shaping this stress response (Schiavone, Jaquet, Trabace, & Krause, 2013). At rest, the parasympathetic nervous system tonically inhibits sympathetic activation but withdraws during stress responses. Extensive research has demonstrated how chronic and acute stressors interact to impact autonomic and cardiovascular reactivity to stress (Chida & Hamer, 2008). Moreover, sustained elevations in catecholamines are known to contribute to cardiovascular pathology via oxidative stress (Costa et al., 2011). For example, animal evidence shows that catecholamines such as norepinephrine may undergo auto-oxidation and thereby promote oxidative damage with adverse cardiovascular effects (Neri et al., 2007). Others have also reviewed the evidence that psychological distress at work promotes cardiovascular disease via oxidative stress (Siegrist & Sies, 2017).

­System allostasis: Robustness and resilience The notion of homeostasis proposed that the goal of a system—for example, the systems tasked with regulating our body’s response to psychological distress—is to maintain a given set point. In 1988, Sterling and Eyer (1988) argued this is a flawed notion, instead proposing the theory of allostasis—or “stability through change.” Whereas homeostasis using an “error-correct by feedback” principle, allostasis tries to anticipate needs and prepare for them before they arise (Sterling, 2012). In allostasis, the brain is recognized as a master controller of a feedback-regulated system, and hence, the treatments that will logically help restore a eustress state can be opposite of those made according to the predictions of homeostasis (Sterling, 2012). For example, theories of allostasis might suggest that the body anticipates greater energy needs and therefore increases ROS production. Allostatic theory predicts that acute increases might “jolt” the system back to a eustress state. This prediction is arguably consistent with the eustress effects of moderate exercise (Wisløff et al., 2005) and acute psychological stress among individuals free from chronic stress (Aschbacher et al., 2013).

­Mind-body pathways and oxidative stress markers In this section, we provide an overview of the literature investigating associations between psychological distress and oxidative stress. Table 1 provides a brief overview of common markers used to study oxidative and nitrosative stress in relation to psychological distress among humans.

­Mind-body pathways and oxidative stress markers

Table 1  An overview of oxidative stress biomarkers used in psychobiological studies. Biomarker abbreviation

Biomarker(s) full name

Marker type

8-OHdG 8-oxoG F2-Iso MDA

8-hydroxydeoxyguanosine 8-oxo-7,8-dihydroguanosine F2-isoprostanes Malondialdehyde

eNOS, iNOS, nNOS NF-KB

Endothelial, inducible, and neuronal nitric oxide synthases Nuclear factor kappa-B

HIF-1a

Hypoxia inducible factor 1-alpha (HIF-1a encodes a subunit of HIF-1) Nuclear factor erythroid-derived 2 like factor

DNA damage RNA damage Lipid damage Lipid damage, polyunsaturated fats Enzymes that synthesize nitric oxide Gene expression; master regulator of inflammation Gene expression; cellular response to hypoxia Gene expression; regulator of active defense enzymes Antioxidant enzyme Antioxidants

NRF2

SOD GPx, GR, and the GSH/GSSG ratio mtDNAcn; MHI

Superoxide dismutase Glutathione peroxidase, glutathione reductase, the oxidized glutathione ratio Mitochondrial DNA copy number; mitochondrial health index (composite of enzymatic activity and mtDNAcn)

Mitochondrial DNA and enzymatic activity

­Oxidative damage markers Direct measurement of ROS is difficult because of their short half-lives. Hence, commonly studied biomarkers in the mind-body literature include markers of biomarkers of oxidative damage to lipids, such as F2-isoprostanes (F2-iso) and malondialdehyde (MDA). Also, common are oxidative damage to the genome, such as 8-­hydroxydeoxyguanosine (8-OHdG; oxidative damage to DNA), and 8-oxo-7,8dihydroguanosine (8-oxoG; oxidative damage to RNA). Oxidative damage markers have been alternately measured in blood serum, plasma, urine, cerebrospinal fluid, erythrocytes, and leukocytes with varying methodology. Interestingly, a recent ­autopsy-based study of over 100 individuals with severe mental illness (SMI) found that measures of 8-OHdG (but not 8-oxoG) in urine were significantly correlated with those in cerebrospinal fluid (r = 0.50), substantiating the notion that urinary markers may reflect oxidative damage within the nervous system (Christensen et al., 2018).

­Mitochondria Psychological stress is a cascade of responses that essentially reallocate energy from long-term functions (e.g., digestion and restoration) to short-term survival functions (e.g., fight or flight response). Thus, mitochondria are crucial determinants of the

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response to psychological stress. In animal models, mutations or deletions of mitochondrial genes yield whole body phenotypes with dysfunctions in the reactivity and recovery to psychological stress exposures, as well as alterations in inflammation, the HPA axis, and redox balance (Picard et al., 2015). A recent review of animal and human research concluded that acute and chronic stressors influence myriad aspects of mitochondrial biology (Picard & Mcewen, 2018). Studies in humans have investigated mtDNA as a biomarker, because the loss of mitochondrial genome integrity is a potential mechanism of accelerated biological aging (Sharma & Sampath, 2019). However, these associations have yielded inconsistent results, can be driven by changes in the underlying immune cell subsets, and are difficult to interpret (Picard & Mcewen, 2018).

­Nitric oxide and the nitric oxide synthase enzymes Nitric oxide (NO) plays a particularly important role in the cardiovascular, gastrointestinal, and immune systems, and in the brain (Beckman & Koppenol, 1996). Due to rapid signaling and diffusional capacity, NO can locally integrate information regarding vascular shear stress or neural reflexes. Cells generate NO by catalyzing L-arginine using nitric oxide synthase enzymes, including the endothelial (eNOS), neuronal (nNOS), and inducible (iNOS) forms (Santo, Zhu, & Li, 2016). NO is recognized as the primary mediator of vasodilation, which underlies adaptive vascular responses to psychological distress and the accompanying needs for changes in blood flow and oxygenation. iNOS is not normally produced in the brain, but can be detected following injury or inflammation, and may play a role in mental health conditions linked with inflammation such as depression (Gałecki et al., 2012).

­Antioxidants Superoxide dismutase (SOD) enzymes catalyze the partitioning of superoxide (O2−) radicals to oxygen (O2) or hydrogen peroxide (H2O2). SOD may be a mechanism that protects against oxidative stress in some contexts such as diabetes (Kowluru, Kowluru, Xiong, & Ho, 2006) and by which caloric restriction extends life span in some model organisms (Mesquita et al., 2010). Glutathione (GSH) protects the brain (Rae & Williams, 2017), and postmortem human evidence suggests that GSH levels may be decreased in individuals with depression and other mental health conditions (Andreazza, Gawryluk, Wang, Shao, & Young, 2011).

­A brief overview of the literature ­Depression, anxiety, and oxidative/nitrosative stress Several recent reviews have examined the relationship between depression, anxiety, PTSD, and markers of oxidative/nitrosative stress and antioxidants in humans (Hovatta et al., 2010; Lopresti et al., 2014; Maurya et al., 2016). On the whole, this body of literature confirms the existence of significantly greater oxidative stress in

­A brief overview of the literature

clinical groups relative to healthy controls. It also offers some evidence of doseresponse relationships with symptom severity. The findings on cytoprotective or antioxidant mechanisms appear somewhat less consistent; they may be depleted, but they may also sometimes be increased, arguably as a counterregulatory and potentially adaptive response to oxidative stress and inflammation. Oxidative stress can lead to mitochondria dysfunction, inflammation, and impaired neurogenesis. Hence, oxidative stress may be both a consequence and cause of psychological distress.

­PTSD and oxidative/nitrosative stress Studies have also investigated markers of oxidative stress and mitochondrial function in the context of PTSD. PTSD is unique relative to other mental health disorders in being the only one that, by definition, is triggered by exposure to an external stressor. An animal model of PTSD suggests that increases in systemic oxidative stress and inflammation are involved in the development of this disorder (Wilson et al., 2013). Consistent with these findings, transcriptomics analyses in humans also show that transcripts relevant to inflammation and oxidative stress, particularly in monocytes, are among those most altered in PTSD (Breen et al., 2017; O’Donovan et al., 2013). Epigenetic and metabolomic data further converge, suggesting that dysregulations in cortisol stress reactivity and glucocorticoid sensitivity contribute to the etiology of PTSD, via oxidative, inflammatory, and metabolic aberrations (Somvanshi et al., 2018; Turecki & Meaney, 2016).

­A transdiagnostic perspective There is also a relevant literature outside of a DSM-based clinical context. Studies of chronic caregiver stress, acute laboratory stress tasks, or self-reported perceived stress reveal associations of psychological distress with markers of oxidative damage and mitochondrial health (Aschbacher et al., 2013; Aschbacher et al., 2014; Aschbacher et al., 2014; Picard et  al., 2015; Picard et  al., 2018; Picard & Mcewen, 2018). Moreover, the redox-sensitive transcriptional regulator nuclear factor-kappa B (NF-kB) features prominently at the intersection of psychological distress, oxidative stress, and inflammation. Many human studies of social genomics report that NF-kB transcriptional control pathways expressed in peripheral monocytes specifically or mixed leukocyte samples are upregulated in relation to acute psychological stress tasks, depression, PTSD, and various forms of social adversity (Bierhaus et al., 2003; Cole, 2013; O’Donovan et al., 2011). These pathways appear to represent a conserved transcriptional response social adversity, which may transcend particular diagnoses (Fredrickson et al., 2015).

­Translational approaches to eustress Further translational research is needed to bridge the largely associational findings from the clinical literature (reviewed earlier) with the mechanistic understandings offered by basic science. Cellular models of stress adaptation (eustress) provide

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one promising route. For example, cultured mammalian cells and model organisms (Drosophila) adapt to repeated environmental stressors by upregulating cytoprotective systems to mitigate oxidative stress (Pickering, Vojtovich, Tower, & A Davies, 2013). These adaptions temporarily enhance resilience to oxidative stress (e.g., by H2O2) (Pickering et al., 2013). Furthermore, they address the crucial question of how to close the gap between responses to a single acute stress exposure and chronic or repeated exposures. Interestingly, successful adaptation (resilience to oxidative stress) occurred only when acute oxidative exposures were separated by a sufficient interval for recovery. Furthermore, successful short-term adaptations shortened longevity in the model organism (Pickering et al., 2013). Hence, future research might explore whether similar cellular adaption models might elucidate eustress responses in the context of cells (e.g., circulating leukocytes) from individuals with versus without clinical phenotypes. In addition, stress hormones might be applied during in vitro adaptation trials to draw parallels with in vivo reactivity to acute psychological stress tasks or other paradigms.

­ xidative stress as a mediator of psychosocial stress and O aging There is intense interest in understanding whether psychological distress contributes to aging, and in identifying the precise mechanisms by which this might occur. As discussed, psychological distress (especially when chronic) may increase oxidative stress. In turn, oxidative stress may work through various pathways to accelerate cellular aging, including contributions to telomere shortening, cellular senescence, and deficient autophagy (cellular house cleaning), leading to accumulation of cellular damage.

­Oxidative stress: Explaining the stress-telomere connection In 2004, the revelation that chronic stress in humans, as defined through a maternal caregiving model, was associated with shorter telomeres (Epel et al., 2004), generated explosive interest about the potential for psychological distress to contribute to accelerated biological aging via telomere shortening. In  vitro, oxidative stress shortens telomeres; hence, it may be a mechanism for stress-related telomere attrition (Von Zglinicki, 2002). Notably, Epel et al. (2004) reported a trend-level association in vivo between the F2-iso/vitamin E ratio with perceived stress levels and years of caregiving. Recent meta-analytic research identified an association between perceived psychological stress and leukocyte telomere length, but it is very small (Mathur et al., 2016). However, telomere length dynamics vary by immune cell subsets, which might attenuate correlations in mixed cell samples (Lin et al., 2016).

­Cellular senescence: A mechanism of aging Cellular senescence represents another pathway by which oxidative damage to DNA may contribute to aging. Cellular senescence is triggered by aging and repeated environmental (and possibly psychological) stressors, characterized by

­A causal role for oxidative stress in depression and anxiety?

g­ rowth/­proliferation arrest, gene expression changes, and proinflammatory cytokine and chemokine secretion (Kang et al., 2015). Mechanisms involved in initiating senescence involve activation of NF-kB (a redox-sensitive transcription factor) combined with GATA4-induced reductions in autophagy (which can be initiated by ROS) (Kang et al., 2015). In contrast, activation of the transcription factor Nuclear factor erythroid 2-related factor (NRF2) protects against oxidative stress-induced activation of the p53/senescence pathway (Volonte et al., 2013) and might therefore be a promising biomarker or target for future eustress research.

­Deficient autophagy: A promising frontier for eustress research Many pathways relevant to psychological distress and oxidative stress converge upon autophagy. Autophagy is a cellular stress response, activated by various cellular damage signals, that breaks down and clears away cellular debris and toxic by-products (Tang, Kang, Coyne, Zeh, & Lotze, 2012). Psychological distress may intensify the energetic demands and hence the wear and tear on cells, promoting increased accumulation of intracellular debris and thereby creating greater need for autophagy. For example, one study of pigs found that social stress provoked a stronger initial increase in autophagy and antioxidants in muscle tissue, which was then also more quickly depleted within 24 h (Rubio-González et al., 2015). This suggests the possibility that the acute initial burst represents a eustress effect, but that with chronic stress, deficits might occur, with relevance for aging. Furthermore, mitochondrial ROS, which are modulated by psychological distress (Picard & Mcewen, 2018), provide crucial signals to initiate autophagy (Filomeni, De Zio, & Cecconi, 2014). Hence, future research might examine whether stressinduced mitochondrial dysfunction adversely impacts autophagy, thereby contributing to signs of premature aging. Interestingly, some of the beneficial effects of antidepressants may be explained by their ability to induce autophagy, which, in turn, contributes to decreases in psychological distress symptoms and inflammatory proteins (e.g., interleukin(IL)-1B) (Alcocer-Gómez et al., 2017; Gulbins et al., 2018). Finally, caloric restriction (CR) and CR mimetics are considered the most robust and reproducible way to slow aging in animal models (Roth & Ingram, 2016), although the findings of CR in humans are mixed (Fontana, Meyer, Klein, & Holloszy, 2004; Tomiyama et al., 2017). Some evidence suggests that autophagy may be an essential mechanism of CR’s antiaging effects (Bergamini, Cavallini, Donati, & Gori, 2007).

­A causal role for oxidative stress in depression and anxiety? ­Neuroinflammatory depression & anxiety Mind-body circuits are bidirectional, thereby enabling allostatic adaptations to stress. Not only does psychological distress impact health, but the peripheral immune system can impact the brain and our mental well-being (Miller & Raison, 2015). A subset of patients with depression, anxiety, and PTSD exhibit a ­“neuroinflammatory

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phenotype,” marked by elevated peripheral levels of both oxidative stress and inflammation (Hovatta et al., 2010; Lopresti et al., 2014; Passos et al., 2015), which prospectively predict symptom trajectories and treatment responses (Jha & Trivedi, 2018; Van Den Biggelaar et  al., 2007). One unanswered mystery of neuroinflammatory depression and anxiety is as follows: Why does the inflammatory state fail to resolve? When the body is wounded, it mounts an inflammatory response, which resolves naturally as the wound heals. Evidence suggests that oxidative/nitrosative stress can sustain inflammation in a positive feedback loop. If oxidative stress explains the persistence of inflammation in a subset of patients with depression and anxiety, then this could lead to novel treatment targets.

­A neuroinflammatory/oxidative subtype of depression/anxiety One of the major psychiatric discoveries over the past decade is that the peripheral immune system can trigger neuroinflammation, thereby causing symptoms of depression and anxiety (Miller & Raison, 2015). Both afferent nerve and cellular trafficking pathways may mediate these effects (Miller & Raison, 2015). Under nonpathologic conditions, monocytes and other immune cells traffic into the central nervous system (CNS) via the meninges (Derecki et al., 2010; Rua & Mcgavern, 2018), thereby potentially contributing to neuroinflammatory depression (Costa et al., 2011; Miller & Raison, 2015). One proinflammatory cytokine that plays a major role and appears to be intimately interconnected with psychological distress and oxidative stress pathways is IL-1B. Individuals with heightened IL-1B reactivity (stimulated by psychological stress or endotoxins) are at heightened future risk for depression (Aschbacher, Epel, et  al., 2012; Van Den Biggelaar et  al., 2007). Meta-analytic research reveals that IL-1B has the strongest effect size association with trauma exposure (Tursich et al., 2014) and PTSD (Passos et al., 2015), relative to other proinflammatory cytokines, and it is also elevated in depression (Miller, Maletic, & Raison, 2009). IL-1B is the most responsive proinflammatory cytokine to acute laboratory stress tasks (Steptoe et al., 2007). Hence, given the importance of IL-1B among patients with mental health disorders, we use it as a focal point to explore the mechanisms for how oxidative stress may initiate or sustain inflammation.

­Repeated oxidative stress can trigger and sustain IL-1B Ironically, physiological processes that protect the body from repeated bouts of oxidative stress may sustain inflammation. The inflammation in depression is “sterile,” as it is caused by cell-derived signals called damage-associated molecular pattern molecules (DAMPs), which are produced in response to cellular damage, trauma, or ischemia. Mitochondrial ROS can act as a DAMP, leading to inflammasome activation and IL-1B production (Elliott & Sutterwala, 2015). For example, adenosine, a metabolite of ATP, is a cofactor for inflammasome activation of interleukin-1 beta (IL-1B) (Ouyang et al., 2013).

­A causal role for oxidative stress in depression and anxiety?

Adenosine may be a double-edged sword: it both provides resilience to oxidative stress and contributes to sustained sterile inflammation. Adenosine facilitates “ischemic preconditioning,” meaning that a smaller dose of ROS can confer cytoprotection against future repeated oxidative challenges (Ramkumar, Hallam, & Nie, 2001). Similarly, as a means to limit the amount of IL-1B secreted, macrophages (the major producers of IL-1B) that are repeatedly challenged by LPS also develop transient intolerance to further stimulation (Ouyang et al., 2013). However, during this refractory period, ADP can supersede the macrophage’s tolerogenic state, thereby sustaining inflammatory activity and IL-1B production specifically (Ouyang et al., 2013). In the context of an ischemic injury, a more sustained influx of macrophages might help clear away dead cells or molecules damaged by oxidative stress. However, if such DAMP responses were somehow to be activated by psychological distress and anticipatory threat pathways, they might have the “unintended” consequence of contributing to sustained neuroinflammation and depression.

­Evidence of potential clinical relevance Such stress adaptation mechanisms might be relevant to the state of chronic lowgrade systemic inflammation associated with depression and anxiety. First, F2-iso is significantly associated with inflammatory cytokines in individuals with untreated MDD, but is not associated among demographically similar healthy individuals without MDD (Rawdin et al., 2013). Furthermore, this association (of oxidative stress and inflammatory markers) among individuals with MDD is attenuated by successful treatment with SSRIs for 8 weeks (Rawdin et al., 2013). Second, there is reason to believe that the pathways and mechanisms described earlier, which facilitate sustained inflammation, could plausibly be initiated or maintained by psychological distress. There is considerable overlap in the inflammatory response to LPS and exposure to an acute psychological stressor (DAMP), as shown by comparative studies from protein and mRNA assays in blood and peripheral tissues (the liver and spleen) (Fleshner, Frank, & Maier, 2016). There is also overlap in the functional immune response for risk prediction: IL-1B reactivity predicts future depressive symptoms, regardless of whether it is evoked in  vitro by LPS-stimulation of leukocytes (Van Den Biggelaar et  al., 2007) or in  vivo by acute psychological stress (Aschbacher, Epel, et al., 2012).

­Inflammation and antidepressant nonresponse Inflammatory markers also predict poor response to treatment with traditional antidepressants (Strawbridge et al., 2015). Roughly two-thirds of patients with MDD do not respond to the first treatment with selective serotonin reuptake inhibitors (SSRIs), and one-third do not respond after treatment with multiple SSRIs (Rush et al., 2008). Higher levels of the proinflammatory marker C-reactive protein (CRP, >1 mg/L) predict treatment resistance to SSRIs and better response to SNRIs or bupropion (Jha et al., 2017; Uher et al., 2014). Furthermore, SSRIs are also the first-line

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t­reatment for anxiety and PTSD, and neuroinflammatory treatment resistance may well be transdiagnostic (Miller & Raison, 2015). Moreover, five randomized clinical trials (RCTs) have demonstrated that anticytokine treatments, which decrease inflammation, significantly reduce depressive symptoms in antidepressant nonresponders (Kappelmann, Lewis, Dantzer, Jones, & Khandaker, 2016). However, these drugs also carry the potential for undesirable immunosuppression. Nonetheless, these observations support a mechanistic role for inflammation in antidepressant response and are fueling precision psychiatry treatments (Insel, 2014).

­Oxidative stress as a target for adjunctive treatment Oxidative stress may be another promising, though less explored, target for precision psychiatry (Smaga et al., 2015). A study of 105 individuals with and without MDD reports that higher basal F2-iso predicted nonresponse to treatment with SSRIs over 8 weeks (Lindqvist et al., 2017). Moreover, changes in 8-OHdG and IL-6 were associated with response trajectories (Lindqvist et al., 2017). Animal and human studies also report symptom improvements when antidepressant therapy is combined with the adjunctive N-acetylcysteine, which increases the antioxidant glutathione (Minarini et al., 2017; Smaga et al., 2012). Hence, further research is needed to explore treatment adjunctives that target oxidative stress, particularly in the context of treatment-resistant depression. In summary, positive feedback loops between oxidative stress and inflammation have the potential to sustain treatment-resistant depression for a subset of patients. Resolution of oxidative stress may be a promising adjunctive target in the treatment of neuroinflammatory depression, anxiety, and PTSD.

­Eustress, distress, and obesity ­Adipose hypoxia Obesity and habitual overconsumption can produce ROS by various mechanisms reviewed elsewhere (Fernández-Sánchez et  al., 2011). In obesity, adipocytes become larger, reaching the diffusion limits of oxygen and resulting in hypoxia (Sun, Kusminski, & Scherer, 2011). In turn, hypoxia drives insulin resistance via mitochondrial mechanisms (Fazakerley et al., 2018). Furthermore, hypoxia also leads to the infiltration of monocytes, which become adipose tissue macrophages (ATMs) that produce inflammation and further oxidative stress in a vicious cycle (Pasarica et al., 2009). This toxic trio of insulin resistance, inflammation, and oxidative stress can fuel the vascular complications of obesity. Not only does obesity lead to oxidative stress, but losing weight, either through lifestyle or surgery, can reduce oxidative stress (Himbert, Thompson, & Ulrich, 2017). In sum, obesity induces a state of adipose hypoxia, which drives metabolic pathology. Adipose gene expression may provide an indication of the local response to hypoxia. Adipocytes cultured in hypoxic conditions (1% oxygen) exhibit upregulation

­Eustress, distress, and obesity

of genes responsive to hypoxia, including hypoxia-inducible factor (HIF)-1a, vascular endothelial growth factor (VEGF) and IL-6 (Wang, Wood, & Trayhurn, 2007). It is unclear whether this may represent a eustress response. There are evolutionary circumstances where metabolically healthy weight gain is adaptive, such as prior to a long winter or hibernation. As adipose tissue expands in response to increasing nutrition, redox signaling should trigger angiogenesis, thereby increasing the vascularization of adipose tissue and improving oxygen delivery. Hence, limited weight gain (in the nonobese) accompanied by sufficient vascularization should not evoke substantial adverse metabolic effects (Sun et al., 2011). Oxidative eustress may be crucial to determining whether weight gain results in a metabolically healthy or unhealthy profile.

­Brain-to-adipose pathways Psychological distress may drive adipose hypoxia both through direct biological and indirect behavioral pathways. Indirect pathways work through increasing maladaptive eating behaviors—for example, acute psychological distress is sometimes associated with “stress eating” (Epel, Lapidus, Mcewen, & Brownell, 2001; Klatzkin, Baldassaro, & Rashid, 2019). There are also important direct (i.e., nonbehavioral) physiological pathways. For example, cortisol and catecholamines can increase blood glucose (Khani & Tayek, 2001). Thus, psychological distress augments glucose oscillations initiated by food intake. Indeed, acute psychological distress during the postprandial period increase glucose excursions, both in individuals with type I diabetes and in those with PTSD (Nowotny et al., 2010; Wiesli et al., 2005). Exacerbation of glucose oscillations (fluctuations throughout the day) is an important determinant of oxidative stress and diabetic pathology. Specifically, one study of individuals with and without diabetes used a euinsulinemic hyperglycemic clamp to contrast infusion of oscillating glucose concentrations with stable hyperglycemia (Ceriello et  al., 2008). These data demonstrated that oscillations in glucose have a more harmful effect on oxidative stress and endothelial dysfunction than constant high glucose. Another study of patients with and without diabetes, which used continuous glucose monitoring over several days, demonstrated that the dynamics (or mean amplitude of glucose oscillations) predicted oxidative damage better than sustained hyperglycemia (Monnier et al., 2006). Strikingly, this finding held, even after accounting for traditional measures of glycemic control, such as HbA1c (Monnier et al., 2006). Thus, glucose oscillations uniquely predict oxidative stress. Future research is needed to connect the entire pathway, from psychological distress, to glucose oscillations, to oxidative stress, in one overarching study. One promising naturalistic model for such investigations could be chronic work stress in relation to oxidative stress and cardiometabolic disease (Siegrist & Sies, 2017). Chronic psychological stress may also magnify the adverse effects of a poor diet. Translational research demonstrates that, in combination, psychological distress and a high-sugar/high-fat (HSHF) diet have synergistic effects on metabolic risk and oxidative stress (Aschbacher, Kornfeld, et al., 2014; Kuo et al., 2008). In a mouse model,

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chronic psychological stress leads sympathetic nerve terminals to shift from releasing norepinephrine to releasing more of the growth factor neuropeptide Y (NPY), which stimulates preadipocyte proliferation and lipid filling. These mechanisms amplified and accelerated diet-induced obesity. Similarly, in women, chronic stress exposure (caregiving) and HSHF consumption synergistically predict heightened levels of 8-oxoG, poorer insulin sensitivity, and greater abdominal adiposity (Aschbacher, Kornfeld, et al., 2014). Moreover, these chronically stressed women also had higher levels of peripheral NPY than demographically similar low-stress women.

­Adipose-to-brain pathways Not only can psychological distress influence adiposity, but also adiposity may influence mental health and stress arousal. In response to oxidative stress, adipocytes actively produce adipokines (inflammatory cytokines and hormones) that regulate hunger and satiety (e.g., ghrelin and leptin). In turn, adipokines can impact brain function, by crossing the blood-brain barrier or signaling via the vagal nerve (Miller & Raison, 2015; Van Dijk et al., 2015). For example, leptin’s regulation of the HPA axis presents a mechanism to modulate stress adaptation in the context of energy allostasis (Aschbacher, Rodriguez-Fernandez, et al., 2014; Roubos, Dahmen, Kozicz, & Xu, 2012; Tomiyama et al., 2012). Obesity is associated with adverse effects on brain structure and increased dementia risk, which, some hypothesize, are mediated by adipokines (Kiliaan, Arnoldussen, & Gustafson, 2014). Hence, it is interesting to note that treatments targeting ROS may help mitigate adipokine dysregulation. One study, which treated obese mice with an NADPH oxidase inhibitor, showed that reducing ROS production in adipose tissue successfully attenuated adipokine dysregulation and improved metabolic markers (Furukawa et al., 2004). Also, in a parallel human cohort of individuals without diabetes, this study found analogous associations between increasing body mass index, adipokines, and lipid peroxidation markers (Furukawa et al., 2004). Hence, adipose tissue is recognized as an active endocrine organ, secreting adipokines that are modulated by oxidative stress and affect both brain and behavior. Interventions to reduce obesity can improve cognitive function and mental health. Multiple meta-analytic studies of intentional weight loss by diet, physical activity, or surgical means have found significant improvements in cognitive function, particularly in the domains of executive function and attention (Thiara et al., 2017; Veronese et al., 2017). Dietary interventions can also improve symptoms of depression (Opie, O’neil, Itsiopoulos, & Jacka, 2015), and some evidence suggests that this may occur in part via reductions in oxidative stress (Perez-Cornago et al., 2014). Lean individuals diagnosed with anxious or depressive disorders had inflammatory gene expression levels in visceral adipose tissue that were higher than their counterparts without a mental health disorder (Coín-Aragüez et al., 2018). Unfortunately, this study did not assess genes more directly responsive to hypoxia, such as HIF-1a. Hence, further research is needed to understand when and how adipose-derived factors influence the brain and mental health and the specific role of oxidative stress.

­Eustress, distress, and obesity

­Neuroinflammation in obesity and metabolic disease Obesity is associated with a systemic and local inflammatory state, potentially initiated by oxidative stress (Furukawa et  al., 2004; Sun et  al., 2011). Furthermore, obesity-associated inflammation, or “metabolic inflammation” disrupts glucose homeostasis, leads to insulin resistance, and contributes to hyperlipidemia and hypertension (Purkayastha & Cai, 2013; Van Dijk et al., 2015). Metabolic inflammation can provoke neuroinflammation in the hypothalamus, a master regulator of energy homeostasis. Animal models show that overconsumption (particularly high sugar consumption) (Gao et al., 2017) is the driver of hypothalamic neuroinflammation, which, in turn, dysregulates systemic glucose control (Purkayastha & Cai, 2013). NF-kB lies at the center of this hypothalamic neuroinflammation, thereby suggesting a role for redox signaling (Gao et al., 2017). Inflammation, needed to fight infectious agents or heal wounds, comes at a high metabolic cost. The classic pyrogenic inflammatory cytokine interleukin-1B (IL-1B), secreted during an infection and during psychological distress (Steptoe et al., 2007), plays a major role in initiating a fever and governing symptoms of sickness fatigue that overlap with depression (Roerink, Van Der Schaaf, Dinarello, Knoop, & Van Der Meer, 2017). Fever is metabolically expensive, requiring an additional 7%–13% increase in caloric consumption per degree (°C) (Hotamisligil & Erbay, 2008). Perhaps not coincidentally, depression is associated with both a dysfunction of thermoregulatory cooling mechanisms and proinflammatory cytokine increases (Raison, Hale, Williams, Wager, & Lowry, 2015). Hence, under chronic inflammatory conditions such as obesity and depression, the brain redirects metabolic resources to the immune system, potentially at a cost to other cognitive functions. Finally, in individuals with obesity relative to lean individuals, ingestion of a high-glucose beverage (75 g) and the associated spikes in blood glucose were both associated with greater attentional food bias, greater glucose-induced relief from psychological distress, and increased nonhomeostatic eating of sweets (Mason et al., 2019). In sum, a picture emerges in which overconsumption drives oxidative stress, promoting inflammation in the periphery and in the brain. In turn, metabolic inflammation leads to worsened glucose homeostasis, cardinal features of metabolic syndrome, and behavioral changes resembling depressive or anxious symptoms.

­Clinical implications In sum, overconsumption in the modern age is degrading many facets of health, and oxidative stress may play a role. This holds implications for interventions targeting metabolic pathology, and it may call for a new approach to behavioral interventions. If the cognitive functions and brain regions required for “willpower” or self-­regulation are hijacked by obesity-induced inflammation and oxidative stress, it may not be enough to count calories or macronutrient percentages. We may need to additionally focus on how the food we eat impacts pathways and mechanisms driving oxidative stress and inflammation. The presence of bidirectional brain-adipose pathways leads to the conclusion that a multicomponent approach is needed, which

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addresses both behavior and the physiological milieu in tandem. Neglecting to target inflammatory and oxidative signaling across the brain-adipose axis may overlook a key component of weight-loss intervention approaches. Consequently, individuals may be unable to sustain behavior changes and resultant weight loss. Hence, the future of interventions targeting metabolic dysregulation may soon evolve toward precision medicine interventions that identify relevant phenotypes using both biomarker panels and behavioral assessments and then target these subgroups with integrative interventions.

­Conclusions Psychological distress and oxidative stress are intertwined through multiple bidirectional pathways that connect the brain with the body. Herein, we synthesized several theories of psychological stress and physiological stress arousal in terms of their impact on oxidative eustress or damage. The dose likely determines the poison: the intensity, temporal characteristics, and quality of stressor exposure may determine whether psychological distress leads to oxidative resilience or damage. Clinical diagnoses such as depression and anxiety are, in general, associated with heightened levels of oxidative damage. However, the correlational nature of this research and the inherent heterogeneity of these disorders make it difficult to leverage the findings toward clinical application. Hence, we explored three areas of focus (cellular aging, treatment of depression/anxiety, and obesity) with a greater focus on pathways and mechanisms that may help bridge basic and clinical research in this area. Further research is needed that employs parallel in vivo and in vitro designs targeting stress pathways (Aschbacher et al., 2016), animal-to-human (Aschbacher, Kornfeld, et al., 2014; Furukawa et al., 2004; Kuo et al., 2008) and human-to-animal translation (Picard et al., 2018; Picard & Mcewen, 2018). Finally, the application of cellular stress adaptation models (Pickering et al., 2013) within the context of clinical samples or in the presence of stress-arousal hormones may be crucial for better understanding how psychological factors influence oxidative eustress.

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Reactive oxygen species and cancer

30

Hyewon Konga,b, Navdeep S. Chandela,b a

Department of Medicine, Division of Pulmonary and Critical Care, Northwestern University Feinberg School of Medicine, Chicago, IL, United States, bDepartment of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, Chicago, IL, United States

Abstract Reactive oxygen species (ROS) were once considered only as a toxic by-product of aerobic metabolism. Many studies done in the past several decades, however, revealed that ROS have necessary physiological and pathological functions. In the context of cancer, there has been a persistent interest in whether ROS have a tumor-supportive or a tumor-suppressive role. Hydrogen peroxide (H2O2) conducts signaling pathways essential for the survival, proliferation, and metastasis of cancer cells. H2O2, however, can also induce the production of cytotoxic lipid ROS and trigger cancer cell death, such as ferroptosis. As a result, cancer cells increase not only the rate of H2O2 production to hyperactivate the protumorigenic signaling but also their antioxidant capacity to evade the lipid ROS-induced cell death. This unique reliance of cancer cells on both the pro- and antioxidative capacities may provide opportunities to specifically target them via ROS manipulation. In this chapter, we review the major findings that lead to the current understanding of the redox environment in cancer cells and the strategies of redox therapies against cancer. ­Keywords: Cancer, Signaling, Ferroptosis, Lipids, Mitochondria, Therapy

­Introduction High levels of reactive oxygen species (ROS) can induce oxidative damage to cellular macromolecules, such as DNA. Therefore, it was widely considered that ROS are oncogenic by promoting genomic instability (Ames, Shigenaga, & Hagen, 1993). Indeed, patients with diseases associated with increased rates of oxidative DNA damage, including cystic fibrosis, chronic hepatitis, and Fanconi’s anemia, are exposed to significantly higher risks of cancer (Brown, McBurney, Lunec, & Kelly, 1995; Hagen et  al., 1994; Takeuchi & Morimoto, 1993). The majority of ROSinduced DNA damages involve oxidative modifications of guanine (G) to 8-oxo7-hydrodeoxyguanosine (8-oxodG), which cause G to thymine (T) transversions Oxidative Stress. https://doi.org/10.1016/B978-0-12-818606-0.00030-4 © 2020 Elsevier Inc. All rights reserved.

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(Shibutani, Takeshita, & Grollman, 1991). Such mutagenic capacity of oxidative stress can enhance the frequency of cancer-causing mutations, including activation of oncogenes or loss of tumor suppressor genes (Du, Carmichael, & Phillips, 1994; Higinbotham et al., 1992). Though ROS facilitate cancer initiation and progression in part by acting as a mutagen, studies in the past two decades highlight a role of ROS as signaling molecules that support cancer cell proliferation, survival, and metastasis. Hydrogen peroxide (H2O2) is the most stable and membrane-penetrable form of ROS and thereby has the highest potential as a secondary messenger in cellular signaling (Reczek & Chandel, 2015). Indeed, H2O2 is essential for the sustained activation of some of the most wellestablished protumorigenic signaling cascades, including phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR), mitogenactivated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), and hypoxia-inducible transcription factor (HIF) (Cao et al., 2009; Chandel et al., 1998; Weinberg et al., 2010). Such tumor-supportive functions of ROS have fueled the interest in harnessing antioxidants as therapeutic or prophylactic agents against cancer. Much of the early excitement was sparked in the 1970s when Linus Pauling and Ewan Cameron reported that administration of high dose of antioxidant, vitamin C (10 g/day), in terminal cancer patients significantly prolongs their survival (Cameron & Pauling, 1976). In a chemoprevention trial carried out in Linxian county of China, where the residents have the world’s highest risk of esophageal and gastric cancer, dietary supplementation with a combination of beta-carotene, vitamin E, and selenium significantly lowered the risk of stomach cancer (Blot et al., 1993). However, the excitement about using antioxidant in cancer therapy or prevention was short-lived, as contradictory evidence has emerged: two large-scale clinical trials conducted by Mayo clinic during the 1970s and 1980s failed to demonstrate any anticancer efficacy of vitamin C (Creagan et al., 1979; Moertel et al., 1985). Other antioxidants, including vitamin A, N-acetylcysteine (NAC), beta-carotene, vitamin E, folic acid, and vitamin D, also showed no protective effect against cancer in multiple clinical trials (Fortmann et al., 2013; van Zandwijk, Dalesio, Pastorino, de Vries, & van Tinteren, 2000). Instead, beta-carotene and vitamin E significantly increase risks of lung and prostate cancer (Alpha-Tocopherol, Beta Carotene Cancer Prevention Study Group, 1994; Fortmann et al., 2013; Goodman et al., 2004; Klein et al., 2011). Since then, many studies using animal models have demonstrated that antioxidants rather promote cancer progression. NAC and vitamin E markedly accelerates ­tumor growth in mouse models of Kras- and Braf-induced lung cancer (Sayin et al., 2014). NAC also increases lymph node metastasis of endogenous melanoma in mice (Le Gal et al., 2015). Collectively, as a result, an alternative concept on the role of ROS in cancer has risen—ROS may be tumor suppressive. After several decades of investing many resources into defining whether ROS support versus suppress cancer, there is overwhelming evidence backing either argument. This controversy presents a challenge in comprehending the redox biology of

­Introduction

cancer and importantly accessing ROS in cancer therapy: Shall we attenuate it? Or foster it? In an attempt to reconcile the dispute, we review major findings advocating the tumor-supportive or tumor-suppressive functions of ROS and discuss how the two seemingly opposite observations are indeed compatible in a context of redox homeostasis in cancer cells. We propose that mitochondria and NADPH oxidases (NOXs) generate localized production of H2O2 to activate signaling pathways that promote the development and progression of cancer. However, H2O2 can potentially generate lipid hydroperoxides that induce cell death; thus, cancer cells have high levels of enzymes that specifically decrease the levels of lipid hydroperoxides. This allows cancer cells to have elevated levels of H2O2 to promote tumor growth without succumbing to cell death (Fig. 1).

FIG. 1 The state of redox homeostasis in cancer cells is shown. Common features of tumors, including activation of oncogenes, loss of tumor suppressor genes, and adaptation to tumor microenvironment, increase the rate of hydrogen peroxide (H2O2) production by mitochondria and NADPH oxidase (NOX). The H2O2 then selectively and reversibly oxidizes target enzymes, conducting redox signaling essential for the survival, proliferation, and metastasis of cancer cells. Excess H2O2, however, can undergo Fenton reaction, giving rise to hydroxyl radical (OH•). The OH• can react with unsaturated lipid (LH), initiating lipid peroxidation, which generate cytotoxic lipid hydroperoxide (LOOH). Cancer cells, therefore, must upregulate their antioxidant capacities, including glutathione (GSH) and glutathione peroxidase 4 (GPX4) that neutralize lipid hydroperoxides.

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­H2O2 promotes tumorigenesis H2O2 has been shown to transduce cellular signaling pathways by selectively and reversibly oxidizing key cysteine residues within target proteins. In one of the proposed mechanisms of the redox signaling, H2O2 oxidizes the thiol (SH) group of cysteine residues with low acid dissociation constant (pKa), thereby existing as an oxidationsusceptible form of thiolate (S−) under the physiological pH. The process can modify the S− into sulfenic acid (SOH), disulfide bond (S–S), or sulfonamide (S–N), which would result in changes of the structure and thus the enzymatic activities of the target proteins. The oxidized forms of S− can subsequently be reduced back to its original form by antioxidant enzymes, thioredoxin (TRX) and glutaredoxin (GRX) (Finkel, 2012; Reczek & Chandel, 2015). Importantly, the best-characterized targets of such redox regulation are phosphatases and kinases, which take integral parts in protumorigenic signaling pathways (Paulsen et al., 2011; Sohn & Rudolph, 2003). The earliest molecular/cellular evidence implicating H2O2 in the genesis of cancer is an observation from the early 1990s that cancer cells, compared with nontransformed cells, have elevated levels of intracellular ROS (Szatrowski & Nathan, 1991). Such prooxidative environment in cancer cells was later discovered to be essential for tumorigenesis, as H2O2 activates mitogenic signaling pathways (Bae et al., 1997; Irani et al., 1997; Sundaresan, Yu, Ferrans, Irani, & Finkel, 1995). H2O2 is necessary and sufficient for the sustained stimulations of PI3K/AKT/mTOR and MAPK/ERK signaling in cancer cells (Cao et al., 2009; Weinberg et al., 2010). A likely mechanism is the oxidative inactivation of the negative regulators of those pathways, including phosphatase and tensin homolog (PTEN), protein tyrosine phosphatase (PTP), and MAPK phosphatase (Lee et al., 2002; Salmeen et al., 2003; Seth & Rudolph, 2006). The H2O2-induced blockade of the brakes can drive the prosurvival and proliferative PI3K/AKT/mTOR and MAPK/ERK signaling into hyperactivation. The activation of AKT, interestingly, has been shown to increase intracellular H2O2 production (Behrend, Henderson, & Zwacka, 2003; Los, Maddika, Erb, & Schulze-Osthoff, 2009), suggesting an existence of a positive feedback loop to further potentiate the protumorigenic signaling. Additionally, a recent study revealed that H2O2 facilitates another cancer-causing signaling pathway via nuclear factor κ-light chain enhancer of activated B cells (NF-κB). Elevated production of mitochondrial H2O2 is necessary for activating protein kinase D-1 (PKD1) and NF-κB to amplify the proproliferative epidermal growth factor (EGF) signaling, which leads to the development of pancreatic cancer (Liou et al., 2016). The causative impact of H2O2 on the protumorigenic cellular signaling suggests that cancer cells may be selected for an increased generation of H2O2. Indeed, oncogenic transformation leads to elevated levels of intracellular H2O2. For example, expression of HrasV12 oncogene in NIH 3 T3 mouse fibroblasts increases intracellular contents of superoxide (•O2−), which is rapidly converted to H2O2 by superoxide dismutase (SOD) (Irani et  al., 1997). Similar results were found upon transforming ovarian epithelial cells and mouse hematopoietic cells by ectopic expression of HRasV12 or Bcr-Abl, respectively (Trachootham et al., 2006). Since then, several studies have honed in on the

­H2O2 promotes tumorigenesis

sources of the oncogene-driven H2O2. Expression of various oncogenes, myristoylated Akt, HRasV12, or KRasV12, in mouse embryonic fibroblasts increases the H2O2 level specifically in mitochondria, one of the major sites of intracellular H2O2 generation (Weinberg et al., 2010). Similarly, inducing KRasV12 or KRasD12 expression in mouse acinar cells increases the production of mitochondrial H2O2 (Liou et al., 2016). The HRasV12-mediated transformation was also reported to increase H2O2 production by NOX (Ogrunc et al., 2014). Notably, such oncogene-driven increases in H2O2 were detected despite an absence of elevated mitochondrial oxygen consumption (Liou et al., 2016) or an induction of cell senescence (Ogrunc et  al., 2014). Thus, the increased generation of H2O2 by mitochondria and NOX is not just a by-product of the heightened rates of metabolism and proliferation of cancer cells, but rather attributable to the neoplastic transformation or, specifically, the acquisition of oncogenes. The H2O2 production in cancer cells is also stimulated by various elements in the tumor microenvironment, such as growth factors. Addition of EGF to A431 human epidermoid carcinoma cells results in elevated concentrations of intracellular H2O2 (Bae et al., 1997). Platelet-derived growth factor (PDGF) also increases the levels of H2O2 in vascular smooth muscle cells (Sundaresan et al., 1995). Hypoxia, another critical component of tumor microenvironment, has been shown to accelerate mitochondrial H2O2 production (Chandel et al., 1998). The H2O2-rich environment in cancer cells is further potentiated by suppression of cellular antioxidant responses. Many tumor suppressors support antioxidant pathways in nontransformed cells; hence, their loss during oncogenic transformations can lead to elevation of intracellular H2O2. Breast cancer type 1 susceptibility protein (BRCA1), whose loss strongly associates with the risk of breast and ovarian cancers, was found to be essential for activating nuclear factor (erythroid-derived 2)-like 2 (NRF2), known as the master regulator of antioxidant pathways. BRCA1 interferes with the ubiquitination and degradation of NRF2, allowing for NRF2 to stabilize and translocate into the nucleus. There, NRF2 transcribes genes required for the generation and/or utilization of many cellular antioxidants, including glutathione (GSH), peroxiredoxin, thioredoxin, and nicotinamide adenine dinucleotide phosphate (NADPH). As a result, the BRCA1-mutated cancer cells exhibit defective antioxidant responses, hence an accumulation of intracellular ROS, including H2O2 (Cao et al., 2007; Gorrini et al., 2013). Tumor suppressor p53 is another potential activator of NRF2 (Chen et al., 2009; Toledano, 2009). p53 upregulates an expression of a H2O2 scavenger, glutathione peroxidase 1 (GPX1), and production of NADPH (Bensaad et al., 2006; Hussain et al., 2004). As a result, loss of p53 in cancer cells dampens their antioxidant responses and elevates intracellular H2O2 levels. Surprisingly, ablating p53 functions of cell-cycle arrest, apoptosis, and senescence, while leaving its antioxidant function intact, allows p53 to retain its tumor suppressing capacity (Li et al., 2012). Furthermore, dietary supplementation of an antioxidant, NAC, reduces the occurrence and growth of p53-deficient tumors (Sablina et al., 2005). These data suggest that the antioxidant function of certain tumor suppressors is sufficient for their ability to prevent cancer, and the loss of such tumor suppressor drives tumorigenesis mainly as a result of increasing intracellular H2O2 levels.

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Such establishment of H2O2-rich, prooxidative environment in cancer cells is critical for the genesis of cancer. Elevated intracellular H2O2 upon the loss of an antioxidant enzyme, peroxiredoxin 1 (PRDX1), significantly increases the frequency of oncogenic malignancies in mice, including a variety of lymphomas, sarcomas, and carcinomas (Neumann et al., 2003). On the other hand, decreased intracellular H2O2 due to NRF2 overexpression abrogates the in  vivo tumorigenicity of transformed human mesenchymal stem cells (Funes et al., 2014). Interestingly, targeting just the localized H2O2 pool has been shown to be sufficient in impairing tumorigenesis. Reducing the level of mitochondrial H2O2 in transformed cells with mitochondriatargeted chemical antioxidants ablates the oncogene-induced capacity of anchorage-­ independent growth (Weinberg et  al., 2010). Mitoquinone (MitoQ), one of the mitochondria-targeted antioxidants, decreases Kras-induced pancreatic tumorigenesis in  vivo (Liou et  al., 2016). Furthermore, expression of mitochondria-targeted catalase (mCatalase), which converts mitochondrial H2O2 to H2O, in a mouse model of adenomatous polyposis coli multiple intestinal neoplasia (APC(Min/+)) significantly reduces the development of spontaneous colon cancer. In contrast, when the mitochondrial H2O2 level was increased as a result of a heterozygous mutation in mitochondrial transcription factor A, the formation of spontaneous colon cancer increased (Woo et  al., 2012). Together, these data support that the increased levels of intracellular, especially the mitochondrial, H2O2 is necessary and sufficient for tumorigenesis. H2O2 promotes cancer beyond tumorigenesis by supporting metastasis. Indeed, prooxidative status of head and neck squamous cell carcinomas correlates with the presence of lymph node metastasis in patients (Dequanter, Dok, & Nuyts, 2017). Metastasis is a multistep process involving the invasion and migration of primary tumor cells into surrounding tissues, intravasation and survival of the cells in the circulatory or lymphatic systems, extravasation into distant tissues, and establishment of metastatic colonies (Lambert, Pattabiraman, & Weinberg, 2017). The early metastatic steps of migration and invasion occur through a series of cellular changes, including cytoskeletal remodeling and degradation of extracellular matrix. These events are driven by a complex network of signaling pathways in which H2O2 has been identified as an essential conductor (Tochhawng, Deng, Pervaiz, & Yap, 2013). As noted earlier, H2O2 activates MAPK/ERK pathway by oxidizing and inactivating its negative regulators, PTP and MAPK phosphatase. Downstream of the MAPK/ERK pathway, phosphorylated activator protein 1 (AP-1) facilitates the transcription of matrix metalloproteinases, which degrade the surrounding extracellular matrix to pave the way for migrating cancer cells (Ho, Wu, Chang, & Pan, 2011). Additionally, H2O2 induces PI3K signaling, likely by suppressing PTEN as previously described. The pathway leads to activation of protein tyrosine kinase Src and focal adhesion kinase (FAK) that supports the dynamic cytoskeletal remodeling during cell migration (Basuroy, Dunagan, Sheth, Seth, & Rao, 2010). Interestingly, Src and protein tyrosine kinase 2-β (Pyk2), a member of the FAK family, can be activated by mitochondrial ROS (mROS). As a result, mROS, including mitochondrial H2O2, are necessary for the migration and metastasis of multiple types of cancer cells (Porporato et al., 2014).

­Cancer cells limit damaging lipid hydroperoxide accumulation

H2O2 also promotes tumor metastasis by activating HIF. Under the state of ROS homeostasis, HIF is constantly degraded as prolyl hydroxylase enzyme (PHD) hydroxylates critical proline residues within the HIF-α subunit, allowing the VonHippel-Lindau tumor suppressor protein (VHL) to recognize and ubiquitinate HIF-α for proteosomal degradation (Epstein et al., 2001; Kaelin & Ratcliffe, 2008). Under tumor hypoxia, which increases mROS production, mitochondrial H2O2 oxidizes and inhibits the PHD, stabilizing HIF1-α to translocate into the nucleus where it engages in transcriptional activities (Bell et al., 2007; Chandel et al., 1998). Subsequently, hundreds of HIF-driven genes are expressed to confer complex cellular changes promoting tumor metastasis (Rankin & Giaccia, 2016). In human melanoma cells, for example, the mitochondrial H2O2-driven HIF-1α stabilization activates Met protooncogene that enhances variety of prometastatic phenotypes, such as spreading on extracellular matrix, motility, invasion into 3D matrices, growth of metastatic colonies, and ability to form vasculogenic mimicry (Comito et al., 2011). As a result, the H2O2-HIF pathway facilitates multiple steps of the tumor metastasis process.

­ ancer cells limit damaging lipid hydroperoxide C accumulation Cancer cells undergo a constant prooxidative pressure. During tumorigenesis, as discussed earlier, oncogenic transformations and sustained mitogenic signaling elevate the levels of intracellular H2O2 in nascent cancer cells. Subsequently, highly proliferative tumors can outgrow the rate at which the vasculature expands, rendering regions within the tumor to become hypoxic, thereby potentiating mitochondrial H2O2 generation. Tumor cells proliferating outside their matrix niches or intravasating into the circulatory system detach from extracellular matrix, which further increases the levels of intracellular ROS (Schafer et al., 2009). Moreover, the blood and viscera are highly oxidative environments, which elevate the ROS levels in circulating cancer cells (Piskounova et al., 2015). Such accumulation of intracellular H2O2, which facilitates the prosurvival, proliferation, and metastasis signaling, can paradoxically induce cancer cell death. Excess H2O2, not neutralized by oxidizing signaling or antioxidant enzymes, can undergo Fenton reaction, where H2O2 reacts with ferrous ion (Fe2+), leading to the formation of hydroxyl radical (OH•) (Winterbourn, 1995). OH• is essential for initiating lipid peroxidation process, as it abstracts hydrogen (H) from unsaturated lipid (LH), generating lipid radical (L•). The L• rapidly reacts with oxygen (O2), giving rise to lipid peroxy radical (LOO•), which can subsequently abstract H from another LH to produce lipid hydroperoxide (LOOH) and an additional L• (Gaschler & Stockwell, 2017). Such propagation of lipid radicals and hydroperoxides within cellular membranes can substantially damage the lipid bilayers by altering their physical properties, including fluidity, permeability, and thickness (Borst, Visser, Kouptsova, & Visser, 2000; Heffern et al., 2013; Wong-ekkabut et al., 2007). Lipid hydroperoxides can also degrade into malondialdehyde (MDA) or 4-hydroxynonenal (4-HNE), which

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are highly reactive molecules capable of damaging DNA and proteins (Esterbauer, Schaur, & Zollner, 1991). Furthermore, recent studies have identified lipid peroxidation as a hallmark of ferroptosis, an iron and lipid ROS-dependent mode of regulated cell death (Dixon et al., 2012; Yagoda et al., 2007; Yang et al., 2014). Consequently, to exploit high intracellular H2O2 level while evading its downstream cytotoxic effects, cancer cells must bolster their antioxidant capacities that directly or indirectly limit the accumulation of lipid ROS. NRF2, the master regulator of cellular antioxidant responses, is therefore critical for the development and progression of cancer. Expression of oncogenes, KRasD12, Braf, or c-Myc, elevates NRF2 transcription, leading to increased activation of NRF2-regulated antioxidant pathways. Genetic targeting of NRF2 significantly inhibits the KRasD12-induced pancreatic and lung tumorigenesis in  vivo (DeNicola et al., 2011). Such oncogene-induced activation of NRF2 is also necessary for the drug resistance of cancer cells. Chemical targeting of NRF2 with brusatol enhances the efficacy of a chemotherapeutic agent, cisplatin, thereby synergistically reducing the tumor burden of mice with KRasD12-induced lung tumors (Tao et al., 2014). Importantly, inhibiting NRF2 sensitizes cancer cells to ferroptosis-inducing agents, such as erastin, RSL3, and sorafenib, suggesting that NRF2 is essential to protect cancer cells from the lipid ROS-dependent cell death (Fan et al., 2017; Shin, Kim, Lee, & Roh, 2018; Sun et al., 2016). Furthermore, NRF2 supports the mRNA translation and mitogenic signaling required for the proliferation of KRasD12-driven pancreatic cancer cells (Chio et  al., 2016). Such oncogenic advantages of the NRF2 confer many types of human cancer to adopt variety of mechanisms to hyperactivate NRF2. Besides the o­ ncogene-mediated induction, these means include gain-offunction mutations of NRF2 and loss-of-function mutations of its negative regulator, kelch-like ECH-associated protein 1 (KEAP1) (Menegon, Columbano, & Giordano, 2016). Collectively, these data have spurred investigations on targeting the NRF2dependent cancers. Indeed, a recent chemical proteomics approach identified an NRF2-regulated protein, nuclear receptor subfamily 0 group B member 1 (NROB1), as a druggable target that supports nonsmall cell lung cancers (NSCLC) with aberrant NRF2 activations (Bar-Peled et al., 2017). One of the important downstream effectors of the NRF2-activated antioxidant response is glutathione (GSH). GSH is the most abundant cellular antioxidant molecule, consisting of a glycine, a cysteine, and a glutamate. De novo GSH synthesis is first catalyzed by glutamate cysteine ligase (GCL), which ligates a cysteine with a glutamate to produce γ-glutamyl cysteine. The dipeptide is subsequently combined with a glycine by GSH synthetase (GSS), giving rise to a l- γ-glutamyll-cysteinyl-glycine, or GSH. GSH, once scavenges ROS, forms oxidized glutathione or GSSG. GSSG can be reduced back to GSH by glutathione reductase (GR) and NADPH (Bansal & Simon, 2018). Many key mediators of the GSH synthesis and regeneration, including NADPH, GR, GCL, and a cysteine importer, xCT/ SLC7A11, are regulated by NRF2 (Sasaki et al., 2002; Yates et al., 2009). Cancer cells with NRF2 hyperactivation, therefore, would have elevated levels of GSH. Indeed, NADPH, SLC7A11, and GCL are highly upregulated in human tumors

­Cancer cells limit damaging lipid hydroperoxide accumulation

(Harris et al., 2015; Jiang et al., 2015), and increased GSH levels have been observed in tumor tissues from various origins, such as breast, ovarian, head and neck, and lung (Gamcsik, Kasibhatla, Teeter, & Colvin, 2012). The increased GSH content in cancer cells is necessary for the initiation and progression of cancer. Inhibiting the de novo GSH synthesis pathway by genetically and pharmacologically targeting GCL prevents spontaneous tumorigenesis in a mammary tumor (MMTV-PyMT) mouse model (Harris et al., 2015). Limiting cysteine availability by targeting SLC7A11 or administering cysteinase enzyme suppresses the growth of a variety of carcinomas in vivo (Cramer et al., 2017; Gout, Buckley, Simms, & Bruchovsky, 2001; Guo et al., 2011). Importantly, GSH is required for cancer cells to evade ferroptosis. Impairing de novo GSH synthesis by a SLC7A11 inhibitor, erastin, or a GCL inhibitor, buthionine sulfoximine (BSO), initiates ferroptosis in cancer cells (Yang et  al., 2014). Such depletion of GSH causes loss of cellular antioxidant capacities, including glutathione peroxidases (GPXs). GPXs work in concert with GSH to relay multiple redox reactions, which scavenge H2O2: cysteine or selenocysteine residues within GPXs undergo oxidation in return for reducing H2O2 to H2O. The oxidized but inactivated GPXs can subsequently be reduced back by GSH. Among the isozymes of GPX family, GPX4, intriguingly, has high preference to reduce lipid hydroperoxides (Brigelius-Flohé & Maiorino, 2013). Targeting GPX4 in cancer cells, therefore, induces elevation of intracellular lipid ROS contents and ferroptosis, which can be rescued by a lipophilic antioxidant, vitamin E (Yang et al., 2014). Collectively, the data support that GSH facilitates GPX4, which in turn limits accumulation of lipid ROS, conferring cancer cell resistance to ferroptosis. Indeed, the GSH/GPX4 pathway has been discovered to be required for protecting therapy-resistant cancer cells from ferroptotic cell death (Viswanathan et al., 2017). NADPH is a reducing equivalent essential for the antioxidant capacities of cancer cells. Oxidation of NADPH fuels reduction and regeneration of cellular antioxidants, such as GSH. Therefore, availability of this reducing equivalent can affect the intracellular levels of lipid ROS. Indeed, NADPH abundance has been proposed as a promising biomarker of ferroptosis sensitivity across a vast number of cancer cell lines (Shimada, Hayano, Pagano, & Stockwell, 2016). One of the major sources of cytosolic NADPH is oxidative pentose phosphate pathway (PPP), a metabolic pathway branching from glycolysis. Regulatory enzymes of glycolysis, pyruvate kinase isoform M2 (PKM2) and TP53-induced glycolysis regulatory phosphatase (TIGAR), can increase the flux of glucose-derived carbons into the oxidative PPP. Such NADPH-generating capacities of PKM2 and TIGAR were found to be necessary for lung and intestinal cancer cells to suppress oxidative stress, including lipid peroxidation, and establish tumors in vivo (Anastasiou et al., 2011; Cheung et al., 2013). Furthermore, direct or indirect inhibition of glucose-6-phosphate dehydrogenase (G6PD), an enzyme that catalyzes the rate-limiting step of oxidative PPP, reduces intracellular NADPH, followed by an increase in ROS, leading to defects in survival, proliferation, and invasion of multiple types of cancer cells (Du et al., 2013; Lucarelli et al., 2015; Mele et al., 2018).

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One carbon metabolism, also branching from glycolysis, is another major source of NADPH. A glycolytic intermediate 3-phosphoglycerate is first converted to serine via phosphoglycerate dehydrogenase (PHGDH). The serine subsequently donates one carbon unit to tetrahydrofolate (THF), through a reaction catalyzed by serine hydroxymethyltransferase (SHMT). The product 5,10-methyl-THF is then oxidized by methylene THF dehydrogenase (MTHFD), converting NADP+ to NADPH. Importantly, this pathway produces NADPH not only in the cytosol but also in the mitochondria (Fan et  al., 2014; Lewis et  al., 2014). A recent study revealed that metabolic activities of mitochondria, which result in generation of H2O2, contribute to the accumulation of sufficient lipid ROS to initiate ferroptosis (Gao et  al., 2019). Therefore, one‑carbon metabolism that provides a unique opportunity to support mitochondrial antioxidants may protect cancer cells from lipid peroxidation and cell death. Indeed, MYC-transformed cells subjected to hypoxia, which elevates mitochondrial H2O2 generation, significantly upregulate mitochondrial isoform of SHMT, SHMT2. Inhibition of the SHMT2 impairs cancer cell survival under hypoxia, which can be rescued by an antioxidant, NAC (Ye et al., 2014). Additionally, targeting PHGDH in breast cancer cells was shown to disturb the mitochondrial redox homeostasis, leading to reduced rate of survival under hypoxia (Samanta et al., 2016). The cytosolic NADPH generation by one‑carbon metabolism, nonetheless, is also critical for cancer cell survival. Metastasizing human melanoma cells require cytosolic folate pathway enzymes, such as MTHFD1, to survive the prooxidative environment during circulation (Piskounova et al., 2015).

­Targeting the redox biology for cancer therapy H2O2 promotes the development and progression of cancer by mediating cellular signaling pathways necessary for the survival, proliferation, and metastasis of cancer cells. However, excess H2O2 can initiate lipid peroxidation that produces lipid ROS, which are cytotoxic. Therefore, while maintaining an elevated rate of H2O2 production and the H2O2-mediated protumorigenic signaling, cancer cells bolster their antioxidant capacities to battle accumulation of lipid ROS. A recent study demonstrated that in APC-deficient intestinal cells, Wnt signaling upregulates both Ras-related C3 botulinum toxin substrate 1 (RAC1) and TIGAR. RAC1 promotes generation of H2O2 by NOX, while TIGAR supports the NADPH-mediated antioxidant defense. Simultaneous elimination of both, compared with removing RAC1 or TIGAR alone, surprisingly induces more profound proliferative defects of the APC-null intestinal crypts in  vivo (Cheung et  al., 2016). These data are consistent with the model in which both prooxidative and antioxidative capacities are indispensable for cancer. Based on this current understanding of the redox balance in cancer cells, successful redox therapy must attenuate the signaling H2O2 while fostering the toxic lipid ROS. This reveals a challenge in using dietary antioxidants against cancer, as they have limited access to the protumorigenic signaling H2O2 yet can reduce the antitumorigenic lipid ROS (Chandel & Tuveson, 2014). Therefore, recent efforts of

­Targeting the redox biology for cancer therapy

using antioxidants for cancer therapy have focused on targeted antioxidants, which can specifically remove localized pools of the signaling H2O2. Indeed, mitochondriatargeted antioxidants have been shown to be more effective in inhibiting cancer cell proliferation than the same antioxidants that reside in the cytosol (Weinberg et al., 2010). Additionally, MitoQ and MitoTempo suppress in vivo tumorigenesis and metastasis, respectively (Liou et al., 2016; Porporato et al., 2014). This therapeutic approach is made even more probable by recent developments of precision-targeted antioxidants that scavenge ROS at specific sites of mitochondrial electron transport chain without affecting bioenergetic functions of the mitochondria (Brand et  al., 2016; Orr et al., 2015). Given the reliance of cancer cells on their antioxidant capacities to evade cell death, antioxidant pathways that directly or indirectly limit lipid ROS are attractive targets for cancer therapy. Inhibiting NADPH synthesis by genetically targeting TIGAR has been reported to elevate lipid peroxidation and suppress tumorigenesis in vivo (Cheung et al., 2013). Targeting NRF2 sensitizes various types of cancer cells to chemical agents that induce ferroptosis (Fan et al., 2017; Shin et al., 2018; Sun et al., 2016). Moreover, pharmacological targeting of the GSH/GPX4 pathway induces ferroptotic cell death in cancer cells (Yang et al., 2014). Importantly, this therapeutic approach has displayed selective lethality toward cancer cells. Erastin, which inhibits SLC7A11, induces ferroptosis in transformed cells expressing HrasV12, but not in isogenic cell lines without the oncogenic Hras (Yang et al., 2014). Additionally, SLC7A11-deficient mice are healthy (Sato et al., 2005), supporting that targeting the protein in patients may have minimal to no adverse effect. Furthermore, inhibition of GPX4 has been identified to selectively induce ferroptosis in therapy-resistant cancer cells (Hangauer et al., 2017). Collectively, the data encourage development of molecules that potentiate accumulation of the cytotoxic lipid ROS in cancer patients. Interestingly, another therapeutic strategy to amplify the antitumorigenic ROS in cancer cells is to use vitamin C, due to its newly exposed role as a prooxidant. Oxidized form of vitamin C, dehydroascorbate (DHA), enters cells through a glucose transporter, GLUT1. The intracellular DHA is then reduced back to vitamin C by GSH. This process depletes the GSH pool, resulting in elevated oxidative stress and perhaps a lipid ROS accumulation. To switch on this prooxidative mode of vitamin C, it is critical to build high, millimolar concentrations of vitamin C in the blood plasma, which is achieved only by intravenous administration and not via oral administration. Additionally, the intravenous route exposes vitamin C to the oxidizing environment of circulatory system, possibly facilitating oxidation of vitamin C to its therapeutically active form of DHA. As a result, intraperitoneal injections of high dose of vitamin C in mice were found to be effective in diminishing the growth of colon cancer (Yun et al., 2015). Moreover, in a recent phase I/IIa clinical trial, intravenous administration of vitamin C, in combination with a conventional regimen of paclitaxel and carboplatin, showed a therapeutic benefit among a small group of ovarian cancer patients, without significant toxicity (Ma et al., 2014). The data provide a strong justification to conduct larger clinical trials testing efficacy of the high-dose intravenous vitamin C as a single or combinatory agent against various types of cancer.

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­Conclusion Cancer redox therapy has long been under the spotlight of oncology. Historically, the data just appeared convoluted: Sometimes antioxidants suppress cancer; sometimes, they promote it. Based on the current understanding of the redox biology of cancer cells, the paradox has most likely stemmed from the qualitative differences between the tumor-supportive and tumor-suppressive ROS, which coexist within a cancer cell. Therefore, now an intriguing task remains: how to target the tumor-supportive ROS while fostering the tumor-suppressive ROS. Answers to this question will provide significant medical advances that reduce cancer incidence and improve therapeutic outcomes.

­Acknowledgments This work was supported by National Institute of Health grants 5P01HL071643 and 5P01AG049665 to N.S.C. H.K. is supported by National Institute of Health predoctoral training grant T32CA9560.

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Perspectives of TrxR1-based cancer therapies

31 Elias S.J. Arnér

Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden

Abstract The predominantly cytosolic selenoprotein thioredoxin reductase 1 (TrxR1, encoded in human by the TXNRD1 gene) is a key enzyme for protection against oxidative stress and support of many reductive pathways in cells. For several decades, the enzyme has also been studied as a potential drug target for cancer therapy. The notion that TrxR1 may represent a promising target for anticancer therapy was further strengthened in recent years, with several clinical trials using inhibitors of the enzyme currently ongoing and with many novel experimental inhibitors being developed. This chapter will discuss the underlying biochemistry, physiological roles, and functional pathways of TrxR1 that can provide a rationale for targeting this enzyme for cancer therapy. ­Keywords: Selenoprotein, Selenocysteine, Thioredoxin, Thioredoxin reductase, Cancer therapy, Signaling, Antioxidant defense

­Introduction The predominantly cytosolic selenoprotein thioredoxin reductase 1 (TrxR1, encoded in human by the TXNRD1 gene) is a key enzyme for protection against oxidative stress and support of many reductive pathways in cells. For several decades, the enzyme has also been studied as a potential drug target for cancer therapy. The notion that TrxR1 may represent a promising target for anticancer therapy was further strengthened in recent years, with several clinical trials using inhibitors of the enzyme currently ongoing and with many novel experimental inhibitors being developed. This chapter will discuss the underlying biochemistry, physiological roles, and functional pathways of TrxR1 that can provide a rationale for targeting this enzyme for cancer therapy.

Oxidative Stress. https://doi.org/10.1016/B978-0-12-818606-0.00031-6 © 2020 Elsevier Inc. All rights reserved.

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­TrxR1 in relation to other cellular enzymatic reducing systems The representative function of TrxR1 is to reduce the active site disulfide of thioredoxin (Trx1) to a dithiol motif using NADPH, thereby keeping Trx1 in its active form (Holmgren, 1977). Trx1, in turn, has a wide range of functions in cells with both support of several reductive enzyme pathways and exerting redox control in a number of signaling pathways in cells. TrxR1 furthermore has several substrates in addition to Trx1, thus promoting a wide array of functions and reductive pathways. The details of these pathways have been thoroughly reviewed in a number of comprehensive reviews to where the interested reader is referred (Arner, 2009; Arner & Holmgren, 2000; Cebula, Schmidt, & Arner, 2015; Gromer, Urig, & Becker, 2004; Lu & Holmgren, 2014; Miller, Holmgren, Arner, & Schmidt, 2018; Nordberg & Arner, 2001; Ren et al., 2017; Rundlöf & Arnér, 2004; Urig & Becker, 2006). Here, we shall solely conclude that TrxR1 promotes a wide range of functions in cells, by the direct reduction of several substrates of the enzyme and subsequently by the concerted actions of substrates and target molecules of those substrates, as schematically summarized in Fig. 1, with the illustrated substrates also listed in Table 1 for additional information and references to further literature. When considering TrxR1 as a target for cancer therapy, it is first crucial to understand the functional roles of the enzyme in cells and how those relate to

FIG. 1 Cellular and physiological functions of TrxR1. This figure schematically illustrates that TrxR1 using NADPH (blue crescent) reduces a number of cellular substrates (green crescent) that subsequently act on several downstream targets (orange crescent), the outcome of which in a cell- and context-dependent manner will determine the cellular and physiological functions of TrxR1.

Table 1  Examples of substrates of TrxR1 and their cellular target molecules. TrxR1 substrate (for proteins are also given abbreviations and GENE) Thioredoxin (Trx1, TXN1)

Target molecules

Cellular functions

References

Ribonucleotide reductase (RNR) Apoptosis signal regulating kinase 1 (ASK1) Phosphotyrosine phosphatase 1B (PTP1B, PTPN1)

Support of synthesis of dNTPs for DNA replication Suppression of apoptotic signaling and control of the immune system Inhibition of protein phosphorylation cascades triggered by growth factor stimulation Antioxidant defense, regulation of redox signaling pathways, chaperone functions

Zahedi Avval and Holmgren (2009), Nordlund and Reichard (2006) Matsuzawa and Ichijo (2008)

Peroxiredoxins (Prx’s, PRDX's)

Sulfiredoxin (Srx, SRXN1) Glutathione disulfide (GSSG) Methionine sulfoxide reductases (MsRs)

Repair of oxidized methionine residues Control of nitrosylation events and signaling through nitric oxide (NO)

Latimer and Veal (2016), Winterbourn and Hampton (2015), Sobotta et al. (2015), Sies (2014), Du, Zhang, Zhang, Lu, and Holmgren (2013), Rhee, Chae, and Kim (2005), Immenschuh and Baumgart-Vogt (2005) Kil et al. (2012), Noh, Baek, Jeong, Rhee, and Chang (2009) Gromer, Merkle, Schirmer, and Becker (2002)

Continued

641

Lee et al. (2013), Fomenko et al. (2009), Oien and Moskovitz (2008), Kim and Gladyshev (2007), Moskovitz et al. (2001) Wolhuter et al. (2018), Wolhuter and Eaton (2017), Engelman, Ziv, Arner, and Benhar (2016), Benhar (2016), Pader et al. (2014), Sengupta and Holmgren (2013), Engelman et al. (2013), Benhar, Forrester, and Stamler (2009), Hashemy and Holmgren (2008), Benhar, Forrester, Hess, and Stamler (2008)

­Introduction

Nitrosylated Cys residues (−SNO moieties)

Repair of sulfinic acid forms of Prx’s, control of circadian rhythms Reduction of GSSG

Dagnell et al. (2017), Schwertassek et al. (2014), Parsons and Gates (2013), Dagnell et al. (2013)

642

TrxR1 substrate (for proteins are also given abbreviations and GENE)

Thioredoxin-related protein of 14 kDa (TRP14), also called thioredoxin domaincontaining protein 17 (TXNDC17)

Target molecules

Cellular functions

References

Persulfidated Cys residues (Prot-SSH)

Control of persulfidation events and signaling through hydrogen sulfide (H2S) Control of nitrosylation events and signaling through nitric oxide (NO)

Alvarez et al. (2017), Wedmann et al. (2016), Doka et al. (2016), Nagy (2015)

Control of persulfidation events and signaling through hydrogen sulfide (H2S) Reduction of L-cystine, possibly important in relation to cellular cystine uptake Control of NFkappaB signaling

Doka et al. (2016)

Nitrosylated Cys residues (−SNO moieties)

Persulfidated Cys residues (Prot-SSH) L-cystine

Dynein light chain 8 (LC8, DYNLL) Glutaredoxin 2 (Grx2, GXN2)

Low-molecular-weight compounds (LMW’s)

Thioredoxin 1 (TXN1) Glutathionylated protein cysteine residues (Prot-SSG) Ribonucleotide reductase (RNR) Quinone compounds, lipoic acid, ascorbate, and many more

Reducing Trx1 upon loss of TrxR1 activities Control of glutathionylation events Support of synthesis of dNTPs for DNA replication Possible importance in control of redox active metabolite status and redox cycling events

Pader et al. (2014)

Pader et al. (2014)

Jeong, Jung, Kim, Park, and Rhee (2009), Jung, Kim, Min, Rhee, and Jeong (2008), Jeong, Chang, Boja, Fales, and Rhee (2004) Du et al. (2013) Hudemann et al. (2009), Fernando, Lechner, Lofgren, Gladyshev, and Lou (2006), Fernandes and Holmgren (2004) Zahedi Avval and Holmgren (2009) Arner (2009), Xu, Cheng, and Arnér (2016), Cenas, Prast, Nivinskas, Sarlauskas, and Arnér (2006), Cenas et al. (2004), May, Mendiratta, Hill, and Burk (1997), Arner, Nordberg, and Holmgren (1996)

CHAPTER 31  Perspectives of TrxR1-based cancer therapies

Table 1  Examples of substrates of TrxR1 and their cellular target molecules.—cont'd

­Introduction

other functionally overlapping enzyme systems. Virtually all cells express different enzymes that use NADPH, derived mainly from glucose entering the pentose phosphate shunt, as a principal source of reducing equivalents to support NADPHdependent oxidoreductases that act on disulfide substrates. Major enzymes of this class found in mammalian cells include three isoenzymes of thioredoxin reductase together with glutathione reductase, as listed in Table 2 together with references for further information. TrxR1 is mainly cytosolic, while TrxR2 is mitochondrial, both being found in most if not all cells, while TGR is a specialized enzyme Table 2  The major NADPH-dependent reductases in mammalian cells. Reductase (abbreviation, GENE)

Major functions

TrxR1 (TXNRD1)

Reduction of Trx1, TRP14, several other proteins, and lowmolecularweight compounds (see Table 1)

TrxR2 (TXNRD2)

Reduction of Trx2 and likely also several other substrates

TGR (TXNRD3)

Unclear

GR (GSR)

Reduction of GSSG

Localization

References

Mainly cytosol but also nucleus in most cell types; some TrxR1 enzyme variants found in the intermembrane space of mitochondria, at other cell membranes as well as extracellularly Mitochondrial matrix of most cell types

Cebula, Moolla, Capovilla, and Arner (2013), Damdimopoulou, MirandaVizuete, Arner, Gustafsson, and Damdimopoulos (2009), Dammeyer et al. (2007), Arner (2009), Sun et al. (2001), Sun et al. (2014), Zhang et al. (2016), Damdimopoulos, Miranda-Vizuete, Treuter, Gustafsson, and Spyrou (2004), Inarrea et al. (2007) Rigobello, Folda, Baldoin, Scutari, and Bindoli (2005), Rabilloud et al. (2001), Rigobello, Callegaro, Barzon, Benetti, and Bindoli (1998), Pickering, Lehr, Gendron, Pletcher, and Miller (2017), Hellfritsch et al. (2015), Prasad et al. (2014), Arner (2009), Biterova, Turanov, Gladyshev, and Barycki (2005), Patenaude, Ven Murthy, and Mirault (2004), Miranda-Vizuete and Spyrou (2002), Sun, Zappacosta, et al. (2001) Su et al. (2005), Sun, Kirnarsky, Sherman, and Gladyshev (2001)

Cytosol and nucleus, mostly in spermatids of the testis Cytosol and mitochondrial matrix of most cell types

Miller et al. (2018), Deponte (2013), Rogers, Bates, Welty, and Smith (2006)

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CHAPTER 31  Perspectives of TrxR1-based cancer therapies

p­ redominantly found in testis, and GR is channeled to both cytosol and mitochondria of most if not all cells. This expression pattern may at first resemble a simple “division of labor” for the NADPH-dependent reduction of disulfide substrates in cells. However, the reductive pathways are rather complex and, importantly, functionally overlapping with each other. As an example illustrating this complexity, we shall here solely consider how the classical reaction catalyzed by TrxR1, namely, the reduction of the active site disulfide of thioredoxin 1 (Trx1) using NADPH (Holmgren, 1985), relates to the activities of the other enzymes listed in Table 2. It was early recognized that a complete genetic knockout of the gene for either Trx1 or TrxR1 in mice yields early embryonic lethality (Bondareva et al., 2007; Jakupoglu et  al., 2005; Matsui et  al., 1996), but later studies using conditional knockout constructs revealed a surprising resilience of adult cells and tissues in mice to the lack of TrxR1. Conditional knockout of TrxR1 in the liver (Iverson et al., 2013; Prigge et al., 2012; Rollins et al., 2010), heart (Jakupoglu et al., 2005), or the nervous system (Soerensen et al., 2008) does not, in contrast to the embryonic lethality of the full knockout, lead to a complete lack of cells in these tissues, but it triggers some abnormal phenotypes as further discussed in the succeeding text. Interestingly, the sole knockout of TrxR1, either completely in the developing embryos or conditionally in adult cells or tissues, does also not necessarily give any overt signs of excessive oxidative stress (Iverson et  al., 2013; Prigge et  al., 2012; Rollins et al., 2010; Soerensen et al., 2008). Furthermore, often, there are no direct signs of Trx1 oxidation in cells lacking TrxR1 unless subjected to additional oxidative stress (Peng et al., 2016). One important explanation for the lack of Trx1 oxidation in the absence of TrxR1 is that Trx1 can also be kept in its reduced form by the glutathione system through the actions of glutaredoxins directly reducing the Trx1 active site (Du et al., 2013). These findings show that TrxR1 is not the only enzyme that keeps Trx1 reduced and, moreover, that the glutathione system can help to support Trx1 functions. Conversely, Trx1 can efficiently reduce GSSG and thus at least theoretically complement the activities of GR as longs as TrxR1 is active (Gromer et al., 2002). Interestingly, if both the GR and TrxR1 enzymes are knocked out, at least in mouse liver, then all the vital reductive pathways of the cells are still maintained through the use of newly synthesized glutathione, which can be derived from methionine using pathways not requiring NADPH (Eriksson, Prigge, Talago, Arnér, & Schmidt, 2015; Miller et al., 2018; Prigge et al., 2017). These observations collectively show that inhibition or deletion of TrxR1 is not necessarily related to incapacitation of the complete Trx1-dependent enzyme systems or even leading to oxidative stress, at least under normal nonstressed conditions. This is a most important point when considering TrxR1 as a potential drug target for cancer therapy. In other words, drug targeting of TrxR1 will trigger celland context-dependent effects rather than a full incapacitation of reductive pathways in all cells. Before discussing the effects of targeting this enzyme in further detail, we shall next briefly review the cellular and physiological functions that are supported by TrxR1.

­Introduction

­Cellular and physiological functions of TrxR1 It should be safe to assume that the many cellular and physiological functions of TrxR1 de facto are orchestrated through the functions, properties, and downstream pathways that are in turn modulated by the direct substrates of TrxR1 (Table  1). Examples of this type of multilevel functionality includes the synthesis of deoxyribonucleotides by RNR as supported by Trx1 (Nordlund & Reichard, 2006; Prigge et  al., 2012; Rollins et  al., 2010; Zahedi Avval & Holmgren, 2009), antioxidant defense or intracellular signaling through peroxiredoxins or methionine sulfoxide reductases, as supported by Trx1 (Fomenko et al., 2009; Immenschuh & Baumgart-Vogt, 2005; Lee et al., 2013; Lu & Holmgren, 2014; Oien & Moskovitz, 2008; Rhee et al., 2005; Sies, 2014; Sobotta et al., 2015; Winterbourn & Hampton, 2015), or control of PTP1B signaling through Trx1 or TRP14 (Dagnell et al., 2013; Schwertassek et al., 2014). Some additional direct substrates of TrxR1 may perhaps also modulate cellular physiology, including low-molecular-weight substrates of the enzyme such as dehydroascorbate (May et al., 1997) or lipoic acid (Arner et al., 1996) and the sulfenic acid form of PTP1B (Dagnell et al., 2017). TrxR1 is also, directly or indirectly, linked to modulation of the persulfidation status of proteins (Doka et al., 2016; Wedmann et al., 2016), nitrosylation pathways (Benhar, 2015; Benhar et  al., 2009; Engelman et  al., 2013; Engelman et  al., 2016; Hashemy & Holmgren, 2008; Pader et al., 2014; Sengupta & Holmgren, 2013), glutaredoxin 2 functions (Fernando et al., 2006; Hudemann et al., 2009; Zahedi Avval & Holmgren, 2009), or modulation of transcription factors such as Nrf2, NFκB, or HIF (BrigeliusFlohe & Flohe, 2011; Cebula et al., 2015; Hou et al., 2018; Johansson et al., 2017; Kipp, Deubel, Arner, & Johansson, 2017). It should also be emphasized that several reductive pathways in mammals, at least if they are essential for survival, are redundant and can be supported by either the thioredoxin system or by the glutathione system, with major cross talk between the two (Arner, 2009; Du et al., 2013; Eriksson et al., 2015; Prigge et al., 2012; Prigge et al., 2017; Rollins et al., 2010; Zahedi Avval & Holmgren, 2009). The physiological functions of TrxR1, as determined and orchestrated through its many downstream targets (Table 1 and Fig. 1), are thereby complex and fully context dependent. Before discussing the concepts of targeting TrxR1 for anticancer therapy, we shall briefly review different important observations together illustrating how the activities of TrxR1 can be linked to health and disease.

­TrxR1 in health and disease With TrxR1 being an essential enzyme for embryonic development and being tightly linked to a wide range of reductive and redox regulatory pathways, as discussed earlier, it is perhaps not surprising that the status of TrxR1 can also be linked to normal physiology as well as pathophysiological processes. The links between TrxR1 status and health or disease are however complex and can even at first appear paradoxical, such as yielding stronger antioxidant defense systems at certain occasions of lower TrxR1 activities (Cebula et al., 2015; Iverson et al., 2013; Lei et al., 2015). Here, we shall briefly

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CHAPTER 31  Perspectives of TrxR1-based cancer therapies

Increased

Potentially health promoting effects

•

Protection against oxidative damage in healthy cells and tissues

•

Support of normal cellular differentiation and proliferation

•

Redox control of physiological cell signaling pathways

Potentially disease promoting effects

•

Support of cancer progression

•

Distorted embryogenesis

•

Increased propensity for epileptic seizures?

TrxR1

TrxR1 Activity

Decreased

646

•

Strong activation of cell- and tissue-protective Nrf2-driven pathways

FIG. 2 TrxR1 in health and disease. Schematic representation of either how high (top boxes) or low (bottom boxes) activities of TrxR1 can be beneficial for health (green) or promote disease (red) depending upon context, as further discussed in the text.

summarize these different effects of modulated TrxR1 activities on the physiology or pathophysiology of mice or men, as also schematically summarized in Fig. 2.

­Potentially health-promoting effects of high TrxR1 activities

It seems plausible to assume that high TrxR1 activities would be health promoting, provided that the downstream effects of TrxR1-dependent enzyme systems would promote health. This may include health-promoting effects of several of the physiological functions discussed earlier and listed in Table 1 and Fig. 1, such as antioxidant defense, support of deoxyribonucleotide synthesis, or normal control of diverse signaling pathways. There are, however, no direct studies that this author is aware of that have yet conclusively linked a higher TrxR1 activity with increased health or longer healthy life span. It is possible that higher TrxR1 activity could improve the immune response against viral or parasite infections, as the enzyme seems to be required for normal T cell responses to such infections (Muri et al., 2018), or that higher TrxR1 activities may counteract obesity-related disorders, since the enzyme may counteract adipogenesis and is inversely correlated with insulin sensitivity in a human cohort (Peng et al., 2016). In spite of more definite data on the possible links between high TrxR1 activities and increased health, such possible relations should be cautiously considered.

­The intricate roles of TrxR1 in cancer

­Potentially health-promoting effects of low TrxR1 activities

Perhaps counter intuitively, there are rather strong pieces of evidence suggesting that a lowered TrxR1 activity may be linked to increased health or rather to an increased health at instances of challenges with oxidative stress. The molecular mechanisms behind these observations are likely to be involving the strong activation of the Nrf2 transcription factor often seen upon loss or inhibition of TrxR1, as recently discussed elsewhere in further detail (Cebula et  al., 2015). Indeed, upon conditional genetic deletion of TrxR1 in hepatocytes of mice, they survive otherwise lethal doses of acetaminophen (paracetamol). This is due to a combination of a “priming” with activated Nrf2-driven antioxidant and xenobiotic metabolizing defense systems, as well as the fact that TrxR1 in itself is a target for NAPQI that is the hepatotoxic metabolite of acetaminophen (Iverson et al., 2013; Jan et al., 2014; Patterson et al., 2013). TrxR1 can also be deliberately targeted by a large number of electrophilic compounds that will induce a strong, protective, Nrf2 activation (Cebula et al., 2015). This was, for example, shown to yield good protection against hyperoxic lung injuries in mice subsequent to inhibition of TrxR1 (Britt Jr., Velten, Locy, Rogers, & Tipple, 2014; Li et al., 2016).

­Potentially disease promoting effects of high TrxR1 activities

It is plausible that increased TrxR1 activities can promote cancer progression, which is the underlying principle for targeting of TrxR1 as an anticancer therapy. The possible roles of TrxR1 in cancer are, however, still likely to be complex, with possible impacts that differ during the different stages of cancer development, from initiation to progression and possible formation of metastases. It is also likely that different subtypes of cancer may be affected differently by TrxR1 targeting. As this is the main topic of this chapter, these different aspects will be discussed in the succeeding text in further detail.

­Potentially disease promoting effects of low TrxR1 activities

With the full Txnrd1 knockout mice dying at early embryogenesis (see previous text), this infers that drugs targeting TrxR1 may potentially be teratogenic. The embryonic lethality of Txnrd1 knockout is likely linked to functional roles of TrxR1 in control of cell differentiation patterning, as was recently reviewed (Dagnell, Schmidt, & Arner, 2018). It was also found that an SNP in the human TXNRD1 gene resulting in a Pro to Leu substitution in TrxR1 that yields lower activity of the enzyme can give rise to epileptic seizures (Kudin et al., 2017), which may possibly be considered as a caution if using drugs inhibiting TrxR1 that cross the blood-brain barrier.

­The intricate roles of TrxR1 in cancer There is good evidence, as mentioned earlier, that TrxR1 can promote cancer progression, but the functional links between TrxR1 and cancer with its impact in each cancer stage are nonetheless complex. Before discussing how TrxR1 can be targeted

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with inhibitors and what the effects may be, we shall therefore first briefly review the different possible roles of TrxR1 in cancer development and progression.

­Genetic links of TrxR1 to cancer Several genetic analyses have linked TrxR1 with cancer. This includes the observations that high TrxR1 expression (TXNRD1 transcription) associates with poor outcome in such parameters as overall survival, distant metastasis-free survival and disease-free survival in breast cancer patients (Bhatia et al., 2016), and worse prognosis in pancreatic, lung, head and neck, prostate, and colon cancer (Leone, Roca, Ciardiello, Costantini, & Budillon, 2017). It is also an unfavorable factor for patients with hepatocellular carcinoma (Fu et al., 2017). Recent findings suggest that parts of the aberrant expression of TrxR1 in cancer may be related to cancer-specific effects on certain regulatory miRNAs (Degli Esposti et al., 2017; Hao et al., 2017). In addition, it is well known that strong activation of Nrf2, through several different genetic mechanisms, occurs in many cancer forms and contributes to cancer-specific metabolic reprogramming and progression (Chio et al., 2016; Ganan-Gomez, Wei, Yang, Boyano-Adanez, & Garcia-Manero, 2013; Mitsuishi et al., 2012; Mitsuishi, Motohashi, & Yamamoto, 2012; Probst, McCauley, Trevino, Wigley, & Ferguson, 2015), with TXNRD1 being an important Nrf2 target gene that hence becomes overexpressed when Nrf2 is activated (Cebula et al., 2015; Higgins & Hayes, 2011; Surh, Kundu, & Na, 2008). It was also found that some SNPs in the TXNRD1 gene, together with SNPs in other genes and lifestyle factors, correlate with the propensity of developing colorectal cancer (Peters et al., 2008; Slattery, Lundgreen, Welbourn, Corcoran, & Wolff, 2012). Together, these observations suggest that several genetic mechanisms can contribute to increased expression levels or activities of TrxR1 in cancer and that such increased activity seems to correlate with worse overall prognosis and survival.

­Protection against carcinogenesis by TrxR1? Although high TrxR1 expression seems to promote cancer development and relate to worse prognosis in cancer patients, it may still be that TrxR1 expression in normal, noncancerous cells can protect against the initiation stage of carcinogenesis. With TrxR1 being important in support of some of the key cellular antioxidant defense systems, it is likely that this activity may protect normal cells from oxidative damage and thus protect cells from mutations and as such counteract cancer initiation. This would agree well with findings showing that the expression of peroxiredoxins (Egler et  al., 2005; Guo et  al., 2018; Hampton, Vick, Skoko, & Neumann, 2018; Rolfs et al., 2013) and Nrf2 (Satoh, Moriguchi, Takai, Ebina, & Yamamoto, 2013; Tao, Rojo De La Vega, Chapman, Ooi, & Zhang, 2018) in mice protects them from cancer initiation events, and the differences between initiation and progression of cancer with Nrf2 and its selenoprotein antioxidant enzyme targets GPx2 and TrxR1 have also been discussed previously (Brigelius-Flohe, Muller, Lippmann, & Kipp,

­TrxR1 as a selenoprotein oxidoreductase and the effects of its inhibition

2012). Although it is plausible, there is however still a lack of experimental studies that have specifically addressed the assumed protection against carcinogenesis exerted by TrxR1.

­Promotion of cancer progression by TrxR1 It was early found that a wide range of primary human tumors, including breast cancer, thyroid, prostate, colorectal carcinoma, and malignant melanoma, display increased expression of TrxR1 (as well as Trx1) (Berggren et al., 1996; Lincoln, Ali Emadi, Tonissen, & Clarke, 2003). Using a mouse model of lung carcinoma, it was shown that knockdown of TrxR1 hinders tumor development and establishment of metastases (Yoo, Xu, Carlson, Gladyshev, & Hatfield, 2006), and with high TrxR1 expression in tumors correlating with poor prognosis in human (Bhatia et al., 2016; Cadenas et al., 2010; Fu et al., 2017; Leone et al., 2017), these observations underpin the rationale of targeting the enzyme as a principle for cancer therapy. To understand the potential mechanisms and outcomes of TrxR1 drug targeting, it is essential to understand the rather unique properties of this selenoprotein and the effects in cells that may be triggered upon its inhibition. This shall be discussed next.

T­ rxR1 as a selenoprotein oxidoreductase and the effects of its inhibition TrxR1 is a selenoprotein that in its functions as a flavoprotein oxidoreductase utilizes NADPH for reduction of any of its substrates, such as the active site disulfide of Trx1, with a selenocysteine (Sec) moiety in its C-terminal-Gly-Cys-Sec-Gly tetrapeptide motif being the genuine active site residue (Gladyshev, Jeang, & Stadtman, 1996; Lee et  al., 2000; Zhong, Arner, & Holmgren, 2000; Zhong, Arnér, Ljung, Åslund, & Holmgren, 1998; Zhong & Holmgren, 2000). Many in-depth studies of its catalytic mechanism have been conducted (Cheng et al., 2010; Eckenroth, Lacey, Lothrop, Harris, & Hondal, 2007; Eckenroth, Rould, Hondal, & Everse, 2007; FritzWolf, Urig, & Becker, 2007; Johansson, Arscott, Ballou, Williams Jr., & Arner, 2006; Lothrop, Ruggles, & Hondal, 2009; Lothrop, Snider, Ruggles, & Hondal, 2014; Urig, Lieske, Fritz-Wolf, Irmler, & Becker, 2006; Xu et al., 2015; Xu et al., 2016), and several crystal structures were determined (Biterova et al., 2005; Cheng, Sandalova, Lindqvist, & Arnér, 2009; Fritz-Wolf et  al., 2007; Sandalova, Zhong, Lindqvist, Holmgren, & Schneider, 2001; Xu et  al., 2015), but a thorough review of these features lies outside of this book chapter. Here, we shall instead discuss the possible impact of TrxR1, either in its native form, inhibited or when lacking from cells, on cellular H2O2 levels. The conclusions of this discussion are schematically summarized in Fig. 3. As thoroughly discussed earlier, it should be clear that the physiological activities of noninhibited TrxR1, through the complete thioredoxin system (Table 1 and Fig. 1), should mainly act to lower cellular H2O2 levels by means of the reductive

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CHAPTER 31  Perspectives of TrxR1-based cancer therapies

FIG. 3 Effects of targeting TrxR1 on cellular H2O2 levels. This diagram summarizes how cellular steady-state H2O2 levels (green) can be modulated by normal functions of wild-type TrxR1 (blue), inhibition of TrxR1 using several compounds targeting the Sec residue of the enzyme (red), or loss or knockdown of the enzyme (gray, dashed). The interrelations with Nrf2 activities are also indicated (orange). The inhibited forms of TrxR1 that gain NADPH oxidase activities are also called selenium compromised thioredoxin reductase-derived apoptotic proteins (SecTRAPs) and may also redox cycle with quinone compounds (“Q”). (See text for further details.)

antioxidant pathways that are dependent upon this enzyme (Fig. 3, blue pathways). Inhibition of the enzyme is not necessarily resulting in merely a loss or lower activities of such antioxidant pathways. Instead, if inhibitors covalently target the ­solvent-exposed Sec residue of TrxR1, which easily occurs with many electrophilic alkylating or arylating inhibitors such as many Michael acceptors, then enzyme activity is not completely lost, but instead, the enzyme is typically converted into a prooxidant NADPH oxidase. This activity involves the FAD and two Cys residues in the N-terminal domain of the inhibited enzyme and is further facilitated by oneelectron redox cycling compounds such as certain quinone compounds including juglone (Cheng et al., 2010; Eriksson, Prast-Nielsen, Flaberg, Szekely, & Arner, 2009; Salmon-Chemin et al., 2001; Xu et al., 2015; Xu et al., 2016; Xu & Arner, 2012). It was early observed that such prooxidant forms of the enzyme can become toxic to cells by a gain of function, which was also seen with two amino acid truncated enzyme species that lacked the Sec residue, and these TrxR1 derivatives were named SecTRAPs (Anestal, Prast-Nielsen, Cenas, & Arner, 2008; Arner, 2006; Arner, 2009; Cassidy et al., 2006; Cebula et al., 2015; Cheng et al., 2010; Eriksson et al., 2009; Moos, Edes, Cassidy, Massuda, & Fitzpatrick, 2003; Xu et al., 2015; Xu et al., 2016)

­Drugs targeting TrxR1 for use in cancer therapy

(Fig. 3, red pathways). Formation of SecTRAPs and their detrimental effects on cells can be especially pronounced in cancer cells, due to a combination of high TrxR1 levels in cancer cells and their endogenously higher steady-state levels of H2O2 as a result of their abnormal replicative drive and distorted metabolism (Arner, 2009; Trachootham et al., 2006; Trachootham, Alexandre, & Huang, 2009; Watson, 2013). Importantly, the loss of TrxR1 by genetic means will thus obliterate the possibility of producing SecTRAPs, but such loss can nonetheless have major effects for cells due to the lack of TrxR1 support of the many reductive pathways discussed earlier (Fig. 3, gray and dashed pathways). Finally, it must be considered that antioxidant and otherwise protective Nrf2-driven pathways also counteract H2O2 accumulation, which is an activity supported also in a TrxR1-independent manner through the GSH-dependent reductive pathways. Moreover, TrxR1 and Trx1 are Nrf2 target genes, and, as already discussed earlier, Nrf2 is strongly activated upon a loss of TrxR1 activity (Fig. 3, orange pathways). The impact of TrxR1 targeting on cellular H2O2 levels therefore results from the combined effects of all the activities illustrated in Fig. 3, which will likely yield both cell- and context-dependent final outcomes. Nonetheless, it seems as if the net result of TrxR1 targeting is more detrimental against cancer cells and tumors than it is against normal cells and tissues, which is the basis for TrxR1 targeting as an anticancer principle.

­Drugs targeting TrxR1 for use in cancer therapy It has been recognized for at least 20 years that TrxR1 can be targeted by a number of drugs or compounds that also have anticancer properties (Arnér & Holmgren, 2006; Becker, Herold-Mende, Park, Lowe, & Schirmer, 2001; Fang, Lu, & Holmgren, 2005; Gromer et al., 2004; Gromer, Schirmer, & Becker, 1997; Witte, Anestal, Jerremalm, Ehrsson, & Arner, 2005), but it is only during the last couple of years that this concept has been further strengthened and TrxR1 potentially recognized as a bona fide anticancer drug target, rather than only considering its inhibition as a noncausal offtarget effect. Here, we shall first discuss drugs that are already in clinical use for cancer therapy, where targeting of TrxR1 may be part of the mechanisms explaining clinical efficacy, whereupon we shall also discuss experimental compounds that were described in more recent years.

­TrxR1-inhibiting drugs in clinical use for cancer therapy There are several drugs in clinical use that are known to target TrxR1, although it is difficult to know whether TrxR1 inhibition is an off-target effect or if it contributes to either clinical efficacy and/or toxic side effects. Some of these compounds are listed in Table 3 together with further references to relevant literature. An interesting example is cisplatin, a potent and efficient drug in treatment of several cancer types that has normally been considered to cross-link DNA, but it is also recognized that the mechanisms of action of cisplatin are rather complex (Maccio & Madeddu,

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Drug

Indications

Auranofin

Rheumatoid arthritis, in clinical trials against cancer

Arsenic trioxide (often used together with all-trans retinoic acid)

Leukemia and other cancer forms

Cisplatin

Melphalan and other nitrosourea compounds Chlorambucil

Presumed main mechanism of action

Targeting of TrxR1

Targeting of TrxR1, possibly proteasome inhibitor Unknown/ targeting of Pin1

Potent inhibitor of TrxR1 at stoichiometric amounts

Several forms of solid tumor cancers

Cross-linking with DNA

Also inhibitor of TrxR1 with medium potency

Myeloma, melanoma, ovarian cancer, and sarcoma

Alkylating DNA

Inhibiting TrxR1 with medium potency

Leukemias and lymphomas, immunosuppressant

Alkylating DNA

Inhibiting TrxR1 with medium potency

Potent inhibitor of TrxR1

References Stafford et al. (2018), Sachweh et al. (2015), Roder and Thomson (2015), Arner (2009), Cox, Brown, Arner, and Hampton (2008), Marzano et al. (2007), Gromer et al. (2002) Lu, Chew, and Holmgren (2007), Seo, Urasaki, Takemura, and Ueda (2005), Miller Jr., Schipper, Lee, Singer, and Waxman (2002), Kozono et al. (2018)

Dammeyer et al. (2014), Peng, Xu, and Arner (2012), Eriksson et al. (2009), Witte et al. (2005), Arner et al. (2001), Chu (1994), Deneve et al. (1990) Witte et al. (2005), Schallreuter, Gleason, and Wood (1990)

Witte et al. (2005)

Ongoing recruiting trials in cancer (clinicaltrials.com) NCT03456700 NCT01737502

NCT03503864 NCT03624270 NCT01409161 NCT03318016 NCT02339740 NCT02688140 NCT03031249 NCT02899169 NCT02190695 NCT03096496 NCT02788201 NCT02200978 Numerous ongoing trials

Numerous ongoing trials

NCT03462719 NCT02788201

CHAPTER 31  Perspectives of TrxR1-based cancer therapies

Table 3  Examples of clinically used anticancer drugs that also inhibit TrxR1.

­Conclusions and future perspectives

2013; Riddell, 2018). For example, apoptotic programs are triggered by cisplatin also in enucleated cytoplasts independently of DNA targeting (Berndtsson et al., 2007; Mandic, Hansson, Linder, & Shoshan, 2003). Since TrxR1 is also inhibited by cisplatin, albeit with medium efficiency, this enzyme inhibition could potentially contribute to either cisplatin efficacy (Anestal & Arner, 2003; Arner et al., 2001; Marzano et al., 2007; Prast-Nielsen, Cebula, Pader, & Arner, 2010; Sasada et al., 1999; Witte et al., 2005) or to its toxicity such as toward hair cells of the cochlea that are supposedly resting cells being less sensitive to DNA cross-linking but prone to cell death triggered by oxidative stress (Dammeyer et al., 2014). To try addressing the uncertainties of “dirty” compounds, such as cisplatin, which are likely to have many diverse targets in cells, and as a means of trying to probe TrxR1 as a drug target for anticancer therapy, there have been several efforts to develop new anticancer agents targeting this enzyme. These shall briefly be discussed in the next chapter.

­Experimental compounds inhibiting TrxR1 Several gold compounds including auranofin and aurothioglucose are among the most efficient inhibitors of TrxR1 (Becker, Gromer, Schirmer, & Müller, 2000; Gromer, Arscott, Williams, Schirmer, & Becker, 1998; Smith, Guidry, Morris, & Levander, 1999), and thus, there have been attempts to develop gold-based inhibitors of the enzyme improved for better pharmacokinetics, selectivity, or isoenzyme/cell compartment specificity, such as more specifically targeting, or not targeting, the mitochondrial TrxR2 enzyme (Casini & Messori, 2011; Gabbiani & Messori, 2011; Hickey et al., 2008; Omata et al., 2006; Ott et al., 2009; Rigobello et al., 2008; Roder & Thomson, 2015; Urig et al., 2006; Zou et al., 2014). Similar efforts have been done to improve the efficacy of platinum-based drugs targeting TrxR1 (Ahmadi et al., 2006; Becker et al., 2001). Many additional TrxR1 inhibitors have been described, most of which were recently reviewed (Zhang et al., 2016; Zhang, Li, Han, Liu, & Fang, 2017; Zhang, Liu, Li, Xu, & Fang, 2018). Using completely novel experimental compounds that target TrxR1, the concept was recently further strengthened that irreversible inhibition of TrxR1, through targeting of its Sec residue, indeed seems to be promising anticancer therapy principle that is plausible also in vivo, with little toxicity to normal tissues at least in mouse models (Stafford et al., 2018; Zheng et al., 2019). For cancer therapy to be successful, it is, naturally, important to consider the effect of the treatment on both cancerous and noncancerous cells and tissues. We shall therefore finally consider the possible impact of TrxR1 targeting in normal version cancerous tissues in vivo.

­Conclusions and future perspectives If TrxR1 targeting using drug therapy shall be used in cancer treatment, then it is important to consider the effects of TrxR1 targeting also in normal cells. It was shown that cancer cells and tumors in  vivo are highly sensitive to the dual targeting of

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both TrxR1- and the GSH-dependent pathways (Harris et al., 2015; Mandal et al., 2010). However, targeting both of these pathways at once is highly toxic also to normal cells and tissues, whereby such dual reductive pathway targeting should be taken with extreme caution not to yield lethality due to organ failure (Eriksson et al., 2015; Stafford et al., 2018). When targeting merely TrxR1 in normal cells, however, the survival, or even strengthening of such cells toward oxidative stress due to their strong Nrf2 activation (Britt Jr. et al., 2014; Cebula et al., 2015; Iverson et al., 2013; Li et al., 2016; Locy et al., 2011), is interesting from a cancer therapeutic perspective. Could it be that TrxR1 targeting at cancer therapy may, at the same time, yield cancer and tumor cell death while normal cell resistance to oxidative stress and their overall survival becomes strengthened? If true, the question then arises: how this will affect cancer initiation, progression, or treatment outcome, considering the multicellular multiorgan nature of cancer (Casey et  al., 2015; Ruffell & Coussens, 2015). This aspect of TrxR1-targeting cancer therapy still awaits further scrutiny and years of studies, with many uncertain aspects still unanswered. Potential development of resistance of cancer cells toward TrxR1 targeting is also an important aspect that has yet to be addressed, as is the impact of TrxR1 inhibition in combination with other cancer therapy modalities. It seems clear, however, that TrxR1 is an intriguing, interesting, and potentially important cellular target for drug therapy against cancer that deserves further studies.

­Acknowledgments Funding from Karolinska Institutet, the Cancerfonden, the Swedish Research Council, the Knut och Alice Wallenbergs Stiftelse, and Oblique Therapeutics AB is gratefully acknowledged.

­COI Declaration The author has three patents on TrxR1 inhibitors and collaborates with Oblique Therapeutics AB for the development of such inhibitors toward clinical practice in cancer treatment.

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Oxidative/nitrosative stress and hepatic encephalopathy

32 Dieter Häussinger, Boris Görg

Clinic for Gastroenterology, Hepatology, and Infectious Diseases, Heinrich-Heine-University, Düsseldorf, Germany

Abstract Hepatic encephalopathy (HE) is a neuropsychiatric syndrome that frequently occurs in the course of acute or chronic liver diseases. HE is triggered by a heterogeneous group of factors such as ammonia, which is considered a main toxin in HE; hyponatremia; proinflammatory cytokines; and benzodiazepines. Symptoms of HE mainly comprise disturbances of cognitive and motoric function. HE in patients with liver cirrhosis is seen as the clinical manifestation of a low-grade cerebral edema and accompanying cerebral oxidative/nitrosative stress, which trigger a variety of functional consequences. These include posttranslational protein modifications such as tyrosine nitration and O-GlcNAcylation of proteins, oxidation of RNA, gene and protein expression changes, and senescence. It is assumed that these alterations impair the functions of astrocytes and neurons leading to disturbances of glio-neuronal communication in the brain with consequences for neurotransmission and oscillatory networks in the brain. ­Keywords: Astrocytes, Ammonia, Glutamine, Protein tyrosine nitration, RNA oxidation, MicroRNA, Senescence

­Introduction Oxidative stress plays an important role in health and disease (for a review, see Sies, Berndt, & Jones, 2017, Sies, 2017). On the one hand, it serves as a physiological regulator of diverse cell functions through redox signaling (called oxidative eustress), but on the other hand, it can give rise to oxidative damage of organs due to an overshooting imbalance between oxidant generation and removal (called oxidative distress) (Sies, 2017; Sies, 2018). Deregulated oxidative eustress and oxidative distress are involved in the pathogenesis of a variety of disorders such as Alzheimer’s disease, diabetes, and coronary heart disease and drug toxicity. Here, we summarize our current knowledge on the role of oxidative stress in the pathogenesis of hepatic encephalopathy and ammonia toxicity. Hepatic encephalopathy (HE) is a potentially life-threatening neuropsychiatric syndrome that frequently arises in the course of acute or chronic liver disease. Mild Oxidative Stress. https://doi.org/10.1016/B978-0-12-818606-0.00032-8 © 2020 Elsevier Inc. All rights reserved.

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forms of HE were reported to occur in up to 80% of patients with liver cirrhosis. HE in chronic liver disease comprises a broad spectrum of symptoms of varying severity with disturbances of cognitive and fine motor functions being the most prominent (Häussinger & Blei, 2007; Häussinger & Schliess, 2008; Häussinger & Sies, 2013a). While symptoms of HE are in principle reversible, cognitive disturbances may persist after an acute episode of HE (Bajaj et  al., 2010; Riggio et  al., 2011). HE in chronic liver disease is a consequence of a low-grade cerebral edema (Cordoba et al., 2001; Häussinger et al., 1994; Häussinger, Kircheis, Fischer, Schliess, & Vom Dahl, 2000; Shah et al., 2008) and cerebral oxidative/nitrosative stress (Görg et al., 2010; Görg, Bidmon, & Häussinger, 2013; Häussinger & Schliess, 2008), which trigger a variety of functional disturbances including gene and protein expression changes (Görg et  al., 2010; Jördens et  al., 2015; Schliess et  al., 2002; Sobczyk, Jördens, Karababa, Görg, & Häussinger, 2015; Warskulat et al., 2002; Zemtsova et al., 2011; Zhou & Norenberg, 1999), posttranslational protein modifications (Görg et al., 2010; Karababa, Görg, Schliess, & Häussinger, 2014; Schliess et al., 2002), oxidation of RNA, and senescence (Görg et al., 2010; Görg, Karababa, Shafigullina, Bidmon, & Häussinger, 2015) (for reviews see Häussinger & Schliess, 2008, Görg, Schliess, & Häussinger, 2013). As a result, communication between astrocytes and neurons and synaptic plasticity becomes impaired, and oscillatory networks are disturbed, thereby triggering symptoms of HE (Fig. 1). Most importantly, pathogenetic key observations that were derived from animal or cell culture experiments have also been confirmed in the human brain.

­Astrocyte swelling in HE There is general agreement that astrocytes play a key role and ammonia is a major toxin in the pathogenesis of HE (Häussinger & Blei, 2007; Häussinger & Schliess, 2008; Häussinger & Sies, 2013b; Norenberg, 1987). In the brain, ammonia is detoxified in the astrocytes through glutamine synthesis (Norenberg & Martinez-Hernandez, 1979). 1H-MR spectroscopic studies showed that glutamine levels are increased, whereas the levels of the astrocytic organic osmolyte myoinositol are decreased in brains of patients with liver cirrhosis and HE (Cordoba et al., 2001; Häussinger et al., 1994). From these findings, it was proposed that glutamine accumulation triggers in patients with liver cirrhosis the development of a low-grade cerebral edema with impaired volume-regulatory capacity due to a depletion of the myo-inositol pool in astrocytes (Cordoba et al., 2001; Häussinger et al., 1994; Häussinger et al., 2000) (Fig. 2). Direct evidence for an increased brain water content in patients with liver cirrhosis and HE was also provided by quantitative water mapping by magnetic resonance imaging (MRI) of human brains (Shah et al., 2003; Shah et al., 2008). These studies showed that changes in brain water content in patients with liver cirrhosis are brain region-specific and correlate with the severity of HE (Shah et al., 2003, Shah et al., 2008). The most prominent increase in brain water content was observed within the white matter,

­Astrocyte swelling in HE

FIG. 1 Pathogenetic model of hepatic encephalopathy. Factors that trigger episodes of hepatic encephalopathy (HE-precipitating factors) induce astrocyte swelling and the formation of reactive nitrogen and oxygen species (RONS) in astrocytes. Astrocyte swelling and RONS formation are mutually interrelated and self-amplify each other, thereby triggering a variety of functional consequences. These are suggested to induce astrocytic/neuronal dysfunction and thereby to disturb oscillatory networks in the brain (Butz et al., 2010; Butz, May, Häussinger, & Schnitzler, 2013), which is reflected by the symptoms. Modified from Görg, B., Karababa, A., Häussinger, D. (2018). Hepatic encephalopathy and astrocyte senescence. Journal of Clinical and Experimental Hepatology 8, 294–300, Häussinger, D. & Schliess, F. (2008). Pathogenetic mechanisms of hepatic encephalopathy. Gut 57, 1156–65, Häussinger, D. & Sies, H. (2013b). Hepatic encephalopathy: Clinical aspects and pathogenetic concept. Archives of Biochemistry and Biophysics 536, 97–100.

which consists mainly of axon-myelinating astrocytes, thereby strengthening a role of astrocyte swelling in patients with liver cirrhosis and HE (Shah et  al., 2003, Shah et al., 2008). Not only ammonia but also other conditions known to precipitate or to worsen HE episodes in patients with liver cirrhosis, such as proinflammatory cytokines, hyponatremia, and sedatives of the benzodiazepine-type induce astrocyte swelling (Bender & Norenberg, 1998; Lachmann, Görg, Bidmon, Keitel, & Häussinger, 2013; Norenberg et al., 1991; Rama Rao, Jayakumar, Tong, Alvarez, & Norenberg, 2010;

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FIG. 2 Development of a low-grade cerebral edema in HE. Hyperammonemia due to chronic liver dysfunction triggers osmotic stress by elevating the synthesis of glutamine in cerebral astrocytes. This is compensated by the release of other organic osmolytes such as myoinositol. The resulting depletion of the astrocytic osmolyte pool diminishes the volumeregulatory capacity and renders the astrocyte vulnerable for the swelling-inducing effects of HE-precipitating factors, which then exacerbate the low-grade cerebral edema. Modified from Häussinger, D. & Sies, H. (2013b). Hepatic encephalopathy: Clinical aspects and pathogenetic concept. Archives of Biochemistry and Biophysics 536, 97–100.

Reinehr et al., 2007). Astrocyte swelling in response to the HE-precipitating factors such as ammonia and TNFα is suggested to result from an activation and/or upregulation of the Na+-K+-2Cl− cotransporter 1 (NKCC1), respectively (Jayakumar et al., 2008; Pozdeev et al., 2017). Moreover, aquaporin 4, which in the brain is expressed by astrocytes, was suggested as a water entry route in animal models of acute liver failure induced by thioacetamide or acetaminophen (Rama Rao, Verkman, Curtis, & Norenberg, 2014) and in ammonia-exposed cultured astrocytes (Bodega et al., 2012; Rama Rao, Chen, Simard, & Norenberg, 2003). Thus, astrocyte swelling represents a point of convergence of the actions of the remarkably heterogeneous group of HEprecipitating factors. These findings established the paradigm that HE in chronic liver disease is a clinical manifestation of a low-grade cerebral edema that exacerbates in response to HEprecipitating factors after an ammonia-induced exhaustion of the volume-regulatory capacity of the astrocyte (Häussinger et al., 2000) (Fig. 2).

­Astrocyte swelling and oxidative/nitrosative stress in astrocytes in HE

­ strocyte swelling and oxidative/nitrosative stress in A astrocytes in HE As in many cell types also in astrocytes, changes in cell hydration or cell volume were shown to affect metabolic cell function and gene expression (for review, see Häussinger & Lang, 1991, Lang et  al., 1998). Swelling of the astrocytes, either induced by hypoosmotic cell culture medium (Schliess, Foster, Görg, Reinehr, & Häussinger, 2004), ammonia (Schliess et al., 2002), diazepam (Görg et al., 2003), or TNFα (Görg et al., 2006), elevates intracellular Ca2+ levels [Ca2+]i in a N-methyld-aspartate receptor (NMDAR)-dependent way. While the initial channel opening of the ionotropic NMDAR is suggested to be a consequence of plasma membrane stretch (Kloda, Lua, Hall, Adams, & Martinac, 2007) and a depolarization-induced removal of the Mg2+ blockade (Mayer, Westbrook, & Guthrie, 1984), a subsequent prostanoid-dependent release of vesicular L-glutamate amplifies the NMDARdependent elevation of [Ca2+]i. This elevation of [Ca2+]i by HE-precipitating factors rapidly triggers the formation of RONS through protein kinase Cζ-dependent serine phosphorylation of the NAPDH oxidase (Nox) subunit p47phox (Reinehr et al., 2007) and activation of the neuronal nitric oxide synthase (nNOS) (Kruczek et al., 2009; Reinehr et al., 2007; Schliess et al., 2004). Another source for nitric oxide (NO) in astrocytes is the inducible nitric oxide synthase (iNOS), which becomes upregulated by ammonia in a NFκB-dependent way (Chastre, Jiang, Desjardins, & Butterworth, 2010; Schliess et al., 2002; Sinke et al., 2008). Upregulation of nNOS and iNOS was also observed in rat astrocytes and pyramidal neurons in the cerebral cortex (Suarez, Bodega, Arilla, Felipo, & Fernandez, 2006) and in cerebellar Bergmann glia and Purkinje cells (Suarez, Bodega, Rubio, Felipo, & Fernandez, 2005) after portacaval anastomosis, which is frequently used as an animal model for HE. However, no upregulation of iNOS was found in the cerebral cortex after acute ammonia intoxication or after portal vein ligation (Brück et al., 2011). Likewise, iNOS and nNOS protein and mRNA levels were unaffected in postmortem brain samples from the cerebral cortex of patients with liver cirrhosis and HE (Görg et  al., 2010; Görg, Bidmon, & Häussinger, 2013; Zemtsova et  al., 2011). These contradictory findings may reflect species differences or relate to the animal model and/or to an insufficient detection sensitivity when analyzing protein and mRNA changes in brain homogenates by Western blot and qPCR as compared with immunofluorescence analysis. Apart from p47phox, nNOS, and iNOS, the Nox isoform 4 becomes upregulated by ammonia in cultured astrocytes (Görg et al., 2019) in a glutamine synthesis-­dependent way (Häussinger and Görg, unpublished). Of particular interest, Nox4 is constitutively active, and in contrast to other known Nox isoforms such as O2−-producing Nox2, Nox4 is the only isoform that produces H2O2 (Takac et al., 2011) (for review, see Nayernia, Jaquet, & Krause, 2014, Bedard & Krause, 2007, Brandes & Schröder, 2008). Depending on the cell type, Nox4 was found to be expressed in different subcellular compartments such as in the mitochondria (Block, Gorin, & Abboud, 2009), the nucleus (Hilenski, Clempus, Quinn, Lambeth, & Griendling, 2004), and the

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e­ ndoplasmic reticulum (Chen, Kirber, Xiao, Yang, & Keaney Jr, 2008). However, the intracellular localization of Nox4 in the astrocytes remains to be established. While astrocyte swelling is sufficient to trigger RONS formation, H2O2 and the nitric oxide donor spermine-NONOate in turn induce astrocyte swelling (Lachmann et al., 2013; Moriyama, Jayakumar, Tong, & Norenberg, 2010). In view of this mutual interrelationship between osmotic and oxidative/nitrosative stress, it was proposed that HE-precipitating factors engage a self-amplifying cycle in the astrocytes (Schliess, Görg, & Häussinger, 2006). This cycle may further be augmented by the synergistic actions of HE-precipitating factors as demonstrated for proinflammatory cytokines and ammonia, both being triggers for astrocyte swelling (Rama Rao et al., 2010) and formation of oxidative stress (Häussinger & Schliess, 2008) (Fig. 1). Currently, only little is known about the effects of ammonia and other HEprecipitating factors on the formation of reactive oxygen species (ROS) and NO in brain cell types other than astrocytes. Ammonia was shown to trigger ROS formation in cortical neurons (Kruczek et  al., 2011) and cerebellar granule cells (Bobermin et  al., 2015) and to elevate ROS and NO in microglia (Rao, Brahmbhatt, & Norenberg, 2013; Zemtsova et al., 2011) and in endothelial cells in vitro (Jayakumar, Tong, Ospel, & Norenberg, 2012), but the underlying mechanisms are unknown. Interestingly, in vitro studies suggest that RONS derived from ammonia-exposed microglia and endothelial cells may also contribute to the ammonia-induced astrocyte swelling (Jayakumar et al., 2012; Rao et al., 2013) and therefore may further amplify osmotic and oxidative/nitrosative stress in astrocytes. Whether a mutual interdependence between the formation of reactive nitrogen and oxygen species (RONS) and cell swelling, similar to what is observed in astrocytes, also exists in other brain cell types is currently unknown. However, ammonia does not trigger cell swelling in cultured neurons (Lachmann et al., 2013). Ammonia induces swelling of microglia in culture in a glutamine synthesis-dependent way; however, expression of glutamine synthetase under these conditions may represent a cell culture artifact (Lachmann et al., 2013).

­Mitochondria and oxidative stress in HE Studies on ammonia-exposed isolated mitochondria (Niknahad, Jamshidzadeh, Heidari, Zarei, & Ommati, 2017), ammonia-exposed astrocytes (Görg et al., 2015), and animal models for HE (Chadipiralla, Reddanna, Chinta, & Reddy, 2012; Dhanda, Sunkaria, Halder, & Sandhir, 2018; Jamshidzadeh et  al., 2017; Kosenko et  al., 2017) identified mitochondria as another site of ammonia-induced ROS formation (ROSmito), which may due to an induction of mitochondrial permeability transition (mPT) (Rama Rao, Jayakumar, & Norenberg, 2005). Furthermore, ammonia dissipates the mitochondrial membrane potential (ΔΨ), decreases the synthesis of ATP, and triggers swelling and fragmentation of mitochondria in astrocytes (Bai et  al., 2001; Görg et  al., 2015; Pichili, Rao, Jayakumar, & Norenberg, 2007; Rama Rao & Norenberg, 2014). Swelling of mitochondria, ROSmito formation, and impaired

­Mitochondria and oxidative stress in HE

ATP synthesis were also observed in ammonia-exposed isolated brain mitochondria (Niknahad et al., 2017), in animal models for HE (Dhanda et al., 2018; Jamshidzadeh et al., 2017; Laursen & Diemer, 1980; Reddy, Murthy Ch, & Reddanna, 2004) and in astrocytes in postmortem brain samples from patients with acute liver failure (Kato, Hughes, Keays, & Williams, 1992). Interestingly, in thioacetamide-induced liver failure, feeding of the animals with a diet containing the antioxidant and osmolyte taurine fully prevented mitochondrial swelling and ROSmito formation and preserved mitochondrial ATP levels (Jamshidzadeh et al., 2017; Niknahad et al., 2017). While this suggests a close relationship between osmotic and oxidative/nitrosative stress in mitochondria, it remains to be established whether the ammonia-induced swelling of mitochondria is a consequence of ROSmito formation or vice versa and whether ROSmito and mitochondrial swelling mutually depend on each other. Although the ammonia-induced structural and molecular changes in mitochondria are clearly indicative for mitochondrial dysfunction, ammonia does not impair the viability of the astrocytes (Oenarto et al., 2016; Schliess et al., 2002). This may be explained by ammonia-induced mitophagy, which serves as a quality control and degrades defective mitochondria (Polletta et al., 2015). Interestingly, ammonia-induced ROSmito formation in astrocytes in  vitro depends on the synthesis and the import of glutamine into mitochondria (Görg et al., 2015; Pichili et al., 2007). According to the so-called Trojan horse hypothesis, the ­glutaminase-dependent hydrolysis of glutamine will raise intramitochondrial ammonia levels that then trigger ROSmito formation via MPT and the dissipation of ΔΨ and decrease the synthesis of ATP (Rama Rao & Norenberg, 2014). However, it remains to be determined, whether either the glutaminolysis-triggered elevation of intramitochondrial ammonia or glutamate levels or both account for ROSmito formation. In line with this, knockdown of glutaminase in astrocytes prevented ammonia-induced RNA oxidation (Görg and Häussinger, unpublished result). Ammonia and benzodiazepines of the diazepam type, which precipitate HE episodes, trigger ROSmito formation in astrocytes (Görg et al., 2003; Jayakumar, Panickar, & Norenberg, 2002) due to upregulation or activation of the peripheral-type benzodiazepine receptor (PBR) (Kruczek et al., 2011), which is located in the outer mitochondrial membrane. Activation of the PBR may trigger the formation of neurosteroids, which are elevated in brains from patients with liver cirrhosis and HE (Ahboucha, Pomier-Layrargues, Mamer, & Butterworth, 2006; Zaman, 1990). Whether these observations relate to MPT pore opening is yet unclear. Neurosteroids can also activate the membrane-bound bile acid receptor TGR5 (G protein-coupled bile acid receptor, Gpbar-1), which is expressed in astrocytes and neurons and whose activation is coupled to adenylate cyclase activation, elevation of [Ca2+]i, and ROS formation (Keitel et al., 2010). However, TGR5 is downregulated not only in astrocytes by ammonia but also in cerebral cortex from patients with liver cirrhosis and HE (Keitel et al., 2010). Therefore, a significant contribution of TGR5 to ammonia-induced ROS formation is questionable and needs to be established. Fig. 3 summarizes the currently discussed mechanisms triggering ammonia-induced RONS formation.

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FIG. 3 Mechanisms and sources of the ammonia-induced RONS formation in astrocytes. Ammonia induces the formation of RONS in astrocytes through an NMDA receptordependent elevation of the intracellular calcium concentration [Ca2+]i, which is initiated by a depolarization-induced removal of the Mg2+ blockade and membrane stretch and which is amplified by prostanoids and exocytosis of glutamate-containing intracellular vesicles. The elevation of [Ca2+]i stimulates the formation of NO through the activation of nNOS and by upregulating iNOS via degradation of IκBα and activation of Nox2, which generates the synthesis of O2−. Ammonia also triggers the synthesis of H2O2 by increasing the expression of Nox4. Upregulation of the benzodiazepine receptor (PBR) by ammonia stimulates the synthesis of neurosteroids, which are exported from the cell via Mrp4 and which then can activate the GS-coupled bile acid receptor TGR5 in an autocrine and paracrine fashion. TGR5-induced cAMP synthesis contributes to the elevation of [Ca2+]i and RONS formation. Glutaminase-dependent hydrolysis of glutamine inside the mitochondria triggers mitochondrial O2− formation. NO and O2− also combine to form ONOO−, which can trigger protein tyrosine nitration. Modified from Görg, B., Schliess, F., Häussinger, D. (2013). Osmotic and oxidative/nitrosative stress in ammonia toxicity and hepatic encephalopathy. Archives of Biochemistry and Biophysics 536, 158–163.

­Oxidative/nitrosative stress and protein tyrosine nitration in HE

F­ unctional consequences of osmotic and oxidative/ nitrosative stress in HE Since the first reports on cerebral nitric oxide, superoxide, and peroxynitrite formation in animal models for HE (Kosenko et al., 1997; Kosenko, Kaminski, Lopata, Muravyov, & Felipo, 1999; Larsen, Gottstein, & Blei, 2001; Master, Gottstein, & Blei, 1999; Schliess et al., 2002) and in ammonia-exposed astrocytes in vitro (Murthy, Rama Rao, Bai, & Norenberg, 2001; Schliess et al., 2002), several functional consequences were identified. These include covalent protein modifications (Jayakumar et al., 2008; Schliess et al., 2002; Widmer, Kaiser, Engels, Jung, & Grune, 2007), RNA oxidation (Görg et al., 2008), altered protein and gene expression (Jayakumar, Panickar, Murthy Ch, & Norenberg, 2006; Jördens et  al., 2015), signal transduction (Moriyama et al., 2010; Schliess et al., 2002), disturbances of zinc homeostasis (Kruczek et al., 2009; Kruczek et al., 2011), inhibition of proliferation, and induction of senescence (Bodega et  al., 2015; Görg et  al., 2015; Görg et  al., 2018; Oenarto et al., 2016) (Fig. 1). Most importantly, such alterations were also identified in postmortem brain samples from patients with liver cirrhosis and HE, but not those with liver cirrhosis without HE. The first evidence for ongoing oxidative stress in the brain of HE patients was significantly increased levels of heat shock protein 27, a surrogate marker for oxidative stress in the cerebral cortex of patients with liver cirrhosis and HE when compared with controls without liver disease or patients with liver cirrhosis without HE (Görg et al., 2010).

­ xidative/nitrosative stress and protein tyrosine O nitration in HE One consequence of ammonia-induced oxidative-nitrosative stress is protein tyrosine nitration (PTN). In cultured astrocytes, PTN is induced not only by ammonia but also by benzodiazepines and inflammatory cytokines such as TNFα. These effectors act synergistically with regard to PTN. PTN is also induced by hypoosmotic exposure of astrocytes, indicating that astrocyte swelling is sufficient to trigger PTN (Görg et al., 2003; Görg et al., 2006; Schliess et al., 2002; Schliess et al., 2004). Whereas PTN under the influence of ammonia, hypoosmolarity, and TNFα involves the formation of peroxynitrite, benzodiazepine-induced PTN involves activation of the PBR. PTN in the brain was shown to occur in vivo in ammonia- and LPS-intoxicated or ­portacaval-shunted rats (Brück et al., 2011; Görg et al., 2006; Schliess et al., 2002), in hyperammonemic mice due to taurine transporter knockout (Qvartskhava et al., 2019) or liver-specific knockdown of glutamine synthetase (Qvartskhava et  al., 2015). Astrocytes located near the blood-brain barrier show particularly high levels of PTN with potential consequences for blood-barrier permeability and transastrocytic substrate transport (Brück et al., 2011, Görg et al., 2006, Schliess et al., 2002). Tyrosine nitration is a selective process and involves distinct proteins only. Among these, the PBR, glyceraldehyde-3-phosphate, Erk-1, glutamine synthetase,

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and NKCC1 have been identified (Brück et al., 2011; Görg et al., 2006; Jayakumar et al., 2008; Schliess et al., 2002). Whereas nitration of the NKCC1 was suggested to enhance its activity (Jayakumar et al., 2008) and thereby augment astrocyte swelling, PTN of GS inactivates the enzyme and may counteract ammonia-induced astrocyte swelling (Brück et al., 2011; Görg et al., 2006; Görg et al., 2007; Schliess et al., 2002). Knockdown of GS in astrocytes prevents ammonia-induced mitochondrial swelling (own unpublished result) and RNA oxidation (Görg et  al., 2019). Therefore, inactivation of GS by tyrosine nitration may counteract ammonia toxicity. Although PTN interferes with signaling elements and enzyme activities, its exact role in the pathogenesis of HE remains to be defined. However, increased levels of tyrosine-nitrated proteins and glutamine synthetase in particular are also found in postmortem cerebrocortical brain samples from patients with liver cirrhosis and HE, but not from those without HE (Görg et al., 2010).

­RNA oxidation in HE A novel aspect on the pathogenesis of HE arose from the finding that ammonia, TNFα, benzodiazepines, and hypoosmotic swelling can induce RNA oxidation in cultured astrocytes, in the brains from ammonia acetate-treated rats and in ammoniaexposed brain slices (Görg et al., 2008). Here, ROS oxidize guanosine to produce 8-oxo-7,8-dihydro-2′-guanosine (8OHG; also called 8-oxoguanosine), which is detected in astrocytes and in the cytosol of neurons from ammonia-intoxicated rats (Görg et al., 2008). Likewise, RNA oxidation is also found in brains from mice with liver-specific glutamine synthetase knockout (Qvartskhava et  al., 2015) or taurine transporter knockout (Qvartskhava et al., 2019), that is, conditions associated with systemic hyperammonemia. Most importantly, increased RNA oxidation was also found in the brain of patients with liver cirrhosis with HE. Patients with liver cirrhosis but without HE showed levels of RNA oxidation in the brain similar to control patients without liver disease (Görg et al., 2010). 8OHG immunoreactivity was also found in granular structures along the dendrites and in postsynaptic dendritic regions in association with the RNA-binding splicing protein neuro-oncological ventral antigen 2 (NOVA2). Thus, HE-associated oxidative stress apparently modifies RNA species, which participate in the granular RNA transport along the dendrites. Such neuronal RNA granules can contain all elements required for local postsynaptic protein synthesis, which is controlled by synaptic signals and plays a major role for synaptic plasticity, as reflected by late-phase long-term potentiation (L-LTP) (Schuman, Dynes, & Steward, 2006). Postsynaptic protein synthesis is required for learning and the formation of long-term memory (for reviews, see Schuman et al., 2006, Sutton & Schuman, 2005, Martin, Barad, & Kandel, 2000). RNA oxidation in response to HE-relevant conditions (e.g., ammonia, TNFα, benzodiazepines, and hyponatraemia) is a selective process. Among the oxidized RNA species, the mRNA for the glutamate uptake system GLAST and ribosomal (r) RNA were identified (Görg

­Oxidative/nitrosative stress and gene expression changes in HE

et al., 2008). The role of RNA oxidation in ammonia neurotoxicity and HE is not yet clear; however, there is good evidence that rRNA and mRNA oxidation may compromise translation accuracy and efficacy, thereby resulting in the formation of defective or unstable proteins. Oxidation of astroglial GLAST mRNA by ammonia may partly explain the long-known ammonia-induced decrease of GLAST expression and glutamate uptake in cultured astrocytes (Chan, Hazell, Desjardins, & Butterworth, 2000; Jayakumar et  al., 2006) and contribute to the known disturbances in glutamatergic neurotransmission in HE (Vaquero & Butterworth, 2006). To what extent RNA oxidation contributes to the multiple derangements of neurotransmitter receptor systems in HE (for a review, see Häussinger & Blei, 2007) and to HE-associated alterations of gene expression (Görg, Bidmon, & Häussinger, 2013; Oenarto et al., 2016; Sobczyk et al., 2015; Song, Dhodda, Blei, Dempsey, & Rao, 2002) is currently unclear. Neuronal RNA oxidation was associated with mild cognitive impairment in early stages of Alzheimer’s disease (Nunomura et al., 2009; Nunomura et al., 2012). Cognitive impairment without neuronal degeneration is also a hallmark of HE and may involve a disturbed protein synthesis-dependent late-phase long-term potentiation (L-LTP), learning, and memory consolidation due to the oxidation of postsynaptically translated mRNA species. In line with this, learning ability is disturbed in animal models for HE such as bile duct-ligated rats or rats fed with a hyperammonemic diet (Rodrigo et al., 2010). LTP is impaired in mouse brain slices exposed to ammonia or TNFα (Swain, Blei, Butterworth, & Kraig, 1991). However, to what extent the oxidation of locally translated mRNA species contributes to the L-LTP impairment under these conditions remains to be established. Nonetheless, RNA oxidation in response to the oxidative stress as induced by HE-relevant neurotoxins can provide a mechanistic link between cell and mitochondrial swelling and oxidative stress on the one hand and alterations of synaptic plasticity on the other hand. In line with this, portal vein ligation (PVL) in rats triggered hyperammonemia, PTN, RNA oxidation, and impairment of locomotor activity. Indomethacin had no effect on the PVL-induced hyperammonemia, but prevented PTN and RNA oxidation and the disturbances of locomotor disturbances (Brück et al., 2011). These findings underline the importance of oxidative/nitrosative stress in the pathogenesis of HE.

­ xidative/nitrosative stress and gene expression O changes in HE The ammonia-induced RONS formation activates a number of transcription factors, thereby affecting the expression of several genes that are suggested to play a role in the pathogenesis of HE (Fig. 4). In this regard, ammonia- and hypoosmolarity-­ induced nitrosative stress in astrocytes activate the transcription factors specificity protein 1 (SP1) and the metal-responsive transcription factor (MTF)-1 (Kruczek et al., 2009; Kruczek et al., 2011). This was shown to be a consequence of a NOinduced liberation of zinc from Zn2+-thiolate clusters in proteins and the resulting increase of intracellular levels of free zinc ions (Kruczek et al., 2009, Kruczek et al.,

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FIG. 4 Mechanisms of RONS-induced gene expression changes in ammonia-exposed cultured rat astrocytes. Ammonia triggers expression changes of genes relevant for the pathogenesis of HE in astrocytes through ROS- and RNS-dependent activation of the transcription factors NFκB, p53, PPARα, MTF1, and SP1.

2011). As elevated levels of free zinc ions are highly toxic to cells and a MTF-1dependent upregulation of the zinc-chelating metallothioneins MT-1 and MT-2 by ammonia or hypoosmotic media or in the brain in ammonium acetate-treated rats may reflect a protective response (Kruczek et al., 2009, Kruczek et al., 2011). Gene expression levels of six MT isoforms were also significantly upregulated in postmortem brain tissue from patients with liver cirrhosis with, but not in those without HE, and here, expression levels of some MT isoforms positively correlated with the peripheral blood ammonia concentration. These results suggest an important role of ammonia as a trigger for deranged Zn2+ homeostasis in the brain of patients with liver cirrhosis and HE (Görg, Bidmon, & Häussinger, 2013). Apart from MT-1 and MT-2, also SP1 becomes activated by ammonia or hypoosmotic cell swelling in a zinc-dependent way, and the latter was shown to upregulate the mRNA levels of the peripheral-type benzodiazepine receptor (PBR) (Kruczek et al., 2009). In line with an ammonia-induced upregulation of PBR protein in astrocytes (Kruczek et al., 2011), PBR binding sites were found to be elevated in the brain from animal models for HE (Agusti, Dziedzic, Hernandez-Rabaza, Guilarte, & Felipo, 2014; Rao, Audet, Therrien, & Butterworth, 1994) and in brains from patients

­Oxidative/nitrosative stress and gene expression changes in HE

with liver cirrhosis who died in hepatic coma (Lavoie, Layrargues, & Butterworth, 1990). Although ammonia upregulates the PBR protein, the functional consequence remains unclear because ammonia simultaneously triggers PTN of the PBR. As the PBR imports cholesterol into mitochondria and thereby promotes the synthesis of neurosteroids (Papadopoulos, 2003), upregulation of the PBR may account for increased neurosteroid levels in the brain in animal models for HE (Ahboucha et al., 2006; Ahboucha, Layrargues, Mamer, & Butterworth, 2005) and in postmortem brain tissue from patients with liver cirrhosis with HE (Ahboucha et al., 2006; Zaman, 1990). Neurosteroids are substrates of the multidrug resistance protein 4 (Mrp4), which is upregulated through a RONS-induced activation of the peroxisome proliferator-activated receptor-α (PPARα) in ammonia-exposed cultured rat astrocytes (Jördens et  al., 2015). Upregulation of Mrp4 mRNA and Mrp4 protein was also observed in postmortem brain tissue from patients with liver cirrhosis with HE (Jördens et al., 2015). Therefore, increased γ-aminobutyric acid (GABA)-dependent neurotransmission in HE (Ahboucha & Butterworth, 2004; Butterworth, 2016) may be a consequence of PBR-dependent synthesis and Mrp4-mediated release of neurosteroids from astrocytes. However as already mentioned earlier, neurosteroids may also trigger RONS formation through activation of TGR5, which in the brain is strongly expressed by astrocytes and neurons (Keitel et al., 2010). The ammonia-induced RONS formation also triggers mRNA expression changes of a variety of ephrin receptor (EphR) and ephrin (Eph) isoforms, and such changes were also observed in postmortem brain tissue from patients with liver cirrhosis and HE (Sobczyk et al., 2015). Importantly, the EphR/Eph mRNA expression changes found in ammonia-exposed cultured astrocytes were sensitive toward inhibition of NADPH oxidase and nitric oxide synthase (Sobczyk et al., 2015). Interestingly, bidirectional communication between astrocytes and neurons via EphR/Eph strongly affects synaptic transmission by modulating the expression of glial glutamate transporters (Filosa et al., 2009; Yang et al., 2014). Therefore, it was suggested that RONS-induced EphR/ Eph mRNA expression changes may contribute to disturbed glutamatergic neurotransmission and impaired synaptic plasticity in HE (Sobczyk et al., 2015). Further gene expression changes were recently identified by a transcriptome analysis on postmortem brain samples from patients with liver cirrhosis (Görg, Bidmon, & Häussinger, 2013). This study revealed 616 genes whose expression were selectively changed in postmortem brain samples from patients with liver cirrhosis with, but not in those without HE. Importantly, these genes were related to biological processes that have been meanwhile established to play an important role for the pathogenesis of HE such as oxidative stress, microglia activation, and proliferation. Among the upregulated genes that relate to the oxidative stress response, not only HO1 but also selenoprotein V, peroxisome proliferator-activated receptor α, and ­peroxiredoxin-4 were identified (Görg, Bidmon, & Häussinger, 2013). However, gene expression levels of the inducible, neuronal, and endothelial nitric oxide synthase isoforms were not significantly changed. The study also unraveled a number of genes that were selectively changed in brain samples from patient with liver cirrhosis and HE that had not been recognized before to play a role for the pathogenesis of HE

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such as genes that relate to the activation of anti-inflammatory signaling pathways such as the interleukins IL-4 and IL-10β (Görg, Bidmon, & Häussinger, 2013). Due to the cellular complexity of the brain, it remains to be established, which cell types are affected by the observed gene expression changes and whether all of them are also reflected at the protein level (Görg, Schliess, & Häussinger, 2013). Apart from triggering mRNA expression changes, RONS were also shown to account for downregulation of a specific set of microRNAs (miRNA) in ammoniaexposed cultured rat astrocytes (Oenarto et al., 2016). Therefore, these miRNAs were identified as new members of a miRNA subset named “redoximiRs,” whose expression is modulated by RONS. The study further suggested that the downregulation of redoximiRs in ammonia-exposed astrocytes may account for the upregulation of mRNA species, which are predicted to be their targets (Oenarto et  al., 2016). Potential targets of these redoximiRs include mRNAs involved in glutamine transport (solute carrier family 1 member 5 [Slc1a5]), glutaminolysis (kidney-type glutaminase [Gls1]), oxidative stress, and senescence (NADPH oxidase 4 [Nox4], heme oxygenase 1 [HO1]) (Fig. 5). Importantly, miRNAs may decrease protein levels of

FIG. 5 ROS-dependent downregulation of miRNA species in ammonia-exposed astrocytes. The ammonia-induced ROS formation downregulates a subset of miRNA species targeting mRNAs coding for HO1 (gene name: Hmox1) (Oenarto et al., 2016) and predicted to target mRNAs of Nox4, Gls1, and Slc1a5.

­Oxidative stress and astrocyte senescence in HE

a respective target mRNA either by degrading the respective mRNA or by inhibiting its translation. This explains why in the case of HO1, downregulation of miR326-3p is associated with an upregulation of HO1 mRNA and protein (Oenarto et al., 2016), whereas Nox4 protein becomes upregulated despite unchanged Nox4 mRNA levels (Görg et al., 2019). These findings suggest an important role of redoximiRs for gene expression changes relevant to the pathogenesis of HE.

­Oxidative stress and astrocyte senescence in HE Oxidative stress is well-known to trigger premature senescence that arrests the cell cycle and renders cells unresponsive toward growth factors and further signals being crucial for the regulation of many cell functions (Chen, 2000). In several neurodegenerative diseases, cerebral oxidative stress strongly correlates with an upregulation of surrogate markers for senescence in the brain (Chinta et al., 2015; Nagelhus et al., 2013), and cognitive dysfunction in a mouse model for Alzheimer’s disease was recently identified as a consequence of senescence in astroglia (Bussian et al., 2018). In line with a role of oxidative stress for senescence, surrogate markers for senescence were also upregulated in postmortem brain biopsies from patients with liver cirrhosis with HE but not in those without HE (Görg et  al., 2015). As senescent astrocytes lose the ability to stabilize synaptic contacts, astrocyte senescence may disturb synaptic connectivity and thereby impair neurotransmission (Kawano et al., 2012). In this light, astrocyte senescence may explain the recent clinical observation that symptoms of HE may not fully resolve after the resolution of an acute episode of overt HE (Bajaj et al., 2010; Riggio et al., 2011). Studies on ammonia-exposed cultured rat astrocytes confirmed a central role of oxidative stress for the induction of senescence in HE. Here, it was shown that oxidative stress triggers senescence via p38MAPK activation and p53-dependent transcription of the cell cycle inhibitory genes p21 and GADD45α and nuclear accumulation of p21 protein (Görg et al., 2015). Findings from the same study further indicated that these senescent astrocytes become unresponsive toward brain-derived growth factor (BDNF)-dependent actin remodeling, which is thought to be required for synapse stabilization. Further findings indicated a role of HO1 for ammonia-induced senescence in cultured astrocytes (Oenarto et al., 2016). This study also showed that the upregulation of HO1 mRNA is due to a downregulation of the HO1-targeting miRNAs 326-3p, 221-3p, and 221-5p (Oenarto et al., 2016) (Fig. 4). It was postulated that HO1 may induce astrocyte senescence by triggering the Fenton reaction through the liberation of ferrous iron from heme (Görg et al., 2018; Oenarto et al., 2016). The H2O2 required for the Fenton reaction will be supplied by Nox4, which is a predicted target of 326-3p and which is upregulated most likely as a consequence of downregulation of miR326-3p by ammonia (Fig. 4). In line with this, knockdown of HO1 or

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chelation of ferrous iron fully prevented the ammonia-induced RNA oxidation and induction of senescence (Görg et al., 2019). Furthermore, upregulation of HO1 in an animal model for HE was accompanied by oxidative stress and behavioral abnormalities, which were prevented by the HO1 inhibitor zinc protoporphyrin (Wang, Yin, Duan, Guo, & Sun, 2013). Importantly, significantly elevated HO1 mRNA levels were also found in postmortem brain tissue from patients with liver cirrhosis and HE but not in those without HE (Görg, Bidmon, & Häussinger, 2013).

­O-GlcNAcylation and oxidative stress in astrocytes Another covalent protein modification is O-GlcNAcylation, that is, the attachment of N-acetylglucosamine to serine or threonine residues of proteins (for a review, see Yang & Qian, 2017). O-GlcNAcylation is stress and nutrient triggered and a reversible protein modification, which relies on intracellular levels of glucosamine6-­phosphate (GlcN-6P) and which is synthesized from glutamine and fructose6-phosphate by the glutamine/fructose amidotransferases (GFAT) 1 and 2 within the hexosamine biosynthetic pathway (HBP) (Yang & Qian, 2017). Elevated intracellular levels of GlcN-6P (Görg et  al., 2019) and increased O-GlcNAcylation (Karababa et  al., 2014) were also observed in astrocytes in response to ammonia. Apart from other proteins, GAPDH becomes O-GlcNAcylated in ­ammonia-exposed astrocytes (Karababa et al., 2014). Interestingly, O-GlcNAcylation of GAPDH was shown to trigger a nuclear accumulation of GAPDH with so far unknown functional consequences (Park, Han, Kim, Kang, & Kim, 2009). While oxidative stress was shown to stimulate the O-GlcNAcylation of proteins in the ­neuroblastoma-derived SH-SY5Y cell line (Katai et al., 2016), our own studies with rat astrocytes indicate that the ammonia-induced O-GlcNAcylation is not a consequence, but rather a trigger for oxidative stress (Görg et al., 2019). In line with this, knockdown of GFAT 1 and 2 prevented the ammonia-induced RNA oxidation, which is triggered by an ammonia-induced upregulation of HO1 and induction of the Fenton reaction (Görg et  al., 2019). Upregulation of HO1 results from an O-GlcNAcylation-dependent transcription inhibition of the HO1targeting miR326-3p, which may also upregulate Nox4 (Görg et al., 2019). While the underlying mechanism is not yet settled, it was speculated that RNA polymerase II becomes inactivated by O-GlcNAcylation at the pri-miR326-3p transcription site (Görg et al., 2019). Consistent with the central role of HO1 and oxidative stress for the induction of astrocyte senescence by ammonia, knockdown of GFAT1 and 2 fully prevented the ammonia-induced senescence in cultured astrocytes (Görg et al., 2019) (Fig. 6). Importantly, significantly elevated levels of O-GlcNAcylated proteins were also found in postmortem brain tissue from patients with liver cirrhosis and HE but not in those without HE (Görg et al., 2019).

­O-GlcNAcylation and oxidative stress in astrocytes

FIG. 6 Proposed mechanism underlying the ammonia-induced RNA oxidation and senescence in cultured astrocytes. The expression of HO1 and Nox4 in the astrocyte is controlled by miR-326-3p (left side). Ammonia elevates intracellular glutamine and glucosamine-6-P (GlcN-6P) levels by the actions of glutamine synthetase (GS) and glutamine/fructose-6P amidotransferases 1 and 2 (GFAT1/2). Elevated intracellular GlcN-6-P levels trigger the O-GlcNAc-transferase (OGT)-dependent O-GlcNAcylation and inactivation of RNA polymerase II (RNAPII). As a consequence, miR326-p levels decrease, and HO1 and Nox4 levels concomitantly increase. HO1-dependent liberation of Fe(II) and Nox4dependent H2O2 formation triggers the oxidation of RNA and the induction of senescence through activation of the Fenton reaction. Modified from Görg, B., Karababa, A., Schütz, E., Paluschinski, M., Schrimpf, A., Shafigullina, A., Castoldi, M., Bidmon, H. J. & Häussinger, D. (2019). O-GlcNAcylation-dependent upregulation of HO1 triggers ­ammonia-induced oxidative stress and senescence in HE. Journal of Hepatology. doi:10.1016/j. jhep.2019.06.020.

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­Concluding remarks The findings summarized in the present article demonstrate a central role of oxidative/nitrosative stress in the pathogenesis of HE. However, it appears unlikely that antioxidants may represent a therapeutic option for the treatment of HE since they may interfere with physiological RONS-dependent signaling pathways and thereby may produce undesired side effects. Given the important role of HO1 for astrocyte senescence and cognitive impairment in animal models for HE, selective HO1 inhibition could be a potential approach for the treatment of HE.

­Funding Our own studies reported here were supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) through Sonderforschungsbereiche 575 “Experimental Hepatology” and SFB 974 “Communication and Systems Relevance in Liver Injury and Regeneration”, Projektnummer 190586431—(Düsseldorf, Germany).

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ROS signaling in complex systems: The gut

33 Ulla G. Knaus

The UCD Conway Institute of Biomolecular and Biomedical Research, School of Medicine, University College Dublin, Dublin, Ireland

Abstract The gastrointestinal tract is a highly complex ecosystem relying on constant host-microbe interactions. Many modes of dialogue exist in this setting, with chemical mediators serving as a rapid, highly effective way for altering the local environment. Reactive oxygen species (ROS), especially hydrogen peroxide (H2O2), are well suited to relay signals that induce target responses and modify the overall chemical milieu. Here, we discuss the main eukaryotic and prokaryotic ROS sources in the intestine, namely, NADPH oxidases, mitochondria, and lactobacilli, and how primary ROS generation is a prerequisite for intestinal health. In contrast, permanent changes in ROS levels, either due to inactivating NADPH oxidase variants (oxidative hypostress) or induced by persistently elevated ROS generation (oxidative hyperstress) will lead to intestinal disease. The integration of intestinal multi-ROS source conditions into a unified model that accounts simultaneously for rapid turnover of highly reactive species and the presence of various H2O2 gradients, both inducing constantly fluctuating ROS levels, will be required for understanding and therapeutically modifying the gut environment. ­Keywords: Gastrointestinal tract, Reactive oxygen species, ROS, NADPH oxidases, NOX, DUOX, Lactobacillus, Gut barrier, Inflammatory bowel disease, Host defense

­Introduction The gastrointestinal tract is a unique ecosystem characterized by extensive hostmicrobe contact. As bacterial numbers increase along the small intestine (103–108/ mL content), reaching 1011/mL content in the colon (Sender, Fuchs, & Milo, 2016), a single layer of intestinal epithelium not only forms a physical barrier but also provides mucosal immune responses, communicates with microbes, and coordinates innate and adaptive immunity. The gut epithelium discriminates between commensal microbes (microbiome) or luminal antigens by suppressing an immune response (intestinal tolerance) (Mowat, 2018) while mounting defensive responses against pathogenic microbes. This is accomplished by using multifaceted strategies that are based on unique and specialized functions of the many different cell types ­present Oxidative Stress. https://doi.org/10.1016/B978-0-12-818606-0.00033-X © 2020 Elsevier Inc. All rights reserved.

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in ­various segments of the intestinal epithelium. Examples are enterocytes for absorption, ­ mucin-secreting goblet cells, hormone-secreting enteroendocrine cells, chemosensory tuft cells, Paneth cells releasing antimicrobial factors, and M cells for antigen uptake (Allaire et al., 2018). All of these cell types can react to exogenous and endogenous signals and integrate them into a coordinated response. The interconnectivity of epithelium and gut flora delivers benefits for both host and microbe. For instance, the microbiota provides the primary protection against intestinal pathogens, also termed colonization resistance (Sorbara & Pamer, 2019), and supports intestinal epithelial cell (IEC) functions with microbial-derived metabolites (e.g., butyrate, acetate, and lactate). In return, IECs sustain bacterial colonization niches via mucus secretion and proteinaceous/chemical signals and restrict oxygen (O2) availability, which is important for the thriving of obligate anaerobe bacteria and preserves microbiota diversity. Early signs of intestinal instability and disruption of homeostatic processes are changes in the microbiota with a reduction in the bacterial load and loss of diversity (dysbiosis), which is closely connected to an altered chemical environment in the gut. This chemical milieu is modified by many different anorganic and organic compounds derived from bacteria and the host, but the focus here will be on reactive oxygen species (ROS), which are intimately connected to oxygen and nitrogen levels and play an essential role in gut homeostasis and intestinal disease. Sources for generation of primary ROS (O2•−, H2O2) at the gut barrier are host NADPH oxidases, mainly epithelial NOX1 and DUOX2, the mitochondrial electron transport chain (mETC), xanthine oxidase, monoamine oxidases, and lipoxygenases, as well as bacterial sources such as H2O2-producing lactobacilli. In infections or during inflammatory processes, superoxide production will increase due to mitochondrial dysfunction and/or the NOX2-mediated oxidative burst of recruited neutrophils and activated macrophages, which will give rise to secondary ROS (ONOO−, HOCl). Subsequently, the mucosa will become hypoxic, which is required for restitutive functions carried out by neutrophils (Campbell & Colgan, 2015). How neutrophils participate in wound healing is still unresolved, but it may not require downregulating superoxide production (100% O2•− output by neutrophils at 1% O2) (Gabig, Bearman, & Babior, 1979). Changes in O2 availability, primary and secondary ROS, and nitric oxide metabolites at the barrier will alter bacterial niche occupation on several levels (nutrients, mucin attachment sites, intrabacterial signaling, and transcription), thus shifting the composition of the microbiota. These changes in the gut ecosystem can be either beneficial or disease-associated.

­ROS-generating enzymes in the intestinal mucosa The focus of this review will be on ROS in the intestinal epithelium and in the lamina propria, although ROS generation by other cell types in the mucosa including mast cells, fibroblasts, and smooth muscle cells and changes in thiol/disulfide redox systems will impact intestinal homeostasis and disease (Aviello & Knaus, 2018; Circu & Aw, 2011). In terms of intestinal ROS sources, NADPH oxidases are the only

ROS-generating enzymes in the intestinal mucosa

mammalian enzymes solely dedicated to primary ROS generation. Oxidases are usually tightly controlled by the input of multiple regulatory factors, and their biological functions are either evident due to disease-causing human variants or can be addressed by using genetically modified mice. Other enzymes produce primary ROS as a by-product of their main biological function, for example, mitochondrial complex I and III. Specific regulatory mechanisms may exist for the mETC, for example, for altering forward electron flow through complex I (NADH dehydrogenase) or complex III (ubisemiquinone), or for inducing reverse electron flow, which both will increase electron leakage and reduction of O2 to superoxide, but not much is yet known about specialized proteins regulating these events. Mitochondrial ROS (mtROS) participate in immune sensing, redox signaling, and stress-induced responses in the gastrointestinal tract and have been extensively reviewed (Arnoult, Soares, Tattoli, & Girardin, 2011; Berger et al., 2016; Cao & Kaufman, 2014; Clark & Mach, 2017; Dan Dunn, Alvarez, Zhang, & Soldati, 2015; Mehta, Weinberg, & Chandel, 2017; Pinegin, Vorobjeva, Pashenkov, & Chernyak, 2018; Shadel & Horvath, 2015; West, Shadel, & Ghosh, 2011). Xanthine oxidase, which generates O2•− when breaking down hypoxanthine and xanthine to uric acid, has also been associated with immune signaling (Crane, 2013; Ives et al., 2015; Vorbach, Harrison, & Capecchi, 2003). It is not always straightforward to determine the critical ROS generator for regulating a certain cellular pathway or biological process. ROS can regulate other ROS sources, or several ROS sources can act concomitantly, which often impedes identifying the key enzyme. Due to their prominent roles in mucosal immunity and their apparent association with intestinal disease, the focus here is on NADPH oxidases (NOX/ DUOX enzymes). NOX1 is mainly expressed in colonic and rectal IECs (detected as mRNA), either within the lower crypts or on the luminal surface (Geiszt et al., 2003; Szanto et al., 2005). The NOX1 complex translocated in colon cancer cells and human biopsies from internal membranes to the plasma membrane and sites of membrane ruffling when attachment or invasion of bacteria was sensed (Alvarez et al., 2016; Corcionivoschi et al., 2012). The active NOX1 enzyme is composed of a membranebound NOX1-p22phox heterodimer, the adapter NOXO1, the activator NOXA1, and the Rho GTPase Rac1 (Geiszt, Lekstrom, Witta, & Leto, 2003; Takeya et al., 2003). NOX1 generates low levels of O2•− constitutively due to the formation of a low affinity complex between NOXO1 and p22phox (Dutta & Rittinger, 2010). Stimulation of the enzyme will induce a high affinity complex that includes Rac1 in its active, GTPbound form and NOXA1 and requires phosphorylation events to augment superoxide production (Cheng, Diebold, Hughes, & Lambeth, 2006; Miyano, Ueno, Takeya, & Sumimoto, 2006; Streeter et al., 2014; Ueyama, Geiszt, & Leto, 2006). Physiological stimuli of NOX1 in the intestine include bacteria and bacterial ligands acting through toll-like receptors or formyl peptide receptors (Corcionivoschi et  al., 2012; Jones et al., 2013; Lee et al., 2013). DUOX2 mRNA and protein can be found all along the intestine with peak expression in the ileum (El Hassani et  al., 2005; Grasberger et  al., 2015). In infectious disease, Duox2 is transcriptionally upregulated in the colon, and this enhanced

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e­ xpression was under control of a Nox1-Ask1-p38MAPK-Atf2 pathway in a murine Citrobacter rodentium infection model (Pircalabioru et al., 2016). The p38 pathway together with MyD88 signaling was also linked to colonic Duox2 expression using knockout mice and pharmacological inhibitors, while ileal Duox2 expression is regulated by the TLR3/4 adapter TRIF and canonical NF-κB signaling (Sommer & Backhed, 2015). Murine Duox2 expression is dependent on the microbiota, in particular on segmented filamentous bacteria (SFB), as ileal Duox2 expression is negligible in germ-free mice (Grasberger et al., 2015). DUOX2 forms a membrane-bound complex with its partner protein DUOXA2 on the IEC cell surface and is activated by calcium flux and phosphorylation, releasing H2O2 into the extracellular milieu. The NOX2 complex, composed of the NOX2-p22phox heterodimer, p47phox, p67phox, p40phox, and Rac2, is mainly expressed in innate immune cells. In homeostasis, the lamina propria (LP) contains mainly macrophage and dendritic cell subsets. CD103+ dendritic cells are essential for sampling luminal content, either directly by entering the epithelial layer or by uptake after transport through goblet cells (Farache et al., 2013; McDole et al., 2012), and for delivery of these antigens to the mesenteric lymph nodes (MLN). Superoxide production by NOX2 modulates the antigenic repertoire for presentation to CD4+ and CD8+ T cells (Joffre, Segura, Savina, & Amigorena, 2012). Resident LP macrophages and surveying neutrophils protect the host from systemic dissemination if microbes manage to breach the epithelial barrier. Their repertoire entails phagocytosis, NOX2- and NOS2-dependent antimicrobial activity, and cytokine production, while other macrophage subsets express receptors for anti-inflammatory cytokines such as IL-10 or induce a wound healing program when necessary (Davies, Jenkins, Allen, & Taylor, 2013; Gross, Salame, & Jung, 2015; Rivollier, He, Kole, Valatas, & Kelsall, 2012).

­ROS in maintenance of intestinal homeostasis Primary ROS and in particular the less reactive H2O2 participate in many processes essential for gut barrier protection (Aviello & Knaus, 2018; Perez, Talens-Visconti, Rius-Perez, Finamor, & Sastre, 2017). All IEC subtypes are renewed every 2–5 days from intestinal stem cells (Lgr5+ ISC), which are located at the bottom of crypts. This intestinal regeneration is dependent on autophagy and balanced mtROS generation (Asano et al., 2017). On the other hand, Wnt-driven proliferation of IECs has been linked to Rac1-, Nox1-, and Noxo1-dependent O2•− generation (Cheung et al., 2016; Coant et  al., 2010; Moll et  al., 2018; Myant et  al., 2013). While control of junction complexes and epithelial permeability seem not to require Nox1 or Duox2 (Aviello & Knaus, 2018), mucin secretion by goblet cells and thus maintenance of the mucus layer(s) covering the epithelium are dependent on ROS produced by NADPH oxidases. Pharmacological inhibitors revealed that a ROS-dependent step, which connects TLR signaling with the NLRP6 inflammasome, is driving goblet cell exocytosis (Birchenough, Nystrom, Johansson, & Hansson, 2016). Others linked Nox-mediated ROS generation to the autophagy machinery via Atg5 in goblet cells

ROS in maintenance of intestinal homeostasis

(Patel et al., 2013). Addition of exogenous H2O2 resolved the block in mucin granule exocytosis, suggesting that H2O2 released by colonizing lactobacilli may be able to overcome certain goblet cell deficiencies (Ahl et al., 2016; Morampudi et al., 2016). Recently, the catalytic activity of Nox enzymes expressed in epithelium and innate immune cells (Nox1-3) was conclusively linked to dense mucus layer generation in mice (Aviello et al., 2019). Cytoskeletal reorganization and associated cellular functions such as migration and integrin engagement are controlled in IECs, similar as in other cell types, by redox signaling. Nox1 has been linked to phosphorylation of cytoskeletal regulators via H2O2-induced phosphatase inactivation and/or tyrosine kinase activation (Leoni et al., 2013; Sadok et al., 2009). These tasks are important for wound healing and can also be induced by probiotic bacteria (Alam et al., 2014; Swanson 2nd et al., 2011). Gut barrier maintenance involves constant communication between host and microbiota (Fig.  1). Diffusible nanomolar H2O2 released by the host shapes the microbiota community structure by altering O2 availability and secretion of antibacterial lectins and by directly intervening with intrabacterial phosphotyrosine signaling (Alvarez et al., 2016; Corcionivoschi et al., 2012). Commensal bacteria release factors (e.g., anorganic and organic compounds, peptides, lactate, and H2O2) that modulate epithelial junctions, induce secretory processes, and alter host signaling pathways. For example, lactobacilli stimulated Nox1-mediated O2•− generation in the murine colon (Jones et  al., 2013), while presence and attachment of SFB induced Duox2 expression in the ileum (Grasberger et  al., 2015). How lactobacilli activate Nox1 is unresolved as the separation of probiotic bacteria from the colonic epithelium by a dense mucus layer necessitates a soluble, secreted mediator. In Drosophila, Lactobacillus plantarum-derived lactate was implicated in dNox1 activation (Iatsenko, Boquete, & Lemaitre, 2018), but the relevance for the mammalian gut is not certain. Drosophila oxidases seem to respond differently to stimulatory

FIG. 1 Multidirectional ROS signaling in the intestinal mucosa. H2O2 can be generated and released by the immune system, the epithelium, and the microbiota. This multi-ROS source environment leads to short-range and long-range redox signaling, affecting host, microbiome, and metabolome.

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compounds as only dDuox (Ha, Oh, Bae, & Lee, 2005), but not human DUOX2, is activated by uracil (unpublished observations). Maintaining H2O2 at nanomolar concentrations at the epithelial barrier is essential for gut health in mammals. ROS deficiency due to complete Nox1/4 inactivation in the intestinal epithelium or globally (Cyba∆IEC and Cyba−/−) caused mucus alterations that permitted massive overgrowth of endogenous lactobacilli (L. murinus and L. reuteri), which provided H2O2 (and lactate and other secreted compounds) for epithelial homeostasis and host defense functions when Nox activity was absent (Pircalabioru et al., 2016).

­Intestinal disease: Too much ROS Gastrointestinal diseases including inflammatory bowel diseases (IBD; ulcerative colitis [UC] and Crohn’s disease [CD]), colorectal and gastric cancer, alcoholic hepatitis, and pancreatitis have been connected to oxidative distress for many years (Kim, Kim, & Hahm, 2012; Patlevic, Vaskova, Svorc Jr, Vasko, & Svorc, 2016). In the acute phase of UC, the mucosa and submucosa of the colon are inflamed, while CD is characterized by patchy inflammatory foci with granulomas that often affect the terminal ileum and perianal region. Overexpression of ROS sources (e.g., DUOX2) (Aerssens et al., 2008; Csillag et al., 2007; Haberman et al., 2014; Lipinski et al., 2009; Macfie et al., 2014; Mirza et al., 2015), activation of ROS sources (e.g., mETC and xanthine oxidase) (Boda, Nemeth, & Boda, 1999; Mottawea et al., 2016; Novak & Mollen, 2015), and changes in the expression of antioxidant systems (e.g., superoxide dismutase and glutathione peroxidase) (Hoffenberg, Deutsch, Smith, & Sokol, 1997; Ishihara et  al., 2009; Piechota-Polanczyk & Fichna, 2014; Tian, Wang, & Zhang, 2017) have been detected in IBD, but how these changes affect disease severity or progression is not known. Analysis of inflamed patient tissues revealed oxidative modifications on proteins, lipids, and DNA and protein nitration, strongly suggesting that secondary ROS with high reactivity such as hypochlorous acid (HOCl), hydroxyl radical (•OH), and/or peroxynitrous acid (ONOOH) were formed. Mitochondria and hyperactivated neutrophils in connection with inducible nitric oxide synthetase (NOS2) have been suggested as sources of damaging ROS. The presence or upregulation of a ROS-generating enzyme does not per se correlate with oxidative damage. Studies on IBD patient neutrophils or macrophages are inconclusive with some reports indicating an increased capacity to generate superoxide (Alzoghaibi, 2013; Naito, Takagi, & Yoshikawa, 2007), while others reported decreased neutrophil O2•− production in a fairly large subset of CD patients (Denson et al., 2018). In addition, neutrophils are increasingly recognized for participating in the resolution of inflammation, and the loss of NOX2-derived superoxide production in innate immune cells results in hyperinflammation, partially due to deregulated signaling pathways (Campbell et al., 2014; Denning & Parkos, 2013; O'Neill, Brault, Stasia, & Knaus, 2015; Sumagin et  al., 2016). Secondary ROS together with the formation of neutrophil extracellular traps will have a bactericidal effect, thereby decreasing the bacterial load, but the composition of the microbiota will also be altered,

Intestinal disease: Not enough ROS

for example, by local hypoxia that accompanies the oxidative burst. The microbiota of genetically modified mice with partial abrogation of intestinal ROS generation showed distinct changes (Aviello et  al., 2019; Falcone et  al., 2016; Matziouridou et  al., 2018; Pircalabioru et  al., 2016), but patient studies and evaluation of high ROS conditions are still missing. One can assume that both long-term high ROS and persistently low ROS levels in the intestine will change the microbiota composition, which can be harmful or advantageous. As oxidative distress is regarded as one of the key contributors to tissue injury, many pharmacological approaches have been taken to reduce ROS. Antioxidants such as supplying superoxide dismutase or catalase for primary ROS conversion or removal and radical scavengers have been used (Moura, De Andrade, Dos Santos, Araujo, & Goulart, 2015). While protective effects have been observed in mouse models of colitis using some of these drugs, strong data sets with significant IBD patient cohorts and benefit analysis are missing (Khan, Samson, & Grover, 2017; Moura et  al., 2015). Antioxidants have not performed well in other diseases with high ROS burden and are likely detrimental when administered over long periods as these substances will, if effective, deregulate vital redox signaling pathways (Egea et al., 2017, Le Gal et al., 2015, Peris et al., 2019). Newer approaches target a specific compartment, for example, MitoQ, a mitochondrion-targeted derivative of the antioxidant ubiquinone (Dashdorj et al., 2013; Oyewole & Birch-Machin, 2015), or biguanides such as metformin, which inhibit O2•− generation by complex I (Kelly, Tannahill, Murphy, & O'Neill, 2015), although an increase in mtROS production by metformin was also reported (Bridges, Jones, Pollak, & Hirst, 2014; Mogavero et al., 2017). In conclusion, current evidence supports an involvement of damaged or dysregulated mitochondria in generating ROS that may perpetuate inflammation, but the critical cell type(s) and the nature of the reactive species are unresolved. Superoxide production by activated neutrophils constitutes an early event in the inflammation of the gut, but one has to consider that phagocyte NOX2 is controlled by elaborate onoff steps that will limit catalytic output and that its activity is essential for effective antimicrobial host responses. Further, restitutive neutrophil subsets are emerging, suggesting that restricting neutrophil activity may lead to adverse effects.

­Intestinal disease: Not enough ROS The view of oxidative distress uniquely contributing to the development and persistence of intestinal inflammation has been challenged by advances in whole-genome, whole-exome and targeted next-generation sequencing of IBD patients. In particular, sequencing of very early onset IBD (VEOIBD) patients revealed monogenic ­defects, often pathogenic variants in genes associated with the immune response or gut ­barrier function (Leung & Muise, 2018; Uhlig et al., 2014). This work identified in a cohort of VEOIBD patients rare, inherited, or de novo loss-of-function mutations in the NADPH oxidases NOX1 and DUOX2 as risk factor for IBD (Hayes et al., 2015; Lipinski et  al., 2019; Parlato et  al., 2017; Schwerd et  al., 2018). Decreased ROS

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generation by NOX1 or DUOX2 was associated with impaired resistance toward pathogens, suggesting that compromised host defense functions may contribute to IBD. Inactivating variants in components of the phagocyte NOX2 complex (CYBB, CYBA, NCF1, NCF2, and NCF4) were also discovered in VEOIBD (Dhillon et al., 2014). In contrast to pathogenic NOX2 complex variants causing chronic granulomatous disease (CGD), an inherited severe immunodeficiency characterized by life-threatening infections, VEOIBD-associated NOX2 complex variants produced O2•− levels that exceeded the threshold for full onset of CGD (Denson et al., 2018; Kuhns et  al., 2010). Partially inactivating NOX/DUOX variants are currently considered IBD risk alleles, while CGD-associated NOX2 complex variants are causally linked to intestinal inflammation reminiscent of CD (CGD-IBD), which occurs in 30%–80% of CGD patients (Alimchandani et  al., 2013; Huang et  al., 2016; Pellicciotta et al., 2019). Thus, deficiency in ROS generation in the innate immune compartment and/or at the epithelial barrier amplifies susceptibility for intestinal inflammation. Decreased ROS levels will impede intracellular redox signaling, host defense, and cytoskeletal reorganization required for migration and wound healing. CGD patients and Nox2 knockout mice mount an exaggerated immune response with increased numbers of transmigrating neutrophils and enhanced cytokine levels. This hyperinflammatory phenotype is at least partially connected to abrogated redox regulation of phosphatase activity (Meng, Fukada, & Tonks, 2002; O'Neill et  al., 2015). Albeit NOX/DUOX mutants are rare and inactivating mutations of proteins involved in mETC-derived superoxide generation have not yet been identified, these ROS sources represent signaling hubs, activated by various receptors and signaling pathways. Any disturbances of upstream signaling (e.g., pathogenic receptor variants and drug-induced block) will cause inefficient and/or delayed stimulation of ROSgenerating enzymes, thereby increasing the prevalence of disease-associated ROS deficiencies. In light of the essential cellular functions supported by NADPH oxidase-­produced ROS in the intestine, such as redox signaling, mucin secretion, wound healing, and host defense, it is apparent why inactivating NOX/DUOX mutations predispose to intestinal disease. Specifically, host defense-related tasks are an area where permanently generating insufficient ROS levels will weaken defense systems. ROSmediated antimicrobial killing in the phagosome of neutrophils and macrophages constitutes the main host defense system against invading pathogens. However, destruction of intracellular pathogens by autophagy, antigen presentation by dendritic cells, and microbial sensing are also dependent on ROS (Pinegin et al., 2018; Rada & Leto, 2008). Even ROS generation at the epithelial barrier, by host enzymes and by mucus colonizing lactobacilli, contributes to antimicrobial protection. Release of nanomolar H2O2 at the barrier downregulated signaling pathways in bacteria after intrabacterial conversion to secondary ROS took place. For example, nano- to submicromolar H2O2 disrupted the tyrosine phosphorylation network in bacteria by oxidative dephosphorylation in combination with irreversible chemical modification of key phosphotyrosine residues (i.e., protein-bound DOPA), which changed protein activity without affecting bacterial viability (Alvarez et  al., 2016; Corcionivoschi

­Conclusions and future directions

et al., 2012). In vitro and in vivo exposure of enteropathogenic Escherichia coli to nanomolar H2O2 inhibited transcription of the LEE pathogenicity island by a yet unknown mechanism (Pircalabioru et al., 2016). These processes not only are hostand probiotic-initiated antivirulence strategies that will reduce the fitness of pathogens in the extracellular space but also will influence the microbiota community structure. Microbiome studies in CGD patients or IBD patients with low intestinal ROS generation are rare (Sokol et al., 2019), however ROS deficiency in mice alters the microbiota composition. In a mouse model of CGD (Ncf1−/−; p47phox knockout) abundance of mucolytic Akkermansia muciniphila was highly increased in homeostasis (Falcone et al., 2016). Complete inactivation of Nox1/4 catalytic activity by knockout of p22phox (Cyba−/− and Cyba∆IEC) triggered an excessive load of lactobacilli (Pircalabioru et al., 2016). Overgrowth of lactobacilli or supplementation of the endogenous microbiota with L. johnsonii wild type but not with a mutant deficient in H2O2 production (L. johnsonii ∆nfr) protected the host from enteropathogenic E. coli infection (Pircalabioru et al., 2016). Additionally, increasing H2O2 levels in the gut via Lactobacillus administration provided a significant benefit for tissue restitution in the recovery phase of colitis (Singh, Hertzberger, & Knaus, 2018), which is consistent with observations in dermal wound healing (Roy, Khanna, Nallu, Hunt, & Sen, 2006). In conclusion, inducing or supplying physiological concentrations of H2O2 to the intestinal barrier is, independently of the source, beneficial to the organism.

­Conclusions and future directions Primary ROS generated in the gut are mainly connected to redox signaling and communication between and across eukaryote/prokaryote boundaries (intracellular, cell-cell, cell-microbe, microbe-cell, and microbe-microbe). For these tasks, the location of a specific ROS source is critical. For example, mtROS participates in transmitting immune responses that involve mitochondrion-specific events such as the oxidation of newly synthesized mtDNA that, after release into the cytosol, binds and activates the NLRP3 inflammasome (Shimada et al., 2012; Zhong et al., 2018), or the mtROS-induced oligomerization and activation of MAVS, an antiviral signaling and lupus-associated protein, which is located on the outer mitochondrial membrane (Buskiewicz et  al., 2016). Thus, mtROS similar to NOX/DUOX-generated ROS serve as immune system modulator and protective defense system. In contrast, evidence is mounting that damaged mitochondria and a deregulated mETC contribute to intestinal inflammation. Their role in perpetuating oxidative distress and the benefit of drugs removing mtROS in chronic inflammation is increasingly apparent, but mtROS is likely not the initiator of the inflammatory reaction, and potential adverse effects of long-term application of mtROS scavengers and mETC inhibitors will need to be addressed. H2O2 generated by plasma membrane-localized enzymes including certain NOX/DUOX family members will permit outside-in signaling and communication

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o­ utward. Processes such as phagocytosis or bacterial attachment trigger translocation and recruitment of NOX enzymes to membranes at the site of engagement (e.g., phagosome and caveolae). Localization or recruitment of ROS sources to junction complexes or cytoskeletal structures will propagate redox signaling that has profound effects on cell-cell contacts or cell-matrix interactions, thereby generating redox signals that alter physical properties of the cell (Wilson, Terman, GonzalezBillault, & Ahmed, 2016). While it will be important to understand the specialization of ROS sources for transmitting a particular signal, for example, for pharmacological targeting, one can also envision that in some settings, other ROS-generating enzymes can, at least partially, compensate when ROS signals or ROS-regulated pathways are permanently downregulated. Built-in redundancy or in-need upregulation, especially in vivo, may provide enough ROS for vital cellular tasks as long as ROS levels do not fall below a certain threshold. Stimulation of mtROS production by the peroxisome proliferator-activated receptor gamma agonist pioglitazone restored bacterial killing in Nox2-deficient mice and in a CGD patient (Fernandez-Boyanapalli et al., 2015; Migliavacca et al., 2016); this putative therapeutic for CGD patients with severe infections is currently in clinical trials. As mentioned, substitution of ROS sources can even occur between bacteria and the host. Compensatory overgrowth of H2O2-producing lactobacilli substituted for inactivated Nox1/4 enzymes in the colon epithelium, providing ROS for homeostasis and antimicrobial defense (Pircalabioru et al., 2016), a prime example of defensive mutualism. We propose that therapeutics of the future will include drugs or biologicals that induce or supply ROS at physiological concentrations for certain disease conditions. The overarching concept in redox biology is the balance between oxidants and antioxidants, with ROS production and ROS removal maintaining equilibrium. This system can be compared with on-off cycles, which trigger one specific signal for a short time. Tightly regulated enzymes such as many NADPH oxidases contain their own catalytic on-off switches, albeit complexity arises due to ­priming-induced changes in ROS levels (NOX2) or constitutive versus stimulated ROS generation (e.g., NOX4 vs. NOX2). Other enzymes at various cellular locations might be “on” for an extended time, requiring constant conversion and removal of ROS. However, in vivo cells exist in a multi-ROS source environment where each cell is exposed to multiple inputs (chemical, biological, and physical) and is connected to other cells (e.g., transmigration of cells and associated cell types), microbial environments (e.g., the lung and gut) and exogenous substances. Thus, multiple highly divergent ROS signals of different strength and duration will be generated, transmitted, received, converted, and removed. In all likelihood, physiological H2O2 levels will always persists in this cellular environment, not in a steady state but fluctuating across a certain homeostatic range (Fig. 2). We propose that oxidative hyperstress (leading to oxidative distress) and oxidative hypostress caused by primary ROS deficiency will not permit these homeostatic fluctuations or only in a very limited range. Short exposure to high ROS levels will be tolerated due to various protection and removal systems, but permanent exposure will trigger inappropriate signals and reactive ­species conversion to more damaging

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FIG. 2 ROS fluctuations maintain homeostasis in a multi-ROS source environment. In physiological conditions, ROS levels will continuously change over a wide range (oxidative fluctuations), while disease states are characterized by permanent low ROS generation (oxidative hypostress) due to loss-of-function gene variants or drugs or by persistently high ROS levels (oxidative hyperstress) with increased production of secondary ROS.

secondary ROS. In permanent low ROS s­ cenarios ­(oxidative hypostress), certain redox signals persist due to proximity or high affinity of the target, while others are disrupted. As the remaining H2O2 still fulfills some of its signaling function, the system will have less pressure for counterregulation and compensation. Hence, persistently low ROS levels will result in deficiencies and physiological stress responses that will give rise to damaging long-term effects. Evolving knowledge and integration of simultaneous H2O2 gradients into a multicellular context will prove or disprove the validity of our concept.

­Acknowledgment Our work has been primarily supported by the National Institutes of Health (United States), Science Foundation Ireland, and the National Children’s Research Center (both Ireland). I would like to thank PhD students and postdoctoral fellows in my laboratory and colleagues in the NOX and GTPase field who contributed and/or supported our research.

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Oxidative stress in skeletal muscle: Unraveling the potential beneficial and deleterious roles of reactive oxygen species

M.J. Jackson, N. Pollock, C.A. Staunton, C. Stretton, A. Vasilaki, A. McArdle Department of Musculoskeletal Biology, MRC-Arthritis Research UK Centre for Integrated research into Musculoskeletal Ageing (CIMA), Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, United Kingdom

Abstract Skeletal muscle is a highly malleable tissue and responds to various stresses, such as exercise by increasing the generation of superoxide together with nitric oxide. Initially, investigators assumed that these species and the secondary reactive oxygen species (ROS) derived from them were damaging to muscle and other tissues, but subsequently, there has been recognition of both beneficial and deleterious effects of ROS. This review describes studies that have identified and characterized the generation of ROS by muscle, the effects of contractile activity on this generation, redox signaling effects of oxidants in muscle (oxidative eustress), and deleterious oxidative damage processes leading to tissue degeneration (oxidative distress). Increasingly, these oxidation events are recognized as key components of the muscle’s physiological responses contractile activity and to pathophysiological situations, such as denervation and aging. Translation of this knowledge has the potential to identify beneficial interventions to optimize muscle function in health and disease. ­Keywords: Superoxide, Hydrogen peroxide, Transcription factor, Adaptation, Exercise, Contractions, Muscle fiber, Denervation, Ageing

­Introduction The term oxidative stress as it related to oxidative damage to cells and tissues was coined by Helmut Sies and colleagues in the 1980s (for historical review, see Sies, 2018) and is defined as “a disturbance of the prooxidant-antioxidant environment in favor of the former” (Sies, 1985). The implications of this definition were originally that oxidative stress was potentially deleterious to tissues and cells and that inhibition Oxidative Stress. https://doi.org/10.1016/B978-0-12-818606-0.00034-1 © 2020 Elsevier Inc. All rights reserved.

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or reversal of the stress on cells and tissues would generally be beneficial. This could be potentially achieved by a reduction in the promoters of oxidation (usually free radicals or reactive oxygen species [ROS]) or an increase in substances or pathways that decrease oxidation (antioxidant substances or regulatory proteins). These assumptions underlined many of the original studies to investigate this area in skeletal muscle and in exercise (Brady et al., 1979; Davies et al., 1982; Dillard et al., 1978; Jackson et al., 1985). Particularly, prominent in these studies was the assumption that nutritional antioxidants would be beneficial, and many of the early studies included a component to examine the possibility that antioxidant supplementation could be used as intervention to demonstrate cause and effect of free radicals or ROS (Brady et al., 1979; Dillard et al., 1978). As further studies were undertaken, it rapidly became clear that skeletal muscle could, not only, ROS but also could respond to that generation by upregulation of regulatory pathways (Jackson et al., 2002; McArdle, Dillmann, et al., 2004; McArdle, Spiers, et al., 2004), which prevented the potential for subsequent oxidative damage to the tissue. Thus, ROS in this situation were not necessarily damaging but inducing adaptive changes in tissues. These apparent contrasting roles of ROS have subsequently been described as redox signaling effects compared with oxidative stress and more generally by Helmut Sies and colleagues as oxidative eustress and oxidative distress (Sies et al., 2017) and are the subject of this collection of review articles. In this article, our aim is to briefly describe the history and complexities of differentiating beneficial and deleterious effects of ROS in contracting skeletal muscle both at rest and during exercise.

­Sites of ROS generation in skeletal muscle Skeletal muscle fibers generate superoxide and nitric oxide (NO), and it has been shown that these parent molecules can be converted to several secondary reactive oxygen species (ROS) and reactive nitrogen species (RNS). Intracellular generation of superoxide and NO has been demonstrated within muscle fibers, and superoxide (McArdle et  al., 2001; Reid et  al., 1992), hydrogen peroxide (Vasilaki, Mansouri, et  al., 2006; Vasilaki, McArdle, et  al., 2006), and NO (Balon and Nadler, 1994; Kobzik et al., 1994) are also released into the interstitial space of muscle fibers (or generated on the external face of the muscle plasma membrane). Contractile activity increases the intracellular content or activities of superoxide, hydrogen peroxide (H2O2), and NO (Pye et al., 2007; Reid et al., 1992; Silveira et al., 2003) and superoxide, H2O2, hydroxyl radical, and NO in the muscle interstitial space (McArdle et al., 2001; Pattwell et al., 2004; Vasilaki, Mansouri, et al., 2006; Vasilaki, McArdle, et al., 2006). Initial studies suggested that these ROS might be damaging to exercising muscle following contractions, but most contemporary data indicate that the ROS/RNS generated are likely to activate a number of redox-regulated signaling pathways. These redox-regulated pathways are the subject of extensive research, and some specific redox-regulated processes have been shown to stimulate the expression of genes associated with myogenesis (Bakkar et al., 2008), catabolism, and

Sites of ROS generation in skeletal muscle

mitochondrial biogenesis (Bar-Shai et  al., 2005; Van Gammeren et  al., 2009). We have been particularly interested in the role of ROS in the activation of short-term cytoprotective changes in expression of regulatory enzymes and cytoprotective proteins in response to contractile activity (Hollander et al., 2006; McArdle et al., 2001; McArdle, Dillmann, et  al., 2004; McArdle, Spiers, et  al., 2004). This appears to occur through redox-dependent activation of a number of transcriptional pathways including the transcription factors, NFκB, AP-1, HSF-1, and Nrf2 (Jackson et  al., 2002; Ji et al., 2004; Ristow et al., 2009; Vasilaki, Mansouri, et al., 2006; Vasilaki, McArdle, et al., 2006). Most data on ROS-mediated responses to contractile activity have been generated using nonspecific approaches, but techniques have become increasingly sophisticated such that (for instance) new specific, genetically encoded fluorescent probes, such as HyPer, can report changes in single species in defined subcellular compartments. Initial work on responses of muscle to contractions was based on the assumption that mitochondria were the main source of contractile activity-induced increases in ROS in muscle, but many publications now disagree with this possibility (Powers and Jackson, 2008). NAD(P)H oxidase(s) appear to be the most likely alternative sources, and these enzymes have been detected in skeletal muscle plasma membranes (Javesghani et al., 2002), sarcoplasmic reticulum (Xia et al., 2003), and the T tubules (Espinosa et al., 2006). The T tubule-localized enzyme may be particularly relevant since it has been claimed to be specifically activated by contractions (Espinosa et al., 2006; Pal et al., 2013). We have examined the potential contributions of mitochondrial and nonmitochondrial sources to the acute increase in superoxide seen during muscle contractions (Pearson et al., 2014; Sakellariou et al., 2013) and concluded that NAD(P)H oxidase effects predominated over mitochondria during short contraction periods (10–15 min). In summary, current data indicate that a nonmitochondrial NAD(P)H oxidase (likely to be the Nox2 isoform) is the major source of generation of superoxide during short-term contractile activity. The Nox4 isoform of NAD(P)H oxidase has also been reported to be expressed in mitochondria and sarcoplasmic reticulum of skeletal muscle (Sakellariou et al., 2013; Sun et al., 2010), but its role (if any) in contraction-induced superoxide generation is currently unclear. There is some recent evidence that the Nox4 isoform in cardiac muscle plays a crucial role in adaptive changes to exercise training (Hancock et al., 2018), and it will be relevant to determine whether similar effects are seen in skeletal muscle. A number of specific ROS and RNS are detected in the extracellular space of skeletal muscle myotubes or isolated fibers in culture or in microdialysates from muscle interstitial fluid in vivo. Muscle fibers release superoxide into the extracellular space (McArdle et al., 2001; Reid et al., 1992) although substantial diffusion of superoxide (or its protonated form) through the plasma membrane has been reported to be unlikely (Halliwell and Gutteridge, 1989). This is in contrast to other species, such as H2O2 or NO, that can potentially cross membranes, and hence, although they are detected in the extracellular space, they may originate from intracellular sites. Javesghani et al. (2002) reported that a plasma membrane-localized NAD(P)H oxidase could release superoxide to the external face of the membrane,

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and Ward et al. (2014) have described a stretch-activated NAD(P)H oxidase (Nox2 isoform) that plays a major role in contraction-induced ROS generation in cardiac myocytes. This enzyme is also reported to be present in the skeletal muscle plasma membrane and appears to release superoxide to the outside of the cell. Finally in this area, Pal et  al. (2013) have described a novel biosensor to report NAD(P)H oxidase activation in contracting skeletal muscle, which demonstrated colocalization of a NAD(P)H oxidase (Nox2) that was activated by contractile activity with the ryanodine receptor at the triads. Other NAD(P)H-dependent systems for the generation of superoxide have also been suggested to play a role (Jackson, 2008). In muscle in vivo or intact muscle preparations ex vivo, xanthine oxidase enzymes in the endothelium have been proposed to play an important role in contractioninduced release of superoxide (Gomez-Cabrera et  al., 2010), and this enzyme has been claimed to be important in adaptations of muscle to contractile activity (Gomez-Cabrera et al., 2005). This summary has focused on the generation of ROS in skeletal muscle in response to contractile activity, but it is important to note that mitochondria are likely to be important sites for ROS generation in skeletal muscle in other physiological and pathological situations, such as following exposure to proinflammatory stimuli (Li et al., 1999; Lightfoot et al., 2015). Fig. 1 summarizes our current understanding of the key sites for the generation of ROS in skeletal muscle fibers at rest and during contractile activity.

­Functions of physiological ROS in muscle Although there is now some clarity over the sources of ROS in contracting skeletal muscle, the precise mechanisms by which ROS generated during contractions activate relevant signaling processes remain unclear. This is due to the recognition that the increase in specific species of ROS that accompany contractile activity is relatively modest. H2O2 is widely viewed as the only ROS likely to play a major role in signaling, and H2O2 has been shown to activate NF-κB (Zhang et al., 2001), AP-1 (Aggeli et  al., 2006), and other transcription factors (Marinho et  al., 2014). Thus, most authors have assumed that H2O2 interacts with activation pathways for these specific transcription factors leading to their activation. In vitro studies have supported this hypothesis by demonstrating that H2O2 can activate NF-κB and other transcription factors but have used H2O2 concentrations typically in the range 10−4– 10−3 M. It is appropriate to consider whether these concentrations have any in vivo relevance. Helmut Sies has calculated the intracellular H2O2 concentration in most cells to be 10−9–10−8 M (Sies, 2014), and we have calculated from the increase in DCFH oxidation that occurs in muscle during contractions that the increase in H2O2 is likely to be a maximum of 10−7 M (Jackson, 2011). This is a factor of ~1000 below the concentrations reported to activate most transcription factors in vitro. Thus, the concept of H2O2 generated during contractions diffusing through the cell to encounter redox-regulated proteins at sufficiently high concentration to activate them does

­Functions of physiological ROS in muscle

Resting muscle fibre Extracellular H2O2 10–12mM

Action filament

M-disk

Z-line

Myosin filament

Z-line

NADPH oxidase sub-units

Mean intracellular fiber H2O2 10–100nM Mitochondria

(A) Contracting muscle fibre

Potential local area of high ic and ec [H2O2]

O2

Extracellular H2O2 15–20 mM

O2 • − H2O2

NADPH oxidase Action filament

M-disk

H2O2

Myosin filament

[H2O2] [H2O2]

Z-line

[H2O2]

Z-line

? Muscle contractions

NADPH oxidase sub-units

[H2O2]

Mean intracellular fiber H2O2 100–200nM

Mitochondria

(B) FIG. 1 Schematic representation of the key sources and concentrations of H2O2 found in skeletal muscle fibers at rest and during contractions. At rest (A), the flux of H2O2 in the muscle cell appears to be dominated by the large extracellular to intracellular concentration gradient for H2O2. Following contractile activity (B), activation of NAD(P)H oxidase occurs with generation of superoxide and H2O2 that is thought to occur on the outside of the plasma or T-tubule membranes leading to a local increase in H2O2 that increases the flux of H2O2 into the muscle fiber in regions adjacent to the site of H2O2 generation.

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not appear sustainable in the light of the calculated intracellular H2O2 concentrations. Fig. 1 also shows the likely H2O2 concentrations in resting and contracting muscle fibers. An alternative, although unproven, pathway for redox signaling involves the transfer of oxidative equivalents directly from a H2O2-sensitive thiol peroxidase to a specific target protein through direct protein-protein contact allowing the conversion of the oxidizing equivalent from H2O2 into a disulfide bond that can be subsequently transmitted to other substrates through the formation of intermolecular disulfides. It has been shown that thiol peroxidases can transmit oxidizing equivalents to a specific target protein to facilitate H2O2 signaling (Sobotta et al., 2015). This mechanism has been documented in yeast (Delaunay et  al., 2002; Gutscher et  al., 2009) but only recently has been shown to occur in the activation of a transcription factor by H2O2 in animal cells (Sobotta et al., 2015). Key components of such signaling pathways are peroxiredoxins (Prx) and thioredoxins (Trx). Prx are a family of antioxidant enzymes that reduce hydroperoxides to water in the presence of electron donors and are generally considered to be important antioxidant enzymes in the cytosol (Prx1, Prx2, and Prx5), mitochondria (Prx3 and Prx5), and endoplasmic reticulum (Prx4). Importantly and in contrast to the relative poor reactivity of the proteins involved in activating transcription factors discussed previously, Prx are several order of magnitude more reactive with H2O2 (Sobotta et al., 2015) and thus can potentially react with and reduce H2O2 at the low H2O2 concentrations found in muscle fibers. Studies in nonmuscle cells indicate that Prx can function as a signal peroxidase to activate specific pathways. Prx1 has been shown to activate the transcription factor ASK1, (Jarvis et al., 2012), and Prx2 forms a redox relay with the transcription factor STAT3 such that oxidative equivalents flow from Prx2 to STAT3 generating disulfide-linked STAT3 oligomers with modified transcriptional activity (Sobotta et al., 2015). Despite the lack of clear identification of the precise transcriptional pathways involved, it is clear that the ROS and NO generated during contractile activity in muscle mediate the activation of a number of redox-regulated transcription factors, including NF-κB, AP-1, HSF-1, and Nrf2 (Ji et al., 2004; Ristow et al., 2009; Vasilaki, Mansouri, et al., 2006; Vasilaki, McArdle, et al., 2006) leading to an increased expression of regulatory enzymes and cytoprotective proteins (McArdle et al., 2001). It is likely that many other adaptive processes are activated in a similar manner, but the full extent to which redox-dependent systems regulate other adaptations to contractions in muscle is still unclear. There is evidence for a role of redox regulation in the activation of catabolic processes, some aspects of insulin sensitivity, and mitochondrial biogenesis (Powers and Jackson, 2008).

­ROS in muscle degeneration In contrast to these potentially beneficial roles of ROS in adaptive redox signaling (oxidative eustress) in muscle, ROS have also been implicated as pathogenic mediators in many degenerative disorders of skeletal muscle including Duchenne

Oxidative damage and defective redox signaling

muscular dystrophy (Terrill et al., 2013), inflammatory myopathies (Lightfoot et al., 2015), and motor neuron disease (ALS; Pollari et al., 2014). Our work has focused on muscle loss during ageing and more recently on denervation-induced atrophy of muscle, and these disorders will be considered here.

­Age-related loss of skeletal muscle mass and function Ageing leads to a reduction in muscle mass and function that contributes to physical instability and increased risk of falls (Young and Skelton, 1994) such that by the age of 70, skeletal muscle cross-sectional area has declined by 25%–30% and muscle strength by 30%–40% (Porter et al., 1995). This loss of muscle is known as “sarcopenia,” and in both humans and rodents, there is evidence that the age-­related reduction in muscle mass and function is due to decreased numbers of muscle fibers and atrophy and weakening of the remaining fibers (Brooks and Faulkner, 1988; Lexell et al., 1986, 1988). Most of the intrinsic and extrinsic changes regulating muscle ageing in humans have been observed in rodents, and mice and rats have been widely used as models of human sarcopenia (Demontis et  al., 2013). The comparable changes in morphology seen in myofibers of aged rodents and humans also suggest the mechanisms leading to muscle loss and atrophy at the cellular level are comparable (Miller, 2004). Muscles from both older humans and rodents show an increased proportion of more oxidative fibers (Delbono, 2000) and an attenuation of various responses to contractile activity including acute stress responses (Vasilaki, Mansouri, et al., 2006; Vasilaki, McArdle, et al., 2006), mitochondrial biogenesis (Ljubicic and Hood, 2008), and the contraction-induced increase in muscle protein synthesis (Cuthbertson et al., 2005). These changes may mediate potentially important aspects of the multiple age-related deficits in muscle including contributing to slowed reactions and an inability to fine-tune movements. Transgenic studies in mice have also indicated that correction of specific attenuated responses to contractions can preserve muscle force generation in aged mice (Broome et al., 2006; Kayani et al., 2010; McArdle, Dillmann, et al., 2004; McArdle, Spiers, et al., 2004).

­ xidative damage and defective redox signaling in muscle O from old mice and humans An increase in oxidative damage has been reported in tissues (including skeletal muscle) of all aged organisms compared with that found in young organisms (Drew et al., 2003; Sastre et al., 2003; Vasilaki, Mansouri, et al., 2006; Vasilaki, McArdle, et  al., 2006). The possibility that increased oxidative damage plays a key role in age-related loss of tissue function and structure has received considerable attention. In nonmammalian models, interventions designed to reduce the activities of ROS such as overexpression of CuZn, superoxide dismutase (SOD1), catalase, or both

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in Drosophila (Orr and Sohal, 1993, 1994, 2003) or treatment with a MnSOD and catalase mimetic in Caenorhabditis elegans extended life span and thus support the hypothesis, but these effects have not been confirmed in other studies (Melov et al., 2000). In mammals, only a small number of manipulations designed to reduce ROS activities and/or oxidative damage have increased life span (Schriner et  al., 2005; Yoshida et al., 2005). It therefore appears that increased ROS generation is not the fundamental cause of ageing (or more precisely, the fundamental determinant of life span in mammals). Mitochondrial ROS generation has been reported to be increased in skeletal muscle during ageing (Melov, 2000; Van Remmen and Jones, 2009 for reviews) in association with impaired function and oxidative damage to mitochondrial components (Jang and Van Remmen, 2011; Sastre et al., 2003), and a small number of other studies indicate that interventions to reduce mitochondrial H2O2 content (Schriner et al., 2005) or increase cytoprotective proteins that reduce oxidative damage (Broome et al., 2006) may preserve muscle function during ageing. Increased mitochondrial ROS generation has also been proposed to play a key mediating role in some pathological changes in muscle in conditions such as disuse atrophy (Powers et al., 2011).

­ odification of muscle ROS during ageing though knockout M of key regulatory proteins To decipher the effects of ROS in musculoskeletal ageing, a number of studies have examined the effects of deletion of regulatory enzymes for ROS in mammalian models. Despite frequent observations of increased oxidative damage in these models, no clear relationship with skeletal muscle ageing was seen (Jang and Van Remmen, 2009). The exception to this pattern was in mice with a whole-body deletion of SOD1 (Cu, Zn superoxide dismutase), which show neuromuscular changes with ageing and which were described by the discoverer as showing accelerated age-related loss of muscle mass (Muller et al., 2006; Deepa et al., 2019). Adult SOD1KO mice show a decline in skeletal muscle mass, loss of muscle fibers, and a decline in the number of motor units, loss of motor function and contractility, partial denervation, and mitochondrial dysfunction by 8 month old (Jang et al., 2010; Larkin et al. 2011; Vasilaki et  al., 2010). The fiber loss in SOD1KO mice is accompanied by degeneration of neuromuscular junctions (NMJs; Jang et al., 2010). These changes are also seen in old WT mice, but not until after 22 months of age. Hence, we have proposed that SOD1KO mice are a useful model to examine the potential role of ROS in skeletal muscle ageing (Jackson, 2006). It is important to discover why only the SOD1KO mice shows an accelerated muscle ageing phenotype, whereas other models with knockout of regulatory enzymes for ROS or RNS also show an increase in oxidative damage to muscle, but no relevant skeletal muscle phenotype changes. SOD1 is expressed in both the cytosol of cells and within the mitochondrial intermembrane space (IMS) where

­Modification of muscle ROS

it is likely to be present at high concentration compared with cytosolic SOD1 (Kawamata and Manfredi, 2010). Thus, a lack of SOD1 may influence redox homeostasis in the mitochondria in addition to the cytosol, and hence, disturbances in either cytosolic or mitochondrial redox may underlie the accelerated skeletal muscle ageing phenotype seen in SOD1KO mice. We have examined the nature of the reactive species that are generated in mice lacking SOD1. Previous studies of ageing models have suggested that the decline in tissue function that occurs with ageing and the accelerated loss of skeletal muscle fibers in SOD1KO mice may be caused by superoxide toxicity (Jang and Van Remmen, 2009; Melov et al., 2000), but an alternative possibility is that superoxide and NO may react chemically to form peroxynitrite, a reaction that competes with the dismutation of superoxide to H2O2 by SOD (Beckman and Koppenol, 1996). In adult SOD1 null mice, the phenotype may therefore be associated with excess superoxide but may also be due to increased peroxynitrite or a reduction in NO bioavailability. Our studies showed that in muscle fibers from SOD1KO and old WT mice, there was an increase in oxidation of the nonspecific intracellular ROS probe, 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH) at rest compared with fibers from adult WT mice (Vasilaki et al., 2010). Surprisingly, the fibers from SOD1KO mice showed no increase in DCFH oxidation following contractile activity, although an increase in DCFH oxidation was seen in muscle fibers from adult WT mice following contractile activity. The explanation for this is currently unclear, although DCFH is relatively insensitive to oxidation by superoxide but is oxidized by other ROS, including H2O2, hydroxyl radicals, peroxynitrite, and nitric oxide (Murrant and Reid, 2001). Single muscle fibers from flexor digitorum brevis of WT and SOD1KO mice were therefore also loaded with NO-sensitive 4-amino5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM) and superoxidesensitive dihydroethidium (DHE) probes (Sakellariou et al., 2011). Surprisingly, a lack of SOD1 in the fibers from SOD1KO mice did not increase superoxide availability at rest, and no increase in ethidium or 2-hydroxyethidium (2-HE) formation from DHE was seen in fibers from SOD1KO mice compared with those from WT mice. Fibers from SOD1KO mice were found to have decreased NO availability (decreased DAF-FM fluorescence), increased 3-nitrotyrosines (3-NT) in muscle proteins indicating increased peroxynitrite formation, and increased content of peroxiredoxin 5 (a peroxynitrite reductase), compared with WT mice. Following contractile activity muscle fibers from SOD1KO mice also showed substantially reduced generation of superoxide compared with fibers from WT mice. Inhibition of NOS to reduce NO availability and hence the potential for the formation of peroxynitrite did not affect DHE oxidation in fibers from WT or SOD1KO at rest or during contractions. In contrast fibers isolated from nNOS, transgenic mice showed increased DAF-FM fluorescence and reduced DHE oxidation in resting muscle fibers. These data appear to indicate that peroxynitrite is formed in muscle fibers as a consequence of the lack of SOD1 in SOD1KO mice and may therefore contribute to fiber loss in this model. More generally, we proposed that

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these data also support the hypothesis that NO regulates superoxide availability and peroxynitrite formation in muscle fibers (Sakellariou et al., 2011).

­Role of ROS in denervation and link to ageing The SOD1KO mice are deleted of SOD1 in all tissues and show accelerated age-­ related decline in several tissues including motor neurons and skeletal muscle. To specifically examine how changes in muscle SOD1 might influence age-related changes in muscle, a model with muscle-specific deletion of SOD1 (mSOD1KO mice) was examined (Zhang et al., 2013), but these mice show no evidence of premature NMJ degeneration or loss of muscle fibers and, in contrast, showed some muscle hypertrophy (Zhang et al., 2013). We examined whether the changes in ROS generation observed in the global knockout model (SOD1KO mice) were also seen in mSOD1KO mice, but the multiple changes in markers of oxidative damage and adaptation seen in SOD1KO mice and described earlier were not observed in the mSOD1KO mice (Zhang et al., 2013). This included no evidence for the increases in 3-NT and peroxiredoxin 5 previously reported in muscles of SOD1KO mice. It was therefore important to determine the potential role of motor neurons in the loss of muscle mass and function seen in SOD1KO mice, and a transgenic SOD1KO mouse in which human SOD1 is expressed in neurons under control of a synapsin 1 promoter (nSOD1Tg-SOD1KO mice) was established (Sakellariou et  al., 2014). These “nerve rescue” mice expressed SOD1 in central and peripheral neurons but no other tissues. Sciatic nerve CuZnSOD content in nSOD1TgSOD1KO mice was ~20% of WT control mice, but they showed no loss of muscle mass or maximum isometric specific force production at 8–12 months of age, when significant reductions were seen in SOD1KO mice (Sakellariou et al., 2014). Thus, these data appeared to demonstrate that at least 20% of WT CuZnSOD levels in neurons is essential in preserving skeletal muscle and NMJ structure and function in SOD1KO mice and implicated a lack of SOD1 specifically in motor neurons in the pathogenesis of the accelerated muscle ageing phenotype seen in the wholebody SOD1 null mice. Adult mice lacking SOD1 therefore replicate many of the features seen in old WT mice, and further examination of this model and variants of this model with tissue specific modification of SOD1 content may help identify key mechanisms leading to the loss of muscle fibers and function in ageing of WT mice. In particular, the initiating role for the motor neuron in this model provides a means of determining mechanisms by which disruption of redox homeostasis in the motor neuron can cause atrophy and loss of muscle fibers, and we have speculated that this may also be important for ageing in WT mice. It is also important to stress that, although SOD1KO mice are a model in which fundamental questions about mechanisms are highly relevant to the understanding of muscle ageing, there is no evidence that a simple lack of SOD1 contributes to ageing-related loss of muscle in WT mice or humans.

­Denervation induces ROS and adversely affects neighboring fibers

­ enervation of muscle leads to increased muscle D mitochondrial generation of peroxides that stimulates muscle atrophy and loss of muscle and adversely affects neighboring muscle fibers Holly Van Remmen’s group at the University of Texas initially demonstrated that the lack of SOD1 in SOD1KO mice induced the premature muscle ageing phenotype (Muller et al., 2006) and subsequently showed that experimental denervation of skeletal muscle leads to a very large (~100-fold) increase in H2O2 generation by muscle mitochondria (Muller et al., 2007). Their further studies indicated that only ~50% of the peroxide released from mitochondria in denervated muscle was H2O2 and suggested that lipid peroxides were also released and stimulated denervationinduced muscle loss (Bhattacharya et al., 2009; Bhattacharya et al., 2014). In recent studies, our group have examined the effect of transection of the peroneal nerve on the tibialis anterior (TA) muscle of mice, and in this model, mitochondria in the denervated muscle showed increased peroxide generation by 3 days post transection (Pollock, Staunton, Vasilaki, McArdle, & Jackson, 2017). Partial denervation of the TA muscle by transection of one branch of the peroneal nerve was also examined and provided an experimental model in which fibers in some areas of the muscle were denervated while fibers in other areas retained innervation. Surprisingly, a substantial increase in mitochondrial peroxide generation was found to occur in both denervated and innervated fibers (Pollock et al., 2017). The increase in mitochondrial peroxide production following denervation precedes subsequent muscle fiber atrophy and has been proposed to disrupt proteostasis and stimulate apoptosis and other degenerative pathways (Adhihetty et al., 2007; Muller et al., 2007) and Bhattacharya et al. (2014) showed that inhibition of lipid peroxide generation reduced denervation-induced muscle atrophy. Increased local activities of ROS have also been shown to suppress neuromuscular transmission in NMJ reducing the efficiency of stimulation of contractions (Giniatullin et  al., 2005; Shakirzyanova et al., 2016), and chronic exposure to excess ROS has been shown to disrupt NMJ structure and function (Shi et al., 2014). Thus, it appears that the presence of denervated fibers in muscle leads to substantial release of H2O2 and other peroxides by mitochondria in the denervated fibers that may stimulate degenerative pathways leading to loss of muscle mass and negatively feeds back on neighboring innervated muscle fibers potentially causing increased mitochondrial release of peroxides, suppression of NMJ function, and attenuation of redox-regulated adaptive responses to contractile activity. Increased muscle mitochondrial peroxide release also occurs in the SOD1KO mouse model and in old mice (Jang and Van Remmen, 2009). The mechanisms underlying the increase in peroxide generation and the nature of the changes in NMJ structure or function that are required to cause increased muscle mitochondrial ROS generation are unknown. Data from our recent published and unpublished studies indicate that peroxide release occurs from 3 to 21 days post

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denervation and the acetylcholine receptor (AChR) remains intact at these times despite complete loss of presynaptic neuronal structures (Pollock et al., 2017). In mature mammals, repeated cholinergic synaptic transmission is required to maintain stability of the AChR (Avila et  al., 1989). Whether a failure of synaptic transmission also precipitates, increased muscle mitochondrial peroxide generation is unknown, although that possibility is supported by data showing that increased generation of ROS by muscle mitochondria occurred following a lack of repeated cytosolic calcium transients in the muscle (Karam et al., 2017). Significant changes in NMJ structure have been reported in ageing rodent models (Pollock et  al., 2017; Valdez et  al., 2010, 2012; Vasilaki et  al., 2016 [e.g., Fig. 2]). In human ageing, electrophysiological studies indicate that substantial motor unit remodeling occurs in older individuals and is associated with muscle fiber-type grouping (Piasecki et  al., 2016). Some publications have described disruption of NMJ in muscles from ageing humans (Oda, 1984; Wokke et  al. 1990), but a recent study has questioned this (Jones et al., 2017), although it is noteworthy that in this latter paper, the elderly subjects studied also did not show the anticipated muscle fiber atrophy and may reflect a very healthy group. It is clear from comparisons of the small deficit in specific force production from muscles of old mice with the substantial proportion of disrupted NMJ observed on microscopy that the majority of NMJ showing structural disruption during ageing must support synaptic neurotransmission (Larkin et  al., 2011). This is compatible with NMJ having a large “safety factor” (Wood and Slater, 2001) to maintain successful neurotransmission and blockade, or removal of ~80% of AChR may be required to significantly reduce the activation of muscle contractions (Pennefather and Quastel, 1981). It is unknown whether similar safety factors are present to minimize other deleterious effects of NMJ disruption and neither the degree nor the nature of the NMJ disruption required to stimulate muscle mitochondrial peroxide production is known.

FIG. 2 Representative images of neuromuscular junctions from EDL muscles of Thy1-YFP mice showing axons (green) and acetyl choline receptors (AChRs, stained red with αbungarotoxin). NMJ from adult mice showing full innervation (A); fully denervated muscle at 7 days post nerve transection (B); old (24 month) mouse showing blebbing of axons and incomplete occupancy of AChRs (C). Modified from Pollock et al. (2017).

­Implications of the increased mitochondrial peroxide production

I­mplications of the increased mitochondrial peroxide production for prevention of muscle loss with ageing and following denervation Although Bhattacharya and colleagues have demonstrated that reduction of enzymatic generation of lipid peroxides can reduce muscle loss following denervation (Bhattacharya et al., 2014), there are few other relevant published studies that support a role for interventions designed to specifically reduce mitochondrial generation of peroxides as a means of preventing muscle loss in ageing or denervation. Our group have examined the possibility that mitochondria-targeted antioxidants can reduce age-related loss of muscle mass in mouse models and although these studies showed some effect of the antioxidants in preservation of mitochondrial function in old mice, no protective effect against age-related loss of muscle mass was seen (Sakellariou et al., 2016a, 2016b). It is relevant that the mitochondria-targeted antioxidants may have been particularly effective against H2O2 or superoxide but relatively unreactive with lipid peroxides, which may represent a considerable proportion of the peroxide released from mitochondria of denervated muscle fibers. Greater understanding of the precise nature of the changes that occur in mitochondria of denervated muscle fibers and the released factors is therefore required to fully explore the potential of targeted interventions to maintain muscle mass and function in this area (Fig. 3).

FIG. 3 Schematic representation of the effect of acute denervation on muscle redox status. Muscle redox appears unchanged until there is loss of presynaptic integrity that leads to large increases in mitochondrial peroxide generation. This appears to act on neighboring fibers to modify mitochondrial peroxide production in those fibers, and we speculate that this is initially a protective process, which stimulates axonal sprouting and other repair processes. Ultimately, continued release of peroxides will lead to the activation of degenerative processes that are regulated within the mitochondria, such as cytochrome C release leading to apoptosis, and if sufficiently sustained to overcome cytosolic regulatory, enzymes will cause increased cytosolic oxidation and stimulate multiple degenerative pathways leading to disturbed muscle proteostasis.

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F­ uture directions in dissection of the roles of redox signaling from oxidative stress in skeletal muscle Crucial to further understanding of this area is the development and identification of techniques and approaches that can distinguish the modest temporal and spatial increases in ROS activities that mediate redox signaling, from those associated with gross oxidative damage to proteins, lipids, or DNA. Analytical developments that allow assessment of the extent of differential oxidative modifications of target proteins may provide one route forward. Thus, some redox proteomic approaches allows analysis of the extent of oxidation of specific cysteine molecules in proteins (McDonagh, Sakellariou, Smith, Brownridge, & Jackson, 2014), while comparisons of the relative extent of reversible oxidation (during redox signaling) versus hyperoxidation (during oxidative stress) of specific proteins such as peroxiredoxins (Veal et al., 2018) may also be feasible. A number of new fluorescent probes have also been developed to allow monitoring of specific ROS, particularly H2O2. These include the HyPer family of probes (Bilan and Belousov, 2016) and roGFP1-Orp2 probes (Gutscher et al., 2009). These are genetically encoded and can be localized to specific cell compartments. Data on the use of these probes in skeletal muscle is currently limited, but detection of the change in ROS activities that mediate signaling is challenging, and indeed, it has been argued that even these state-of-the-art approaches may be insufficiently sensitive to reliably compete with endogenous redox signaling proteins such as Prx (Young et al., 2019).

­Summary There has been a great deal of progress since the first substantial interest in the role of ROS in skeletal muscle and exercise almost 50 years ago. From initial studies that were limited by the availability of techniques with sufficient specificity and sensitivity to obtain definitive data, it rapidly became clear that skeletal muscle can generate a specific ROS (superoxide) together with nitric oxide (NO). At that stage what was known about the chemical properties of ROS implied that their likely biological roles were damaging to muscle and other tissues, but since then, there has been increasing recognition of both beneficial and potentially deleterious effects of ROS. We have briefly reviewed studies undertaken by the authors and others that have identified and characterized the generation of ROS by muscle, the effects of contractile activity on this generation, redox signaling effects of oxidants in muscle (oxidative eustress), and deleterious oxidative damage processes that play a role in tissue degeneration (oxidative distress). Despite this progress, there is still a great deal to be learned to allow the translation of this knowledge into beneficial interventions to optimize muscle function in health and disease.

­References

­Acknowledgments The authors would like to acknowledge the ongoing generous financial support from the UK Medical Research Council, Arthritis Research UK, US National Institute on Aging (P01 AG 051442), and UK Space agency.

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Shakirzyanova, A., Valeeva, G., Giniatullin, A., et  al. (2016). Age-dependent action of reactive oxygen species on transmitter release in mammalian neuromuscular junctions. Neurobiology of Aging, 38, 73–81. Shi, Y., Ivannikov, M. V., Walsh, M. E., et  al. (2014). The lack of CuZnSOD leads to impaired neurotransmitter release, neuromuscular junction destabilization and reduced muscle strength in mice. PLoS One, 9, e100834. Sies, H. (1985). Oxidative stress: Introductory remarks. In H. Sies (Ed.), Oxidative stress (pp. 1–8). London: Academic Press. Sies, H. (2014). Role of metabolic H2O2 generation: Redox signaling and oxidative stress. The Journal of Biological Chemistry, 289, 8735–8741. Sies, H. (2018). On the history of oxidative stress: Concept and some aspects of current development. Current Opinion in Toxicology, 7, 122–126. Sies, H., Berndt, C., & Jones, D. P. (2017). Oxidative stress. Annual Review of Biochemistry, 86, 715–748. Silveira, L. R., Pereira-Da -Silva, L., Juel, C., et al. (2003). Formation of hydrogen peroxide and nitric oxide in rat skeletal muscle cells during contractions. Free Radical Biology & Medicine, 35, 455–464. Sobotta, M. C., Liou, W., Stöcker, S., et al. (2015). Peroxiredoxin-2 and STAT3 form a redox relay for H2O2 signaling. Nature Chemical Biology, 11, 64–70. Sun, Q. A., Wang, B., Miyagi, M., et al. (2010). Oxygen-coupled redox regulation of the skeletal muscle ryanodine receptor/Ca2+ release channel (RyR1): Sites and nature of oxidative modification. The Journal of Biological Chemistry, 288, 22961–22971. Terrill, J. R., Radley-Crabb, H. G., Iwasaki, T., et al. (2013). Oxidative stress and pathology in muscular dystrophies: Focus on protein thiol oxidation and dysferlinopathies. The FEBS Journal, 280, 4149–4164. Valdez, G., Tapia, J. C., Kang, H., et al. (2010). Attenuation of age-related changes in mouse neuromuscular synapses by caloric restriction and exercise. Proceedings of the National Academy of Sciences of the United States of America, 107, 14863–14868. Valdez, G., Tapia, J. C., Lichtman, J. W., et al. (2012). Shared resistance to aging and ALS in neuromuscular junctions of specific muscles. PLoS One, 7, e34640. Van Gammeren, D., Damrauer, J. S., Jackman, R. W., et  al. (2009). The IkappaB kinases IKKalpha and IKKbeta are necessary and sufficient for skeletal muscle atrophy. The FASEB Journal, 23, 362–370. Van Remmen, H., & Jones, D. P. (2009). Current thoughts on the role of mitochondria and free radicals in the biology of ageing. The Journals of Gerontology. Series A, Biological Sciences and Medical Sciences, 64, 171–174. Vasilaki, A., Mansouri, A., Van Remmen, H., et al. (2006). Free radical generation by skeletal muscle of adult and old mice: Effect of contractile activity. Aging Cell, 5, 109–117. Vasilaki, A., McArdle, F., Iwanejko, L. M., et al. (2006). Adaptive responses of mouse skeletal muscle to contractile activity: The effect of age. Mechanisms of Ageing and Development, 127, 830–839. Vasilaki, A., Pollock, N., Giakoumaki, I., et al. (2016). The effect of lengthening contractions on neuromuscular junction structure in adult and old mice. Age (Dordrecht, Netherlands), 38, 259–272. Vasilaki, A., Van Der Meulen, J. H., Larkin, L., et al. (2010). The age-related failure of adaptive responses to contractile activity in skeletal muscle is mimicked in young mice by deletion of Cu,Zn superoxide dismutase. Aging Cell, 9, 979–990.

­Further reading

Veal, E. A., Underwood, Z. E., Tomalin, L. E., et al. (2018). Hyperoxidation of peroxiredoxins: Gain or loss of function? Antioxidants & Redox Signaling, 28, 574–590. Ward, C. W., Prosser, B. L., & Lederer, W. J. (2014). Mechanical stretch-induced activation of ROS/RNS signaling in striated muscle. Antioxidants & Redox Signaling, 20, 929–936. Wokke, J. H., Jennekens, F. G., van den Oord, C. J., et al. (1990). Morphological changes in the human end plate with age. Journal of the Neurological Sciences, 95, 291–310. Wood, S. J., & Slater, C. R. (2001). Safety factor at the neuromuscular junction. Progress in Neurobiology, 64, 393–429. Xia, R., Webb, J. A., Gnall, L. L., et al. (2003). Skeletal muscle sarcoplasmic reticulum contains a NADH-dependent oxidase that generates superoxide. The American Journal of Physiology, 285, C215–C222. Yoshida, T., Nakamura, H., Masutani, H., et al. (2005). The involvement of thioredoxin and thioredoxin binding protein-2 on cellular proliferation and ageing process. Annals of the New York Academy of Sciences, 1055, 1–12. Young, D., Pedre, B., Ezerina, D., et  al. (2019). Protein promiscuity in H2O2 signaling. Antioxidants & Redox Signaling, 30, 1285–1324. Young, A., & Skelton, D. A. (1994). Applied physiology of strength and power in old age. International Journal of Sports Medicine, 15, 149–151. Zhang, Y., Davis, C., Sakellariou, G. K., et al. (2013). CuZnSOD gene deletion targeted to skeletal muscle leads to loss of contractile force but does not cause muscle atrophy in adult mice. The FASEB Journal, 27, 3536–3548. Zhang, J., Johnston, G., Stebler, B., et  al. (2001). Hydrogen peroxide activates NFkappaB and the interleukin-6 promoter through NFkappaB-inducing kinase. Antioxidants & Redox Signaling, 3, 493–504.

­Further reading Brown, W. F., Strong, M. J., & Snow, R. (1988). Methods for estimating numbers of motor units in biceps-brachialis muscles and losses of motor units with ageing. Muscle & Nerve, 11, 423–432. Campbell, M. J., McComas, A. J., & Petito, F. (1973). Physiological changes in ageing muscles. Journal of Neurology, Neurosurgery, and Psychiatry, 36, 174–182. Chai, R. J., Vukovic, J., Dunlop, S., et al. (2011). Striking denervation of neuromuscular junctions without lumbar motoneuron loss in geriatric mouse muscle. PLoS One, 6, e28090. Haddad, J. J. (2002). Antioxidant and pro-oxidant mechanisms in the regulation of redox(y)sensitive transcription factors. Cellular Signalling, 14, 879–897. Janssen-Heininger, Y. M., Mossman, B. T., Heintz, N. H., et al. (2008). Redox-based regulation of signal transduction: Principles, pitfalls, and promises. Free Radical Biology & Medicine, 45, 1–17. Quintanilha, A. T., & Packer, L. (1983). Vitamin E, physical exercise and tissue oxidative damage. Ciba Foundation Symposium, 101, 56–69. Wang, Z. M., Zheng, Z., Messi, M. L., et al. (2005). Extension and magnitude of denervation in skeletal muscle from ageing mice. The Journal of Physiology, 565, 757–764. Willadt, S., Nash, M., & Slater, C. (2016). Age-related fragmentation of the motor endplate is not associated with impaired neuromuscular transmission in the mouse diaphragm. Scientific Reports, 6, 24849.

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Redox mechanisms in pulmonary disease: Emphasis on pulmonary fibrosis

35

Yvonne Janssen-Heininger, Vikas Anathy Department of Pathology and Laboratory Medicine, University of Vermont, Larner College of Medicine, Burlington, VT, United States

Abstract Lung fibrosis is a disease associated with changes in the redox environment. Diminished survival and altered plasticity of lung alveolar epithelial cells are accompanied by an overpopulation of lung alveolar regions with fibroblasts and extracellular matrix. Disruptions in redox processes within the endoplasmic reticulum and mitochondria or activation of NADPH oxidases within epithelial cells and/or fibroblasts is believed to contribute to disease pathogenesis. This chapter reviews the recent literature, emphasizing alterations of glutathione biochemistry. We place a spotlight on glutathione-dependent protein oxidation, known as Sglutathionylation, based upon recent studies demonstrating that S-glutathionylation chemistry is a strong contributing factor to lung fibrosis. ­Keywords: Fibrosis, Endoplasmic reticulum, Mitochondria, S-glutathionylation, Glutaredoxin, Glutathione S-transferase P

­Introduction The lung is a unique organ, as it is constantly exposed to air and airborne pollutants including oxidant gases such as highly reactive nitrogen dioxide and ozone, particulates, viruses, other microbes, immune activators, and irritants. Defense from these pollutants is provided by a number of cell types that include epithelial cells and macrophages. Differentiated ciliated airway epithelial cells contain cilia, specialized organelles that beat in waves to propel pathogens and inhaled particles trapped in the mucous layer out of the airways (Bustamante-Marin & Ostrowski, 2017). Secretory epithelial cells produce mucins and an array of antimicrobial products (Hiemstra, 2015). Airway epithelial cells also express hydrogen peroxide (H2O2)-producing dual oxidases (DUOX) 1 and 2 providing defense from microbes and other irritants (Van Der Vliet, Danyal, & Heppner, 2018). Located in the distal alveolar lung regions, type II epithelial cells produce surfactant proteins with antimicrobial or surfactant Oxidative Stress. https://doi.org/10.1016/B978-0-12-818606-0.00035-3 © 2020 Elsevier Inc. All rights reserved.

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lowering activities (Mulugeta, Nureki, & Beers, 2015). Type II epithelial cells have been estimated to constitute 60% of alveolar epithelial cells and 10%–15% of all lung cells. These cells are highly bioenergetically active due to their role in surfactant production, barrier protection, metabolism of xenobiotics, and as progenitor cells to replace damaged alveolar epithelial cells. The function of distal epithelial cells and other cell types allow the lung to function to exchange oxygen and carbon dioxide to allow for normal aerobic function (Petersson & Glenny, 2014). Changes in the redox environment have long been implicated in the pathophysiology of a myriad of lung diseases that include cancer, asthma, acute respiratory distress syndrome, chronic obstructive pulmonary disease, and pulmonary fibrosis. While an increased oxidative burden originally was believed to contribute to lung injury, for instance, by damaging epithelial cells, changes in the redox environment are now believed to promote lung disease via the oxidation of proteins, notably via the oxidation of reactive cysteines. Dysregulation of protein cysteine oxidations is speculated to contribute to the pathogenesis of lung diseases by affecting cellular pathways that govern death, proliferation, migration, plasticity, and production of proinflammatory mediators and profibrogenic factors (Janssen-Heininger et  al., 2008, 2010, 2013). Pulmonary fibrosis is an unrelenting progressive disease, characterized by a loss of normal alveolar architecture, repopulation of alveolar spaces with extracellular matrix, an overpopulation of activated myofibroblasts, and loss of alveolar epithelia (Martinez et al., 2017). While the causes of pulmonary fibrosis might be suspected in some cases, for instance, as a result of exposure to asbestos or silica, in other cases, the causes remain unknown. The diagnosis, idiopathic pulmonary fibrosis (IPF), is made when other potential etiological factors have been excluded and is based upon strict radiological criteria, sometimes also requiring histopathological confirmation of a pattern of usual interstitial pneumonia. Pulmonary fibrosis presents in patients in their 50s–70s and affects more men than women (Martinez et al., 2017). Each year, pulmonary fibrosis kills 40,000 people in the United States alone, and it affects over 5 million people worldwide (Mulugeta et al., 2015). The age-associated enhanced susceptibility to fibrosis is believed to reflect the lack of proper repair in an aging individual following repeated cumulative stresses (Martinez et al., 2017). Defective autophagy, telomere attrition, altered proteostasis, and cell senescence have been observed in epithelial cells and fibroblasts from IPF lungs, as compared with agematched controls (Mora, Bueno, & Rojas, 2017). IPF is a dismal diagnosis, and current FDA-approved therapies, pirfenidone and nintedanib (marketed as Esbriet and OFEV, respectively), have limited effectiveness to halt progression of idiopathic pulmonary fibrosis (IPF), leading to death of patients with IPF within 3–5 years from the time of diagnosis (Martinez et al., 2017). A number of environmental insults have been shown or speculated to cause pulmonary fibrosis that include inhalation of the aforementioned particulates, asbestos or silica, smoking, viral infections, radiation, etc. The chemotherapeutic drug, bleomycin, causes pulmonary fibrosis as a side effect, which limits its use as a chemotherapeutic modality. Radiotherapy for the treatment of lung cancer can also induce pulmonary fibrosis, one of its most common

­ER stress, mitochondrial dysfunction

side effects (Son et al., 2017). Pulmonary fibrosis also can have a genetic component, and in cases of familial IPF, patients are known to have germline mutations in certain genes, including surfactant protein C (SFTPC) (Kropski & Blackwell, 2018; Lawson et al., 2008). These genes encode proteins highly expressed in epithelia, and mutations in these genes result in defects in protein folding, leading to ER stress and mitochondrial dysfunction as will be further discussed in the succeeding text (Korfei et al., 2008). ER stress is now recognized as a feature of both familial and sporadic IPF. Relevant to the topic of this book, it is important to recognize that the environment in the ER is highly oxidizing to allow protein folding. Changes in the oxidative environment of the ER (ER redox stress) may therefore contribute to fibrogenesis, as will be discussed in the succeeding text. Besides IPF, a number of other disorders can result in lethal fibrosis, including systemic sclerosis (SS) and Hermansky-Pudlak syndrome (HPS). This chapter intends to summarize the current knowledge about dysregulation of redox-based processes, notably protein cysteine oxidations, in settings of lung fibrosis. It is important to note that other organs, notably the heart, kidney, and liver also are prone to the development of fibrosis, and the presence of fibrosis is in fact linked to organ failure (Rockey, Bell, & Hill, 2015). The processes, mediators, and pathways described herein in the context of the lung are likely relevant to fibrogenesis in other organs, and in some cases, we have referred to studies conducted in those organs.

E­ R stress, mitochondrial dysfunction, and the age-associated enhanced susceptibility to lung fibrosis The endoplasmic reticulum (ER) is an organelle essential to normal cellular function and plays a key role in the regulation of proteostasis, calcium storage, lipid synthesis, mitochondrial function, among others. Chronic ER stress is associated with the development of fibrotic disorders in the lung, liver, and kidney. ER stress within epithelial cells has been strongly implicated in the pathogenesis of lung fibrosis, based upon discoveries of germline mutations in genes expressed exclusively in epithelial cells that result in defects in folding and/or processing of a nascent peptide, causing prolonged ER stress and subsequent fibrosis (Kropski & Blackwell, 2018; Romero & Summer, 2017). Protein misfolding and ensuing ER stress triggers the activation of a signaling network known as the unfolded protein response (UPR) through distinct effector pathways that involve activation of PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1α (IRE1α) that are controlled by a chaperone protein, immunoglobulin heavy-chain-binding protein (Bip). Activation of these pathways is restricted by binding of Bip to the three aforementioned ER sensors (PERK, ATF6, and IRE1α). Bip also binds to misfolded proteins in the ER. As misfolded proteins accumulate, Bip binding to the three UPR sensors is reduced, and resultant UPR signaling in turn modulates new protein synthesis, increases production of ER chaperones, and induces components of the ER-associated

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degradation (ERAD) system (Kropski & Blackwell, 2018; Senft & Ronai, 2015). The UPR response is triggered to rectify the stress, but severe stress can result in cell death. In epithelial cells, ER stress can lead to apoptosis, proinflammatory signaling, and epithelial-­mesenchymal transition (EMT), features that have all been linked to lung fibrosis (reviewed in Kropski & Blackwell, 2018). Evidence that ER stress occurs in patients with IPF was first reported by the laboratory of Dr. Timothy Blackwell in 2008, documenting that expression of various markers of ER stress was increased in airway epithelial cells of patients with IPF, in association with the presence of herpes virus (Lawson et al., 2008). These markers include ATF4, ATF6, and CCAATenhancer-binding protein homologous protein (CHOP), Bip, and X-box binding protein 1 (XBP-1) (Kropski & Blackwell, 2018). Besides environmental factors, pulmonary fibrosis also can have a genetic component, and some families of IPF are known to have germline mutations in surfactant protein C (SFTPC), surfactant protein A2 (SFTPA2), or the lipid transporter, ATP binding cassette class A3 (ABCA3) genes. These genes encode proteins highly expressed in epithelia, and mutations in these genes result in defects in protein folding, leading to ER stress (Kropski & Blackwell, 2018). Some affected individuals with familial IPF shared a rare missense variant (L188Q) containing a mutation adjacent to a cysteine residue in pro-SPC that is required for proper folding (Mulugeta et al., 2015). Similarly, a mutation of cysteine 121 within the so-called BRICHOS domain of pro-SPC has been reported in a child with interstitial lung disease (Katzen et al., 2019). Misfolded pro-SPC arising from these and other mutations aggregates in the ER and activates the UPR. The relevance of these mutations for the pathogenesis of lung fibrosis has been corroborated in mouse models where it was demonstrated that transgenic overexpression the L188Q mutant variant of SFTPC in type II alveolar lung epithelial cells increased the sensitivity to the development of fibrosis (Lawson et al., 2011). Knock-in mice expressing of an isoleucine-to-threonine substitution at codon 73 (I73T) of SFTPC, or a cysteine-toglycine substitution at codon 121 (C121G), developed spontaneous fibrosis (Katzen et al., 2019; Nureki et al., 2018). These findings strongly support the concept that ER dysfunction in alveolar type II cells plays a key role in driving the pathogenesis of IPF. ER stress is not limited to those individuals with familial IPF, and ER stress is now recognized as a common feature of sporadic IPF. Links between ER stress, loss of proteostasis, and mitochondrial dysfunction in settings of IPF also have become apparent (Mora et  al., 2017). The ER tightly controls the calcium pool available for mitochondrial uptake through a number of proteins that include the mitochondrial calcium uniporter via sarcoendoplasmic reticulum Ca2+ ATPase and the inositol triphosphate receptor, providing a mechanism whereby the ER regulates cellular bioenergetics (reviewed in Kropski & Blackwell, 2018) and cell death (Marchi et  al., 2018). Specialized regions of interaction and communication between ER and mitochondria have been demonstrated and represent an area of active investigation (Csordas, Weaver, & Hajnoczky, 2018). Notably, aging and enhanced ER stress during aging have been shown to lead to mitochondrial dysfunction in type II alveolar epithelial cells (Mora et al., 2017). A higher frequency of enlarged mitochondria with a bias toward mitochondrial fusion and an increased

­Death of lung epithelial cells in fibrosis

mitochondrial area has been shown in aging type II alveolar epithelial cells (Bueno et al., 2015). Additional findings of mitochondrial abnormalities in the aging lung include increases in mitochondrial reactive oxygen species, decreases in mitophagy, impaired respiration, mitochondrial DNA deletions, and decreased expression of sirtuin 3 (Bratic & Larsson, 2013; Mora et al., 2017). ER stress in epithelial cells has been linked to mitochondrial perturbations via decreases in levels of PTEN-induced putative kinase 1 (PINK1), a regulator of mitochondrial homeostasis. A role for PINK1 in lung fibrosis was corroborated based upon findings demonstrating that PINK1-deficient mice exhibited increased susceptibility to apoptosis, and spontaneous transforming growth factor beta (TGF-β)-dependent fibrosis, in association with abnormal mitochondria in type II alveolar epithelial cells (Bueno et al., 2015). Epithelial cells expressing the I73T-mutant variant of human SPC showed defects in autophagy in association with increases in mitochondria biomass and decreases in mitochondrial membrane potential (Hawkins et  al., 2015), showing that ER stress and attendant disruptions in proteostasis are linked to mitochondrial dysfunction in settings relevant to pulmonary fibrosis. Mitochondrial dysfunction and ER stress also have been linked to the senescent fibroblast phenotype present in the IPF lung (Schuliga et al., 2018). Fibroblasts from IPF patients have shorter telomers, express a number of senescence-associated proteins, and produce cytokines related to the senescence-associated secretory phenotype. Upon TGFB stimulation, markers of ER stress increased to greater extents in IPF fibroblasts compared with age-matched cells from control lungs (Alvarez et al., 2017). TGFB was furthermore shown to stimulate increases in mitochondrial mass in human lung fibroblasts via the SMAD2/SMAD3 (SMAD represents an acronym from the fusion of Caenorhabditis elegans Sma genes and the Drosophila Mad, Mothers against decapentaplegic) and CCAAT/enhancer binding protein beta (CEBPB) pathways (Sun et al., 2019). Interestingly a role of oxidants has been suggested in promoting the senescence phenotype in fibroblasts, and this had been linked to the activation of signal transducer and activator of transcription (STAT)-3, a transcription factor implicated in premature senescence (Kojima, Inoue, Kunimoto, & Nakajima, 2013; Waters et al., 2019). Human lung fibroblasts exposed to H2O2 displayed increases in expression of senescence markers and expression of interleukin-6 (IL6), in association with translocation of STAT3 to the nucleus and mitochondria. These observations were accompanied by increased basal respiration, proton leak, and an associated increase in oxidant production in senescent fibroblasts. Using a small molecule inhibitor of STAT3, the authors demonstrated attenuated IL6 production, senescence markers, and restoration of normal mitochondrial function (Waters et al., 2019).

­Death of lung epithelial cells in fibrosis A critical role of epithelial cells in the pathogenesis of pulmonary fibrosis has clearly emerged. In fibrotic lung disease, including idiopathic pulmonary fibrosis (IPF), loss of or damage to distal conducting airway, or alveolar epithelial cells

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represent common histopathological features that are believed to contribute to diminished lung function. Chronic injury leads to death of epithelial cells, and lack of normal epithelial restitution is a cardinal trigger for fibrosis, with concomitant activation and proliferation of myofibroblasts (Kuwano et al., 2001; Matute-Bello et al., 2007). Despite earlier studies that downplayed the importance of epithelial cells in lung fibrogenesis, numerous preclinical studies strongly support the importance of epithelial cell death in subsequent fibrogenesis (Selman & Pardo, 2006). For example, upon administration of diphtheria toxin to transgenic mice expressing the diphtheria toxin receptor in type II epithelial cells or club cells (epithelial cells with secretory functions that exist in the proximal airways of the lung were gas exchange does not occur), respectively, marked death of these cells occurred with resultant prolonged fibrosis (Perl, Riethmacher, & Whitsett, 2011; Sisson et  al., 2010). The functional role of the death receptor, FAS (also known as CD95) in the development of pulmonary fibrosis is evident from studies showing that agonistic FAS antibody (which mimics the crosslinking between FAS ligand [FASL] and FAS), results in apoptosis of bronchial and alveolar epithelial cells leading to fibrosis. Conversely, bleomycin-induced fibrosis could be prevented using soluble anti-FAS, or anti-FASL antibodies, and did not occur in mice that lack functional FAS or FASL (Kuwano et  al., 1999; Matute-Bello et  al., 2007; Wallach-Dayan, Golan-Gerstl, & Breuer, 2007). Apoptosis of epithelial cells has been shown following coculture with myofibroblasts isolated from patients with IPF, which express FASL, while these myofibroblasts themselves are resistant to FASL-induced apoptosis (Golan-Gerstl et al., 2012; Wallach-Dayan et al., 2007). Epithelial cell death by IPF-derived fibroblasts has been linked to H2O2 produced by myofibroblasts (Waghray et  al., 2005). Low immunoreactivity of FAS was found in fibroblasts within fibroblastic foci, and upregulation of surface FAS in fibroblasts sensitized them to FASL-induced apoptosis (Wynes et al., 2011). A recent study showed that expression of protein tyrosine phosphatase-N13 mediated the resistance of human lung (myo)fibroblasts to FAS-induced apoptosis and promoted pulmonary fibrosis in mice (Bamberg et al., 2018). Collectively, these findings demonstrate the importance of FAS and epithelial cell apoptosis in the pathogenesis of lung fibrosis and suggest that avenues that dampen the extent of epithelial cell death and target the biology of the FAS receptor may attenuate fibrotic remodeling. Besides apoptosis, other modes of epithelial cell death also may contribute to lung fibrosis. Notably, a role of necroptosis controlled by receptor-interacting protein kinase −3 (RIPK3) and subsequent release of the danger signal, high-mobility group box-1 (HMGB1), and interleukin-1 beta (IL1B) in the pathogenesis of idiopathic pulmonary fibrosis was recently suggested (Lee et al., 2018). Aside from death, other processes affecting the plasticity of epithelial cells also may contribute to fibrosis. Epithelial cells can take on features of mesenchymal cells in a process known as epithelial to mesenchymal transition (EMT), mentioned earlier, and TGFB has been shown to be important inducer of EMT (Alcorn et al., 2008). Epithelial cells themselves are an important source of collagen production, evidenced in preclinical models of fibrosis (Yang et al., 2013). Single cell RNA sequencing of

­Oxidative stress in lung fibrosis: glutathione biochemistry

epithelial cells sorted from lungs from IPF patients are strongly suggestive of altered epithelial plasticity, based upon the appearance of cell populations that simultaneously express multiple markers of alveolar type II epithelial, alveolar type I cells, and conducting airway-selective markers, demonstrating “indeterminate” states of differentiation (Xu et al., 2016).

­Oxidative stress in lung fibrosis: Glutathione biochemistry Numerous studies in patients with IPF and rodent models have demonstrated that perturbations of redox status occur and are linked to disease pathogenesis (Cantin, Hubbard, & Crystal, 1989; Cantin, North, Fells, Hubbard, & Crystal, 1987; Kinnula, Fattman, Tan, & Oury, 2005). Disruptions in mitochondria and, as mentioned earlier, the ER have been implicated in the pathogenesis of IPF (Kropski & Blackwell, 2018; Lederer & Martinez, 2018; Mora et al., 2017), although the extent to which redox perturbations originating from dysfunctional mitochondria or ER, respectively, or other sources contribute to IPF remain unclear. In rodent models of fibrosis, genetic ablation of the antioxidant enzyme extracellular superoxide dismutase increased the susceptibility to fibrogenesis (Fattman, Tan, Tobolewski, & Oury, 2006), and similar findings were observed in mice lacking the transcription factor, nuclear factor erythroid 2-related factor (NRF2) (Cho, Reddy, Yamamoto, & Kleeberger, 2004), which regulates numerous antioxidant and cytoprotective responses including expression of enzymes important in GSH synthesis. Conversely, administration of various antioxidants has been suggested to be protective against the development of fibrosis (for review, see Day, 2008, Liu, 2008). The nonphagocytic NADPH oxidase 4, (NOX4), which produces H2O2, has been shown to play a causal role in myofibroblast activation and resultant fibrosis in the bleomycin and fluorescein isothiocyanate models (Hecker et al., 2009). NOX4 dampens mitochondrial bioenergetics and biogenesis via an NRF2-dependent pathway. Interestingly, NOX4 silencing increased the levels of NRF2 mRNA, while NRF2 silencing led to a decrease in NOX4 mRNA expression, showing counterregulation between NOX4 and NRF4. A NOX4/NRF2 redox imbalance also was demonstrated to contribute pulmonary fibrosis in aged mice where persistent fibrosis was linked to upregulation of NOX4 and failure to induce expression of NRF2 (Hecker et  al., 2014). Levels of the low MW thiol antioxidant glutathione (GSH) were reported to be markedly decreased in lung lining fluids, induced sputum and plasma of patients with IPF, in association with increases in glutathione disulfide (GSSG, the oxidized state of GSH) in plasma (Beeh et  al., 2002; Cantin et  al., 1989; Rahman et  al., 1999). Increased levels of H2O2 occur in patients with IPF, compared with control groups (Psathakis et al., 2006). To address the potential impact of GSH on the biology of lung fibroblasts, studies as early as 1990 found that extracellular GSH in the 500 μM range, similar to the concentration of GSH in the lung lining fluid, decreased lung fibroblast proliferation, suggesting that lowered levels of reduced GSH might promote fibroblast activation. The effect of GSH was mimicked by other

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­sulfhydryl-containing molecules including cysteine and N-acetylcysteine (NAC), precursors of GSH, 2-mercaptoethanol, and low concentrations of dithiothreitol but not by GSSG (Cantin, Larivee, & Begin, 1990). Transforming growth factor beta-1 (TGFB1) is a growth factor critical to organ fibrogenesis, and overexpression of active TGFB1 is sufficient to induce lung fibrosis in rodent models (Sime, Xing, Graham, Csaky, & Gauldie, 1997). TGFB1 alters the GSH status in the lung. Intranasal instillation of AdTGFB1(223/225), an adenovirus expressing constitutively active transforming growth factor beta 1 (hereafter referred to as AdTGFB1), suppressed the expression of catalytic and modifier subunits of glutamate-cysteine ligase (GCL), the rate-limiting enzyme in GSH synthesis, resulting in decreases of GSH. This resulted in increases in epithelial apoptosis, which occurred prior to the development of lung fibrosis (Liu et al., 2012). The microRNA, miR-433, has been shown to directly target GCL. In  vivo models of renal and hepatic fibrosis were associated with a TGFB1-mediated reduction of GCL levels that were miR-433 dependent, resulting in decreases in GSH levels, increases in S-glutathionylation, and enhanced fibrogenesis (Espinosa-Diez et al., 2015). It remains unclear whether miR-433 has a similar impact on lung fibrosis. TGFB1 is one of the major repressors of GCL expression leading to GSH depletion (Liu et  al., 2012; Ryoo, Shin, Kang, & Kwak, 2015), suggesting that in a profibrogenic environment, depletion and/or oxidation of GSH occurs through multiple mechanisms, which in turn promote fibrosis. Using the bleomycin model of fibrosis, the redox potential (E(h)) of plasma GSH and Cys redox systems was shown to change uniquely as a function of progression of bleomycin-induced lung injury. Plasma E(h) GSH/GSSG was shown to be selectively oxidized during the proinflammatory phase, whereas oxidation of E(h) Cys/CySS occurred at the later fibrotic phase. Increased oxidation of E(h) Cys/ CySS in the lung lining fluid was also detected and was linked due to decreased food intake in mice exposed to bleomycin (Iyer et al., 2009). To address the role of glutathione disruption in pulmonary fibrosis, a number of studies involving the GSH precursor, N-acetyl-l-cysteine (NAC), were conducted with the goal to augment pulmonary GSH levels and to alleviate oxidative stress. NAC was shown to dampen bleomycin-induced inflammation and subsequent fibrosis in mice in association with decreases in lysyl oxidase (Hagiwara, Ishii, & Kitamura, 2000; Li et al., 2012). A number of clinical trials have been also conducted to determine whether increases in GSH in the lung would attenuate the progression of disease in patients with IPF and maintain lung function. One clinical trial that included the GSH precursor, NAC, along with another drug, initially showed therapeutic effects associated with NAC (Behr et al., 2009; Demedts et al., 2005). However, re-evaluation of NAC in a clinical trial with over 130 IPF patients in the NAC or placebo groups failed to show a clinical benefit of NAC on disease progression or lung function decline (Martinez, De Andrade, Anstrom, King Jr., & Raghu, 2014). However, a pharmacogenomic interaction involving toll interacting protein (TOLLIP) and mucin 5B (MUC5B) single nucleotide polymorphisms (SNPs) and a response of IPF patients to NAC has become apparent (Oldham et al., 2015).

Protein S-glutathionylation, the death receptor FAS, and epithelial cell death

­ rotein S-glutathionylation, the death receptor FAS, and P epithelial cell death Cysteines in proteins have emerged as important biological sensors for redox perturbations that in turn regulate biological responses. Reactive, low pKa cysteines can be oxidized in specific, reversible, and regulated manners, to permit cells to sense and respond to redox changes. Protein S-glutathionylation (PSSG) represents a cysteine oxidation wherein GSH is conjugated covalently to the protein cysteine. PSSG has emerged as a critical regulatory mechanism that regulates protein structure and function (Fig.  1). The conjugation of glutathione to protein cysteines changes protein

FIG. 1 Steps in the catalytic cycle of protein S-glutathionylation (PSSG) and deglutathionylation known to be relevant in the pathogenesis of pulmonary fibrosis. A number of biochemical events can induce PSSG. The mode of PSSG is likely to be target and context specific, dependent upon the proximity of oxidant producing enzymes, redox relays, the oxidation state of the GSH/GSSG redox couple etc. In epithelial cells in settings of a profibrotic environment, oxidants originating from multiple sources lead to a sulfenic acid intermediate (SOH), which can be a platform for subsequent (1) PSSG, which can happen spontaneously or catalyzed by glutathione S-transferases (GST). GSTP is highly expressed in lung epithelial cells and promotes PSSG in preclinical models of pulmonary fibrosis. GSTP associates with Fas and contributes to its S-glutathionylation and epithelial cell apoptosis. (2) Conversely, glutaredoxin (GLRX, also known as GRX) acts to deglutathionylate proteins, restoring the original sulfhydryl group. GLRX induces deglutathionylation via the monothiol mechanism requiring only the N-terminal cysteine in the thioredoxin (TXN) domain. In settings of fibrosis, GLRX is downregulated by TGFB. Additionally, GLRX can be inactivated via S-glutathionylation of one or more cysteines outside of the TXN domain (3) and can be directly cleaved by caspases 8 and 3 (4). Evidence of oxidative inactivation of GLRX in lungs from patients with idiopathic pulmonary fibrosis (IPF) has been demonstrated.

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conformation (addition of three amino acids) and charge (glutamic acid), thereby affecting function (Anathy et al., 2012; Gallogly & Mieyal, 2007; Georgiou, 2002; Janssen-Heininger et al., 2008). The exact pathways that lead to PSSG in biological settings are not completely known, but it is thought that an oxidant, including H2O2, can first lead to a sulfenic acid intermediate (-SOH), a gateway oxidation, that can subsequently be S-glutathionylated. While PSSG has been shown to occur spontaneously, PSSG can also be catalyzed by glutathione-S-transferases (GSTs), a class of enzymes critical in phase II drug metabolism. Most of the studies conducted thus far on GST-catalyzed PSSG have centered on glutathione S-transferase π (GSTP) (Tew et al., 2011; Townsend et al., 2009). Conversely, the family of glutaredoxins (GLRX, also known as GRX) under physiological conditions act to specifically deglutathionylate proteins restoring the reduced sulfhydryl group (Lillig, Berndt, & Holmgren, 2008) (Fig. 1). Thus, regulatory pathways that control PSSG and deglutathionylation operate to precisely control the oxidation state of target cysteines. To assess the potential implications of PSSG for the pathophysiology of lung fibrosis, we took advantage of the highly specific catalytic activity of mammalian GLRX toward reduction of PSSG to visualize PSSG in formalin-fixed, paraffinembedded (FFPE) tissues in situ using microscopy approaches (Aesif et al., 2009) (Fig.  2). FFPE lung tissues were deparaffinized and rehydrated, using standard procedures, and tissues were then permeabilized with Triton X100, while reduced thiols were blocked with the thiol-specific blocking agent, N-ethylmaleimide. After washing of tissues, they were incubated with catalytically active GLRX, in the presence of GSH, glutathione reductase, and NADPH. Newly reduced protein thiols were next labeled with biotin-conjugated N-(3-maleimidylpropionyl) biocytin and biotin-tagged protein sulfhydryls where then visualized with streptavidin conjugated to fluorophore. Notable PSSG immunoreactivity was apparent in bronchiolar epithelial cells and alveolar macrophages under physiological conditions. In mice with bleomycin-induced fibrosis, increases in PSSG occurred in bronchiolar epithelial cells and parenchymal regions (Aesif et al., 2009). Increases in PSSG also occurred in lungs from patients with IPF (Fig. 2), and increases in PSSG inversely correlated with lung function in these patients (Anathy et al., 2018). Airway epithelial cells showed noticeable PSSG. Interestingly, GSTP was shown to be highly expressed in bronchiolar epithelial cells and type II alveolar epithelial cells (McMillan et al., 2016), consistent with a role of these cells in protection against environmental insults and metabolism of xenobiotics. In lungs from patients with IPF, GSTP was prominently increased in bronchiolar epithelia as was in the distal lung epithelial cells, in areas of rebronchiolarization, and in reactive type II epithelial cells, including those type II pneumocytes at the leading edge of disease progression (McMillan et al., 2016). In contrast to increases in GSTP immunoreactivity in lungs from patients with IPF, the enzymatic activity of GLRX was decreased in IPF lungs, in a partial dithiothreitol-specific manner, indicative of oxidative inactivation of GLRX (Fig.  1). Intriguingly, increases in GLRX S-glutathionylation were observed in lungs from patients with IPF and in mice with bleomycin-induced fibrosis (Anathy et al., 2018), suggestive of negative feedback inhibition of GLRX

­Protein S-glutathionylation, the death receptor FAS, and epithelial cell death

FIG. 2 Increases in S-glutathionylation in lung tissues from patients with idiopathic pulmonary fibrosis (IPF). Lung sections were deparaffinized, rehydrated, permeabilized, and reduced protein thiols blocked with N-ethyl maleamide (NEM). Sections were then subjected to GLRX-catalyzed protein cysteine labeling to detect regions of PSSG, as described in the text. Red = PSSG, blue = DAPI counterstain. Note the increases in PSSG in lungs from IPF patients (n = 4), compared with non-IPF controls (n = 4). Panels represent individual subjects. Arrowheads point to reactive alveolar type II epithelial cells believed to contribute to disease pathogenesis. This image was first published in Anathy, V., Lahue, K. G., Chapman, D. G., Chia, S. B., Casey, D. T., Aboushousha, R., Van Der Velden, J. L. J., Elko, E., Hoffman, S. M., McMillan, D. H., Jones, J. T., Nolin, J. D., Abdalla, S., Schneider, R., Seward, D. J., Roberson, E. C., Liptak, M. D., Cousins, M. E., Butnor, K. J., Taatjes, D. J., Budd, R. C., Irvin, C. G., Ho, Y. S., Hakem, R., Brown, K. K., Matsui, R., Bachschmid, M. M., Gomez, J. L., Kaminski, N., Van Der Vliet, A., & Janssen-Heininger, Y. M. W. (2018). Reducing protein oxidation reverses lung fibrosis. Nature Medicine 24, 1128–1135. https://doi.org/10.1038/s41591-018-0090-y. Epub 2018 Jul 9, and was reproduced with permission from Nature Medicine.

via S-glutathionylation. Earlier studies reported decreases in GLRX content in lungs from patients with IPF and COPD (Peltoniemi et al., 2004, 2006) and downregulation of GLRX by TGF-β1 (Reynaert, Wouters, & Janssen-Heininger, 2007), consistent with a putative role of enhanced PSSG in promoting lung fibrosis. As was described earlier, the death receptor, FAS, plays an important role in epithelial cell death and the subsequent development of fibrosis (Kuwano et al., 1999, 2001). Upon administration of FASL, rapid death of epithelial cells occurs resulting from activation of caspases 8 and 3 and death via apoptosis. In FASL-treated cells, one predominant immune-reactive band reacting with an anti-GSH antibody was apparent, suggesting a high degree of specificity toward specific proteins targeted via PSSG. Complementary immunoprecipitation strategies revealed that FAS itself was S-glutathionylated in epithelial cells exposed to FASL, and site directed mutagenesis experiments demonstrated that cysteine 294 in the death domain was the target for

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S-glutathionylation and subsequent cell death. S-glutathionylation of FAS coincided with a loss of GLRX enzymatic activity. Inhibition of caspases 8 and 3 or knockdown of caspase 8 prevented the loss of GLRX activity, FAS-SSG, and abolished cell death. Consistent with a role of S-glutathionylation of FAS in promoting apoptosis, the absence of GLRX enhanced FASL-induced apoptosis, in association with more FAS accumulating in lipid rafts and FASL binding to FAS, while overexpression of GLRX protected against epithelial cell apoptosis and diminished accumulation of FAS onto lipid rafts (Anathy et al., 2009). The demonstration that caspases 8 or 3 can directly cleave GLRX (Anathy et  al., 2009) suggests a protease-dependent mechanism toward inactivation of GLRX, in addition to aforementioned mechanism of oxidative inactivation of GLRX (Fig. 1).

­ER redox stress and tissue fibrosis The specificity of redox modifications of target proteins, including S-glutathionylation, is controlled via compartmentalization of oxidant formation and the presence of enzymes that regulate protein cysteine redox status in subcellular domains (Chen, Kirber, Xiao, yang, & Keaney Jr, 2008; Janssen-Heininger et al., 2008; Winterbourn, 2008). The endoplasmic reticulum (ER) is of particular interest because it represents a highly oxidizing environment (Hwang, Sinskey, & Lodish, 1992). For example, the GSH/GSSG ratio is substantially lower in the ER than the cytosol, and it has been suggested that GSH regulates oxidative folding processes (Appenzeller-Herzog, 2011). Many proteins that enter the secretory pathway, including the aforementioned surfactant proteins, contain cysteines that are oxidatively processed into disulfide bonds in the ER to stabilize the protein. The family of protein disulfide isomerases (PDI) catalyzes this disulfide bridge formation in their client proteins. Oxidized PDI (SS) will covalently bind its reduced client protein (SH) to catalyze disulfide bridge formation, via a sulfhydryl-disulfide exchange reaction in which the client protein will be oxidized (S-S), while PDI will be reduced (SH). To regenerate catalytically active PDI, it has to be reoxidized by ER oxidoreductin-1 (ERO1). During this regeneration cycle of PDI by ERO1, H2O2 is formed (Kakihana, Nagata, & Sitia, 2012). Thus, oxidative processing of proteins in the ER leads to the production of H2O2. Steady-state levels of H2O2 are precisely controlled in part via oxidation of GSH to GSSG and the action of the H2O2 scavenger, peroxiredoxin-4 (PRDX4, also known as PRX4), which is localized within the ER. Besides ERO1, other mechanisms have emerged to regenerate oxidized PDI, which are speculated to involve H2O2 produced by NOX4, acceptance of electrons by oxidized PRDX4, Vitamin K epoxide hydrolase (VKOR), quiescin-sulfhydryl oxidase (QSOX), and glutathione peroxidases (GPX) 7 and 8 (Kakihana et al., 2012; Nguyen et al., 2011; Tavender, Springate, & Bulleid, 2010). Similarly, additional quality control systems exist the ER. For example, Selenof is an ER-resident thioredoxin-like oxidoreductase that acts as a gatekeeper for immunoglobins and likely other disulfide-rich glycoproteins, thereby providing redox quality control of secreted glycoproteins (Yim et al., 2018). The respective roles of these and

­ER redox stress and tissue fibrosis

other ER proteins in redox regulation of ER processes in vivo and their roles in tissue fibrosis remain to be elucidated. A few studies have been conducted to suggest a link between dysregulated oxidative processing in the ER and altered extracellular matrix biology. The protein disulfide isomerase A3 (PDIA3) can associate with Bak and promote its oligomerization, thereby triggering intrinsic apoptosis under conditions of ER stress. We recently demonstrated in a house dust mite model of allergic inflammation that genetic ablation of PDIA3 specifically in lung epithelial cells decreased proapoptotic Bak oligomerization and caspase-3 activity in mice, in association with decreases in airway fibrosis (Hoffman et al., 2016), and similar results were obtained following siRNA-mediated ablation of ATF6-alpha (Hoffman et al., 2013). Ablation of PDIA3 resulted in more exposed sulfhydryls (–SH) in the glycoproteins periostin and epidermal growth factor, both of which are involved in airway remodeling, suggesting a potential mechanism, whereby PDIA3 promotes airways remodeling. Mice with combined loss of function mutations in genes encoding ERO1α, ERO1β, and PRDX4 display a compromised extracellular matrix due to defects in the intracellular maturation of procollagen. Combined absence of these enzymes resulted in increased cysteinyl sulfenic acid modification (sulfenylation) of ER proteins. Intriguingly, the increased cysteinyl sulfenic acids led to oxidation of ascorbate to dihydroascorbate. Consistent with the requirement of ascorbate in maintaining the function of ER-localized prolyl-4 hydroxylases, the decreases in ascorbate in the ER of mouse embryonic fibroblasts lacking ERO1α, ERO1β, and PRDX4 resulted in a strong decrease in procollagen 4-hydroxyproline content (Zito, Hansen, Yeo, Fujii, & Ron, 2012). Thioredoxin domain containing 5 (TXNDC5), an ER-localized protein disulfide isomerase, was shown to facilitate ECM protein folding and to contribute to the stability of ECM proteins. Enhanced expression of TXNDC5 promoted redoxsensitive activation of cardiac fibroblasts and augmentation of cardiac fibrosis (Shih et al., 2018). Quiescin sulfhydryl oxidase 1 (QSOX1) is an atypical disulfide catalyst, as it is localized to the Golgi and can be secreted from cells. QSOX1 activity was shown to be essential for the incorporation of laminin into the extracellular matrix synthesized by fibroblasts (Ilani et al., 2013). A role of VKOR in organ fibrosis has is emerging based upon a study showing that inhibition of VKOR with 3-acetyl5-­methyltetronic acid (AMT) attenuated the progression of cisplatin-induced renal fibrosis in rats (Uchida, Miyoshi, & Miyamoto, 2017). Lastly, overexpression of PRDX4 elicited protective effects against nonalcoholic steatohepatitis and type 2 diabetes in a mouse model (Nabeshima et al., 2013). Based upon these observations, it is highly plausible that dysregulation of proteostasis in the lung, linked to altered functions in ER-redox processes, contribute to the pathogenesis of lung fibrosis. A functional link between FAS, ER redox stress, and lung fibrosis is emerging. In this regard, we have demonstrated that the molecular events that culminate in FAS-SSG originate within the ER (Anathy et al., 2012). FAS contains 24 cysteines, 20 of which occur as disulfide bridges, formed in the ER that stabilize the receptor and enable binding of FASL. We discovered that epithelial cells contain a pool of latent FAS, not in the disulfide-bonded state, and that administration of FASL triggers a calcium-dependent signal that promotes disulfide bridge formation of FAS

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within the ER. PDIA3 was shown to be responsible for disulfide bridge formation in FAS. Absence of PDIA3, or its pharmacological inhibition, maintained FAS cysteines in a sulfhydryl state, and resulted in attenuated FAS-SSG and attenuated epithelial cell death. Incubation of cells with the sulfenic acid trapping agent, dimedone, also prevented FAS-SSG, suggesting that formation of a sulfenic acid intermediate preceded the formation of FAS-SSG. In epithelial cells stimulated with FASL, GSTP was found to bind to FAS, and interaction between Fas and GSTP was first observed in the ER-enriched fraction (Anathy, Roberson, Cunniff, et al., 2012). In lung tissues from patients with IPF, an interaction between FAS and GSTP was also observed (McMillan et al., 2016), along with increases in FAS-SSG (Anathy et al., 2018). SiRNA-mediated ablation of GSTP or its pharmacological inhibition attenuated FAS-SSG and decreased cell death. The active GSTP inhibitor, TLK117, is a GSH analog and is the active metabolite of TLK199, which has completed phase II clinical trials for myelodysplastic syndrome (Raza et al., 2009, 2012). TLK117 is a highly specific GSTP inhibitor, with a binding affinity greater than GSH itself and a selectivity for GSTP over 50-fold greater than the GSTM and GSTA classes (inhibition constant [Ki] = 0.4 μM) (Laborde, 2010; Morgan, Ciaccio, Tew, & Kauvar, 1996). We showed this inhibitor attenuated both Fas-SSG and cell death (Anathy, Roberson, Cunniff, et al., 2012; McMillan et al., 2016). To address the functional role of GSTP in lung fibrosis, mice that lacked functional GSTP1 and GSTP2 were subjected to bleomycin or AdTGFB1-induced fibrosis and were shown to have attenuations in FAS-SSG, decreased caspases 8 and 3 activity, and diminished fibrosis, compared with WT controls. Direct administration of TLK117 into airways of mice with existing bleomycin- or AdTGFB1-induced fibrosis blocked the progression of fibrosis, in association with decreases in overall S-glutathionylation, decreases in S-glutathionylation of Fas, and decreased activities of caspases 3 and 8, compared with mice receiving vehicle control (McMillan et al., 2016). The importance of S-glutathionylation in lung fibrosis was further corroborated by studies that addressed the role of GLRX, using transgenic mice overexpressing GLRX in lung epithelia, as well as mice that globally lack GLRX. Mice that lack GLRX were more sensitive to AdTGFB1- or bleomycin-induced fibrosis, whereas mice that overexpress GLRX in lung epithelial cells showed increased resistance to fibrosis. Absence of GLRX augmented FAS-SSG in mice with bleomycin or AdTGFB1-induced fibrosis, compared with WT mice, in association with enhanced caspase 3 activities, consistent with increases in cell death. Overexpression of GLRX in epithelial cells dampened Fas-SSG and caspase 3 activation. Furthermore, ablation of caspase-8, which is activated following activation of the FAS pathway, also attenuated Fas-SSG, caspase-3 activation, and fibrosis (Anathy et al., 2018). Collectively, these data demonstrate that attenuation of epithelial cell death confers protection from bleomycin- or AdTGFB1-induced fibrosis and that S-glutathionylation of FAS in epithelial cells is an important death-inducing signal that promotes fibrogenesis. We next assessed whether direct administration of GLRX into airways of mice with existing bleomycin- or AdTGFB1-induced fibrosis would elicit similar protective

­Concluding comments and challenges for future research and development

responses. Indeed, administration of recombinant GLRX augmented GLRX activity in the lung tissue, dampened PSSG, and reversed increases in collagen content, while inducing collagenolytic activity within the lung. Similar protective effects of GLRX were observed in lungs from aging mice, which were more prone to bleomycininduced fibrosis. A mutant of GLRX lacking cysteine 23, which is critical in the deglutathionylation reaction, failed to elicit protective responses, demonstrating that the catalytic activity of GLRX is important in the antifibrotic responses (Anathy et al., 2018). These collective observations point to the importance not of GSH per se but a unique facet of GSH chemistry, which involves its covalent incorporation into proteins, catalyzed by GSTP, and reversed by GLRX, a biochemical pathway regulated by enzymes that had not previously been recognized in settings of pulmonary fibrosis (Fig. 1). Thus, the consideration of clinical studies utilizing the clinically relevant GSTP inhibitor, TLK199, or a variant thereof, in conjunction with active GLRX, appears well warranted and may yield new insights into the functional importance of S-glutathionylation chemistry in the pathogenesis and progression of this deadly disease.

­ oncluding comments and challenges for future research C and development The development of fibrosis is an insidious process that develops over age in response to repeated stresses and lack of adequate repair. A loss of proteostasis, linked with defective autophagy, accumulation of defective mitochondria, and as of yet unknown processes in the aging fibrosis-prone lung culminate in an environment wherein alveolar type II epithelial cells cannot adequately give rise to type I cells that line the alveolar surface area, due to altered plasticity, enhanced death and/or senescence. Defective epithelia in turn send signals that promote myofibroblasts recruitment and activation, leading to the massive remodeling that occurs in patients with lung fibrosis (Fig. 3). Based upon these findings, redox perturbations relevant to fibrogenesis may originate from different organelles that may be controlled by multiple oxidant sources. Changes in the redox environment likely contribute to the altered biology of epithelial cells and fibroblasts and thereby the sequelae that culminate in fibrosis. As we described, changes in glutathione homeostasis occur in settings of fibrosis, evidenced by increased oxidation of the GSH/GSSG redox couple, increased protein S-glutathionylation, increased expression of GSTP in affected epithelia, downregulation of GCL and GLRX by TGFB, and oxidative inactivation of GLRX. Attempts to augment GSH via oral administration of NAC have failed to impact the progression of disease in IPF patients, highlighting an urgent need for improved strategies to normalize redox homeostasis in settings of fibrosis. Based on our knowledge about the processes described herein that govern the development of fibrosis, such approaches may warrant avenues to alleviate ER redox stress, dampen ROS produced

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Healthy

Fibrotic

3 2 ¯ Epi. ¯

750

Fibr.

Alveoli disrupted

Production of surfactants, etc. Progenitor function

1

ER Stress Mitochondrial dysfunction Loss of proteostasis Altered cell plasticity and enhanced death

FIG. 3 Proposed model highlighting potential sources of redox perturbations in the aging fibrosis prone lung. 1: A loss of proteostasis, due to ER stress in alveolar type II epithelial cells, linked with defective autophagy, and accumulation of defective mitochondria, and as of yet unknown processes in the aging fibrosis prone lung culminate in an environment, wherein alveolar type II epithelial cells cannot adequately give rise to type I cells that form the alveolar surface area (2). Altered plasticity, enhanced death, and/or senescence are processes reported in epithelial cells from the IPF lung. Defective epithelia (epi↓) in turn send signals that promote myofibroblasts (fibr↑) recruitment, activation, and senescence, leading to massive remodeling and honeycombing (3). Redox perturbations originating from the ER, defective mitochondria, potentially driven by NOX4, which has been localized to the ER or mitochondria, potentially contribute to disease pathogenesis. Defective expression of NRF2, TGFB-mediated downregulation of GLRX or GCL, contribute to altered homeostasis of GSH or PSSG. Similarly, enhanced expression of GSTP coupled with (oxidative) inactivation of GLRX promotes an environment of enhanced PSSG in settings of fibrosis. While most of the proteins targeted for PSSG in fibrosis remain to be discovered, S-glutathionylation of Fas is a driver of epithelial cell apoptosis and resultant fibrosis.

­References

from mitochondria, inhibit NOX4, or diminish S-glutathionylation through inhibition of GSTP or augmentation of GLRX. A number of questions and concerns arise regarding the development of redox-based therapeutics to combat lung fibrosis. For example, will a singular approach to normalize redox homeostasis suffice to combat fibrosis? What will be the impact of redox-based therapies be against a back drop of existing pirfenidone and/or nintedanib therapies? Should redox-based therapies selectively target affected organelles, such as the ER or mitochondria? What are the redox-modified target proteins, and do they play dominant roles in fibrogenesis? How narrow or broad should the reactivity of the redox based-drug be, and what macromolecules should they target? These questions are not trivial, and addressing them has implications that reach far beyond lung fibrosis. Scientists working in the domain of redox biochemistry should take note, as the past failures of antioxidant therapies raise questions about how druggable redox processes in fact are. The recent FDA approval of the electrophile, dimethyl fumarate (Tecfidera), for the treatment of multiple sclerosis (English & Aloi, 2015) nonetheless provides a hopeful message. Our hope is that the recent discoveries pertaining to glutathion(e)ylation chemistry provide a rapid path to drug development to increase the survival and quality of life of the millions of patients diagnosed with this deadly disease.

­Acknowledgments This work was supported by grants NIH R35HL135828 (Y-JH), an ATS unrestricted grant, and NIH R01HL122383 (VA). The authors thank Dr. Isabelle Tian for creating Fig. 3.

­Conflict of Interest Yvonne Janssen-Heininger and Vikas Anathy hold patents: U.S. Patent No. 8,679,811, “Treatments Involving Glutaredoxins and Similar Agents” (YJ-H, VA), U,S. Patent No. 8,877,447, “Detection of Glutathionylated Proteins” (YJ-H), and U.S. Patent, 9,907,828, “Treatments of oxidative stress conditions” (YJ-H, VA). Yvonne Janssen-Heininger and Vikas Anathy have received consulting fees from Celdara Medical LLC for their contributions with the commercialization of glutaredoxin for the treatment of pulmonary fibrosis.

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CHAPTER

Dicarbonyl stress and the glyoxalase system

36

Naila Rabbania, Mingzhan Xueb, Paul J Thornalleyb a

Clinical Sciences Research Laboratories, Warwick Medical School, University of Warwick, University Hospital, Coventry, United Kingdom bDiabetes Research Center, Qatar Biomedical Research Institute, Hamad Bin Khalifa University, Qatar Foundation, Doha, Qatar

Abstract

Dicarbonyl stress is defined as the abnormal accumulation of reactive α-oxoaldehyde, dicarbonyl metabolites leading to cell and tissue dysfunction implicated in aging and disease. There is increased formation of arginine-derived hydroimidazolone adducts of proteins and ­guanosine-derived imidazopurinone adducts of DNA, linked to protein misfolding and mutagenesis, respectively. Methylglyoxal is a dominant mediator of dicarbonyl stress in  vivo. It is metabolized by glutathione-dependent glyoxalase 1 (Glo1). The main drivers of dicarbonyl stress are increased formation of methylglyoxal by glycolytic overload and decreased metabolism of methylglyoxal by downregulation of Glo1. Proteins susceptible to dicarbonyl modification are enriched in protein folding, protein synthesis, and glucose metabolism. Dicarbonyl stress activates the unfolded protein response and downstream proinflammatory and prothrombotic responses. The pathobiology of dicarbonyl stress is most evident in obesity, vascular complications of diabetes, cardiovascular disease, renal failure, and aging. It may be corrected by dicarbonyl scavengers and inducers of Glo1 expression. ­Keywords: Glyoxalase, Methylglyoxal, Glycation, Glycolytic overload, Unfolded protein response, Inflammation, Proteomics, Diabetes, Cardiovascular disease, Aging

­Introduction Dicarbonyl stress is the abnormal accumulation of dicarbonyl metabolites leading to increased modification of protein and DNA contributing to cell and tissue dysfunction in aging and disease. The dicarbonyl metabolites are α-oxoaldehydes such as glyoxal, methylglyoxal (MG), 3-deoxyglucosone, and glucosone—as reviewed (Rabbani & Thornalley, 2012a). Acyclic α-oxoaldehyde metabolites are the most reactive, and the metabolite of this type formed at the highest flux in physiological systems is MG. Dicarbonyl stress is damaging in physiological systems because it leads to increased formation of adducts with proteins and DNA. The major adducts of proteins are hydroimidazolones formed by the reaction of α-oxoaldehydes Oxidative Stress. https://doi.org/10.1016/B978-0-12-818606-0.00036-5 © 2020 Elsevier Inc. All rights reserved.

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with arginine residues, and the major adducts formed by reaction with DNA are imidazopurinone derivatives such as MG-derived MGdG (Ahmed, Dobler, Dean, et al., 2005; Thornalley, Waris, Fleming, et al., 2010)—Fig. 1. Other minor adducts are Nε-(1-carboxyethyl)lysine (CEL), MG-derived imidazolium lysine cross-link (MOLD), argpyrimidine, N2-carboxyethylguanosine (CEdG), and others (Rabbani and Thornalley, 2012, 2014a). The formation of these adducts is suppressed to low, tolerable levels by metabolism of MG by glyoxalase 1 (Glo1) of the glyoxalase system present in the cytosol of all cells. MG is metabolized to S-d-lactoylglutathione by Glo1, and this is further metabolized to d-lactate by glyoxalase 2, reforming GSH consumed in the Glo1-catalyzed step (Xue, Rabbani, & Thornalley, 2011)—Fig. 2. Dicarbonyl stress is different to oxidative stress in that it occurs without challenge by oxidants and is relatively insensitive to antioxidants. It is also different from oxidative stress in that it targets primarily arginine residues of proteins that have the highest probability of all amino acid residues to be located in a functional domains of proteins, and modification by dicarbonyls produces functional impairment (Gallet, Charloteaux, Thomas, et al., 2000). The modification reaction is also nonoxidative: a dehydration reaction. It produces loss of positive charge and hence

CO

NH2

O

CH3

HN

NH

Arginine residue

O

H

Methylglyoxal

N

NH MG-H1 residue

(A) N

O

N

NH N

NH2

O

CH3

O

H

N

O

N N

CH3 OH OH N H H

+ O

dG N N

(B)

H

Hydroimidazolone (MG-H1) O

N

CH3

HC (CH2)3 NH

HC (CH2)3 NH NH

CO

H N

N

OH OH N CH3 H

Imidazopurinone MGdG

FIG. 1 Protein and nucleotide glycation and glyoxalase metabolism in dicarbonyl stress. (A) Formation of hydroimidazolone MG-H1 from arginine residues in protein. (B) Formation of imidazopurinone isomers in DNA. The common 2′-deoxyribosyl group has been omitted for clarity.

­Introduction

FIG. 2 Metabolism of methylglyoxal by the glyoxalase system.

loss of all ­electrostatic interactions with neighboring groups and interacting ligands. In some cases, this may affect the ionization of neighboring groups, for example, increasing the pKa of the activated tyrosine phenolic group in tyr-411 of human serum albumin (Ahmed et al., 2005). With DNA, the modification reaction by MG is again nonoxidative: an addition reaction. Dicarbonyl stress produces the highest steadystate levels of adducts produced by a physiological spontaneous modification—MGderived imidazopurinones. The level of imidazopurinones is linked to mutagenesis and potentially carcinogenesis (Thornalley et al., 2010). Indeed, independently, the major enzyme preventing their formation, Glo1, was functionally characterized as a tumor suppressor protein (Zender, Xue, Zuber, et al., 2008). Dicarbonyl stress is often caused by mechanisms unrelated to oxidative stress, such as dysregulation of glycolysis, glycolytic overload, ischemia, and low-grade inflammation. It requires a new approach to prevention and treatment—such as dicarbonyl scavengers and inducers of Glo1 expression or Glo1 inducers (Rabbani & Thornalley, 2019a). Dicarbonyl stress may be exacerbated by oxidative stress in that decreased GSH in oxidative stress directly impairs the metabolism of MG and glyoxal, leading to their accumulation (Abordo, Minhas, & Thornalley, 1999). Metabolic dysfunction driving increased MG formation also often concurrently increases formation of reactive oxygen species (ROS). For example, hexokinase-2 (HK2) driven increase in glycolysis in vascular cells in hyperglycemia associated with diabetes produces increased flux of triosephosphates and MG formation. The same process also increases glucose-6-phosphate concentration, displacing HK2 from mitochondria, increasing hyperpolarization of the mitochondrial membrane and increasing ROS formation. Increased glycolytic flux also increases formation of diacylglycerol formation, via increased flux of formation of dihydroxyacetone phosphate, with downstream activation of protein kinase c and NADPH oxidase—also contributing to ROS formation. This process is called HK2-linked glycolytic overload (Irshad, Xue, Ashour, et al., 2019). We also recently reported that HK2-linked glycolytic overload occurs in cell senescence (Hariton, Xue, Rabbani, et  al., 2018) and ischemia reperfusion injury following myocardial infarction where it is a determinant of myocardium infarct size and adverse clinical outcome. In the latter case, substrate for increased glycolysis is produced by ischemia-stimulated glycogenolysis (Rabbani & Thornalley, 2019b).

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­ eactive metabolites of dicarbonyl stress: Methylglyoxal, R glyoxal, 3-deoxyglucosone, and other α-oxoaldehyde metabolites In mammalian metabolism, MG is mainly formed by spontaneous trace level degradation of triosephosphate intermediates of glycolysis, glyceraldehyde-3-phosphate (GA3P) and dihydroxyacetone phosphate (DHAP) (Phillips & Thornalley, 1993). So, the cellular formation of MG is ubiquitous, and if not metabolized, it crosses membranes and is present in plasma and other physiological fluids. In bacteria, MG was found to be produced enzymatically from DHAP by MG synthase (Grabar, Zhou, Shanmugam, et al., 2006). The concentration of MG in physiological systems is typically 1–5 μM in cells and 100–500 nM in plasma. These estimates also corroborate well with prediction from simple models of the kinetics of protein glycation by MG and protein turnover (Rabbani & Thornalley, 2014b). In aqueous solution at physiological concentrations, MG exists mainly in three forms: unhydrated CH3COCHO, monohydrate CH3COCH(OH)2, and dihydrate CH3C(OH)2CH(OH)2 in the ratio 1:70:29 (Rabbani & Thornalley, 2014b). MG binds rapidly, reversibly, and strongly to cysteine thiol groups with the association equilibrium constant 5 × 106 M−1 (Lo, Westwood, McLellan, et al., 1994). This does not impair redox signaling directly as the extent of thiol modification is low: total thiol concentration in cells is c. 30 mM (GSH and protein thiols), whereas total MG concentration bound is 1–5 μM (Rabbani & Thornalley, 2014b). In plasma, MG binds reversibly to the free cysteinyl thiol of albumin, and in cells, MG binds strongly and reversibly to protein thiols and reduced glutathione (GSH). In cellular metabolism in the steady-state, therefore, the highest concentration of low-molecular-mass form of MG is the hemithioacetal adduct of MG with GSH. For high catalytic efficiency in situ, therefore, Glo1 has a high specificity constant kcat/KM, c. 1.1 × 109 M−1 min−1 for human Glo1 (Allen, Lo, & Thornalley, 1993), and it utilizes GSH-MG hemithioacetal as substrate. MG that escapes metabolism and reaction with protein and DNA crosses the plasma membrane and is then available to react with extracellular matrix and plasma proteins. MG permeates cell plasma membranes by passive diffusion of the unhydrated form. This is rate limited by MG dehydration, giving a half-life of ~4 min (Rabbani & Thornalley, 2014b). The half-life for metabolism of MG in cells in situ is c. 10 min, and the rate of irreversible binding to protein in plasma was c. 3.6 h. This implies that part of the MG formed in cells leaks out from the site of formation and may diffuse through interstitial fluid into plasma and thereafter permeate back into interstitial fluid and cells of other tissues. The reactivity of MG in tissues with proteins was estimated to produce a mean diffusion distance of c. 2–3 cm, suggesting that MG has relatively long range and half-life to locate and modify sensitive sites of proteins, often leading to protein inactivation and dysfunction, cf. ROS, the estimated mean diffusion distance of