Drug-Induced Liver Disease, Second Edition

Drug-Induced Liver DiseasE

Kaplowitz_978-0849398964_TP.indd1 1

6/6/07 4:00:05 PM

Drug-Induced Liver DiseasE Second Edition

Edited by

Neil Kaplowitz Keck School of Medicine, University of Southern California Los Angeles, California, USA

Laurie D. DeLeve Keck School of Medicine, University of Southern California Los Angeles, California, USA

Kaplowitz_978-0849398964_TP.indd2 2

6/6/07 4:00:05 PM

Informa Healthcare USA, Inc. 52 Vanderbilt Avenue New York, NY 10017 q 2007 by Informa Healthcare USA, Inc. Informa Healthcare is an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 0-8493-9896-7 (hb: alk. paper) International Standard Book Number-13: 978-0-8493-9896-4 (hb: alk. paper) This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequence of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Drug-induced liver disease / edited by Neil Kaplowitz, Laurie D. DeLeve. – 2nd ed. p. ; cm. Includes bibliographical references and index. ISBN-13: 978-0-8493-9896-4 (hb : alk. paper) ISBN-10: 0-8493-9896-7 (hb : alk. paper) 1. Hepatotoxicology. 2. Liver–Effect of drugs on. 3. Drugs–Side effects. I. Kaplowitz, Neil. II. DeLeve, Laurie D., 1955[DNLM: 1. Liver Diseases–chemically induced. 2. Liver–drug effects. 3. Preparations–adverse effects. WI 700 D7943 2007] RC848.H48D78 2007 616.3’6–dc22

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Pharmaceutical

2007014965

To our loving families and to the memory of Hy Zimmerman, who inspired us with his intellect and dedication in pioneering this field. We are proud to follow in his footsteps.

Preface to the Second Edition

The high productivity of the pharmaceutical industry has provided exciting, efficacious new drugs. However, with efficacy always comes the potential for toxicity, and the growth in new pharmaceuticals has been accompanied by several new drugs linked to liver toxicity. At the same time, epidemiological studies have found that drugs are now the most common causes of liver failure. This has led to a resurgence of interest in drug-induced liver disease in general and has spurred an influx of clinical researchers into this area. Basic research in this area has also thrived over the last decade, due to innovations in biomedical research that have given us tools that have provided new insights into the mechanisms of drug toxicity. The time was therefore ripe to compile a volume with contributions from scientists around the world with expertise in pathogenesis and clinical presentation, as well as authorities on the various categories of drugs and toxins of importance to this field. We have been gratified by the outstanding reviews of the first edition. However, we believe that in this fast-moving field, a book would only remain of value if it can be revised in a timely enough fashion to keep abreast of recent developments. In the second edition, 16 out of the 36 chapters have new authors or cover new topics. New topics include pharmacogenomics and toxicogenomics, causality assessment, risk factors for drug-induced liver disease, management of drug-induced liver disease, and mushroom poisoning. Pharmacogenomics and toxicogenomics are new fields that provide the hope for rational strategies for the pharmaceutical industry in weeding out toxic drugs earlier in development, but may also devise novel approaches to prevent drug toxicity in the susceptible few without exclusion of new drugs efficacious for the many. The new chapters “Causality Assessment” and “Risk Factors for Drug-Induced Liver Disease” expand the Diagnosis and Management section and provide more background on the fundamentals of the field. We have also added a chapter on management to address issues as such liver transplantation and the use of steroids and ursodeoxycholic acid, and we have addressed therapy in individual chapters as well. The second edition is divided into four sections. Section I focuses on mechanisms of hepatotoxicity, often illustrated by examples of specific drugs. The newly expanded Section II reviews general principles of clinical presentation, histopathology, predisposition to toxicity, diagnosis, and management. Each chapter in Section III examines a class of drugs, toxins, or drugs used within a clinical specialty. This section provides a systematic review of the major xenobiotics associated with drug-induced liver disease and also serves as a reference for clinicians dealing with a possible case of drug-induced liver disease. Section IV contains a completely rewritten chapter on drug toxicity from a regulatory perspective. We are pleased with the second edition, which has allowed us to improve and expand coverage of the field and to update the rapidly advancing knowledge of pathogenesis. We believe this volume will be a great value to hepatologists, physicians in all fields of medicine, toxicologists and pharmacologists, and scientists working in preclinical and clinical drug development in both academia and industry. Neil Kaplowitz Laurie D. DeLeve

Preface to the First Edition

With the ever-increasing exposure to pharmaceuticals, more and more examples of druginduced liver disease have been identified in recent years. At the same time, the basic science of hepatic pharmacology, toxicology, and immunology have exploded in the past 5 to 10 years with exciting new developments and insights. We are now poised at the very end of the 20th century with the opportunity to re-evaluate this important topic as we look to the promise of understanding, predicting, preventing, and healing a common problem in clinical medicine that is of importance to all branches of medicine and to anyone who prescribes pharmaceutical or alternative medications. Therefore, the editors believe that an authoritative, up-to-date volume with contributions by experts in basic pathogenesis, clinical pathology, and use of various categories of agents will be of great interest to a broad spectrum of medicine. In this regard, we have drawn upon worldwide expertise with about one-third of the chapters written by authors outside the United States. Innovations in methodology have had a major impact on research in drug-induced liver injury, and this has led to a greater understanding of the mechanisms involved. A few examples should illustrate the progress that has been made and is described in this book. The explosion of information on apoptosis has provided insight into the subtleties of drug-induced cell death. The use of molecular biological techniques has permitted the cloning of numerous genes encoding for P450 isoenzymes. This has made possible the expression of recombinant P450 enzymes and specific P450 antibodies. The availability of recombinant enzymes and specific inhibiting antibodies has facilitated studies to determine the contribution of individual P450 isoenzymes to the metabolism of specific drugs. Until quite recently, cholestasis was thought to be due to either mechanical obstruction of bile flow or cell toxicity that impeded the handling of bile. Improved techniques for isolating membrane vesicles and the cloning and characterization of hepatocyte membrane transporters have allowed the elucidation of a novel mechanism of cholestasis: drug-induced impairment of bile acid transporters in otherwise intact hepatocytes. As more investigators have taken advantage of relatively new methods to isolate pure nonparenchymal cells, there has been a rapid rise in information on the contribution of Kupffer cells, sinusoidal endothelial cells, and stellate cells to a variety of liver diseases, including drug- and toxin-induced liver injury. The concept of the mitochondrion as a major target of drug-induced toxicity was only raised in the early 1980s. Since then, toxicity of an everincreasing number of drugs has been linked to selective toxicity to the mitochondrion. Although reference is made in these examples to chapters on mechanisms in Section I, Section III reiterates many of these processes in the context of individual drugs that have been linked to one of these modes of toxicity. This book has been divided into three major sections. Section I examines hepatotoxicity from the perspective of the mechanisms, across categories of drugs, so that the principles involved can be explored in depth. Examples of drugs to which these mechanisms apply is provided, but the main focus is on the mechanism. Because the authors are experts who are writing about the current state-of-the-art in their own field, this information is useful to both clinicians who want to gain understanding of the fundamental principles as we understand them today, as well as to knowledgeable clinicians and investigators who wish to read about the newest advances. Section II provides a general outline of the clinical presentation, histopathology, and management of drug-induced hepatotoxicity. Chapter 12 systematically reviews the clinical

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Preface to the First Edition

presentation and pathological picture of the types of liver injury that can be induced by drugs and toxins. Chapter 14 reviews the factors that predispose an individual to drug toxicity, suggests strategies for monitoring patients at risk for toxicity, and provides information on preventive measures. The information provided in this section provides a basic framework for any clinician who might be confronted with xenobiotic-induced hepatotoxicity. Section III systematically reviews specific toxins implicated in drug-induced hepatotoxicity. Each chapter examines the toxicity induced by drugs or toxins within a specific pharmacological class or by drugs used within a clinical specialty. The current understanding of the mechanism of toxicity, risk factors for developing toxicity, histological characteristics, clinical manifestations, and management are discussed for each category of drugs. This section is of value to gastroenterologists and hepatologists who want a systematic review of druginduced liver disease. It also serves as a reference for clinicians in a variety of specialties who are confronted with a patient with liver disease that might be attributable to drug therapy. Neil Kaplowitz Laurie D. DeLeve

Contents

Preface to the Second Edition Preface to the First Edition Contributors

v vii

xiii

PART I. MECHANISMS OF LIVER INJURY 1.

Drug-Induced Liver Disease Neil Kaplowitz

1

2.

Cytochrome P450 Activation of Toxins and Hepatotoxicity F. Peter Guengerich

3.

Antioxidant Defense in Liver Injury: Oxidant Stress, Antioxidant Defense, and Liver Injury 33 Hartmut Jaeschke

4.

Hepatotoxicity Due to Mitochondrial Injury 49 Dominique Pessayre, Bernard Fromenty, Abdellah Mansouri, and Alain Berson

5.

Mechanisms of Cell Death and Relevance to Drug Hepatotoxicity Neil Kaplowitz

6.

Significance of Hepatobiliary Transporters for Drug-Induced Liver Disease Peter J. Meier and Christiane Pauli-Magnus

7.

Immunological Mechanisms in Drug-Induced Liver Injury Dwain L. Thiele

8.

Mechanistic Role of Acyl Glucuronides 125 Hilde Spahn-Langguth, Chunze Li, and Leslie Z. Benet

9.

Nonparenchymal Cells, Inflammatory Macrophages, and Hepatotoxicity Debra L. Laskin and Carol R. Gardner

10. Role of Tissue Repair in Liver Injury Harihara M. Mehendale

13

207

PART II. DIAGNOSIS AND MANAGEMENT 12. Clinicopathological Patterns of Drug-Induced Liver Disease Willis C. Maddrey

97

115

185

11. Genetic Susceptibility to Drug-Induced Liver Disease Mark Russo and Paul B. Watkins

85

223

159

x

Contents

13. Histopathology of Drug-Induced Liver Disease Gary C. Kanel 14. Risk Factors for Drug-Induced Liver Disease Laurie D. DeLeve

237 291

15. Genomics, Proteomics, and Metabolomics in the Diagnosis and Mechanisms of Drug-Induced Liver Disease 307 Andrew A. Stolz 16. Causality Assessment 325 Ma Isabel Lucena, Rau´l J. Andrade, Raquel Camargo, and Miren Garcı´a-Corte´s 17. Management of the Patient with Drug-Induced Liver Disease Thomas D. Boyer

345

PART III. HEPATOTOXICITY OF SPECIFIC DRUGS 18. Mechanisms of Acetaminophen-Induced Liver Disease Sidney D. Nelson and Sam A. Bruschi

353

19. Acetaminophen: Pathology and Clinical Presentation of Hepatotoxicity William M. Lee and George Ostapowicz

389

20. Mechanisms Underlying the Hepatotoxicity of Nonsteroidal Anti-inflammatory Drugs 407 Urs A. Boelsterli 21. Nonsteroidal Anti-inflammatory Drugs and Leukotriene Receptor Antagonists: Pathology and Clinical Presentation of Hepatotoxicity 439 James H. Lewis 22. Mechanism, Pathology, and Clinical Presentation of Hepatotoxicity of Anesthetic Agents 465 J. Gerald Kenna 23. Anticonvulsant Agents 485 Munir Pirmohamed and Steven J. Leeder 24. Hepatotoxicity of Psychotropic Drugs and Drugs of Abuse Dominique Larrey 25. Antibacterial and Antifungal Agents Richard H. Moseley

527

26. Hepatotoxicity of Antituberculosis Drugs Sumita Verma and Neil Kaplowitz 27. Hepatic Toxicity of Antiviral Agents Ulrich Spengler

507

547

567

28. Hepatotoxicity of Cardiovascular and Antidiabetic Medications Sidharth S. Bhardwaj and Naga P. Chalasani 29. Cancer Chemotherapy Laurie D. DeLeve

631

593

xi

Contents

30. Hepatotoxicity of Immunomodulating Agents and the Transplant Situation Timothy J. Davern 31. Methotrexate Controversies Adrian Reuben

663

683

32. Adverse Effects of Hormones and Hormone Antagonists on the Liver Shivakumar Chitturi and Geoffrey C. Farrell

707

33. Mushroom Poisoning: A Clinical Model of Toxin-Induced Centrilobular Necrosis Franc¸ois Durand and Dominique Valla 34. Hepatotoxicity of Herbal Medicines, Vitamins, and Natural Hepatotoxins Lawrence U. Liu and Thomas D. Schiano 35. Occupational and Environmental Hepatotoxicity Keith G. Tolman and Anthony S. Dalpiaz PART IV. REGULATORY PERSPECTIVES 36. Regulatory Perspectives John R. Senior Index

789

771

755

733

723

Contributors

Rau´l J. Andrade

Liver Unit, Hospital Virgen de la Victoria, Ma´laga, Spain

Leslie Z. Benet Department of Biopharmaceutical Sciences, University of California-San Francisco School of Pharmacy, San Francisco, California, U.S.A. Alain Berson E´quipe Mitochondries, INSERM, U773, Centre de Recherche Biome´dicale Bichat Beaujon, Faculte´ de Me´decine Xavier Bichat, Universite´ Paris 7 Denis Diderot, Paris, France Sidharth S. Bhardwaj Division of Gastroenterology and Hepatology, Indiana University School of Medicine, Indianapolis, Indiana, U.S.A. Urs A. Boelsterli Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, Storrs, Connecticut, U.S.A. Thomas D. Boyer Liver Research Institute, University of Arizona, Tucson, Arizona, U.S.A. Sam A. Bruschi Department of Medicinal Chemistry, University of Washington School of Pharmacy, Seattle, Washington, U.S.A. Raquel Camargo

Liver Unit, Hospital Virgen de la Victoria, Ma´laga, Spain

Naga P. Chalasani Division of Gastroenterology and Hepatology, Indiana University School of Medicine, Indianapolis, Indiana, U.S.A. Shivakumar Chitturi Department of Gastroenterology and Hepatology, Australian National University Medical School at the Canberra Hospital, Australian Capital Territory, Australia Anthony S. Dalpiaz Division of Gastroenterology, University of Utah School of Medicine, Salt Lake City, Utah, U.S.A. Timothy J. Davern Gastroenterology Division and Liver Transplant Program, University of California, San Francisco, California, U.S.A. Laurie D. DeLeve Keck School of Medicine, University of Southern California, Los Angeles, California, U.S.A. Franc¸ois Durand Service d’He´patologie, Hospital Beaujon, Clichy, France Geoffrey C. Farrell Department of Gastroenterology and Hepatology, Australian National University Medical School at the Canberra Hospital, Australian Capital Territory, Australia Bernard Fromenty E´quipe Mitochondries, INSERM, U773, Centre de Recherche Biome´dicale Bichat Beaujon, Faculte´ de Me´decine Xavier Bichat, Universite´ Paris 7 Denis Diderot, Paris, France Miren Garcı´a-Corte´s Liver Unit, Hospital Virgen de la Victoria, Ma´laga, Spain

xiv

Contributors

Carol R. Gardner Department of Pharmacology and Toxicology, Rutgers University, Piscataway, New Jersey, U.S.A. F. Peter Guengerich Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee, U.S.A. Hartmut Jaeschke Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas, U.S.A. Gary C. Kanel Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, California, U.S.A. Neil Kaplowitz Keck School of Medicine, University of Southern California, Los Angeles, California, U.S.A. J. Gerald Kenna

AstraZeneca Safety Assessment, R&D Alderley Park, Macclesfield, Cheshire, U.K.

Dominique Larrey Service d’He´pato-Gastroente´rologie et Transplantation, Hoˆpital Saint Eloi, Montpellier, France Debra L. Laskin Department of Pharmacology and Toxicology, Rutgers University, Piscataway, New Jersey, U.S.A. William M. Lee

University of Texas Southwestern Medical Center, Dallas, Texas, U.S.A.

Steven J. Leeder Division of Pediatric Pharmacology and Medical Toxicology, Children’s Mercy Hospital and Clinics, Kansas City, Missouri, U.S.A. James H. Lewis Chunze Li

Georgetown University Medical Center, Washington, D.C., U.S.A.

Merck & Co., Inc., West Point, Pennsylvania, U.S.A.

Lawrence U. Liu Division of Liver Diseases and Recanati/Miller Transplantation Institute, The Mount Sinai Medical Center, New York, New York, U.S.A. Ma Isabel Lucena Departmento de Farmacologia Clinica, Hospital Virgen de la Victoria, School of Medicine, Ma´laga, Spain Willis C. Maddrey Department of Internal Medicine, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, U.S.A. Abdellah Mansouri E´quipe Mitochondries, INSERM, U773, Centre de Recherche Biome´dicale Bichat Beaujon, Faculte´ de Me´decine Xavier Bichat, Universite´ Paris 7 Denis Diderot, Paris, France Harihara M. Mehendale Department of Toxicology, College of Pharmacy, University of Louisiana at Monroe, Monroe, Louisiana, U.S.A. Peter J. Meier

University of Basel, Basel, Switzerland

Richard H. Moseley Ann Arbor Veterans Affairs Healthcare System and Department of Internal Medicine, University of Michigan Health System, Ann Arbor, Michigan, U.S.A. Sidney D. Nelson Department of Medicinal Chemistry, University of Washington School of Pharmacy, Seattle, Washington, U.S.A.

xv

Contributors

George Ostapowicz

University of Texas Southwestern Medical Center, Dallas, Texas, U.S.A.

Christiane Pauli-Magnus

University Hospital Basel, Basel, Switzerland

Dominique Pessayre E´quipe Mitochondries, INSERM, U773, Centre de Recherche Biome´dicale Bichat Beaujon, Faculte´ de Me´decine Xavier Bichat, Universite´ Paris 7 Denis Diderot, Paris, France Munir Pirmohamed Department of Pharmacology and Therapeutics, University of Liverpool, Liverpool, U.K. Adrian Reuben Division of Gastroenterology and Hepatology, Medical University of South Carolina, Charleston, South Carolina, U.S.A. Mark Russo Department of Medicine, University of North Carolina, Chapel Hill, North Carolina, U.S.A. Thomas D. Schiano Division of Liver Diseases and Recanati/Miller Transplantation Institute, The Mount Sinai Medical Center, New York, New York, U.S.A. John R. Senior Office of Surveillance and Epidemiology, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, U.S.A. Hilde Spahn-Langguth

German University in Cairo, New Cairo City, Egypt

Ulrich Spengler Department of General Internal Medicine, Rheinische Friedrich Wilhelms Universita¨t Bonn, Bonn, Germany Andrew A. Stolz Division of Gastrointestinal and Liver Diseases, Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California, U.S.A. Dwain L. Thiele Division of Digestive and Liver Diseases, Department of Internal Medicine, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, U.S.A. Keith G. Tolman Division of Gastroenterology, University of Utah School of Medicine, Salt Lake City, Utah, U.S.A. Dominique Valla

Service d’He´patologie, Hopital Beaujon, Clichy, France

Sumita Verma Keck School of Medicine, University of Southern California, Los Angeles, California, U.S.A. Paul B. Watkins Department of Medicine, University of North Carolina, Chapel Hill, North Carolina, U.S.A.

PART I

1

MECHANISMS OF LIVER INJURY

Drug-Induced Liver Disease Neil Kaplowitz

Keck School of Medicine, University of Southern California, Los Angeles, California, U.S.A.

INTRODUCTION The goal of this chapter is to provide a broad overview of the subject of this book and to introduce a number of concepts that will be expanded upon in subsequent chapters. Drug-induced liver disease represents an important problem for the following major reasons: (1) approximately 1000 drugs have been implicated in causing liver disease at least on rare occasions (1); (2) in the United States drug-induced liver disease is the most common cause of acute liver failure, accounting for one-third to one-half of cases (2,3); although acetaminophen accounts for the bulk of these, other drugs are still a more frequent cause of acute liver failure than viral hepatitis and other causes (4); (3) in addition, drug-induced liver disease represents an important diagnostic/therapeutic challenge for physicians caring for patients presenting with liver disorders, since it can mimic all forms of acute or chronic liver disease (5); the frequency and economic impact of this problem is a major challenge for the pharmaceutical industry and regulatory bodies, especially since the toxic potential of some drugs is not evident in preclinical and phase 1 to 3 clinical testing. The incidence of drug-induced liver injury is not well established in the general population. In a population-based cohort study in France the incidence was 14 cases per 100,000 (0.014%) inhabitants (6), whereas an inpatient study from Switzerland found a higher incidence (1.4%) (7). CLINICAL OVERVIEW Drug-induced liver diseases can mimic all forms of acute and chronic hepatobiliary diseases (Table 1) (5,8). However, the predominant clinical presentations resemble acute icteric hepatitis (hepatocellular jaundice) or cholestatic liver disease. The former is of grave significance as the mortality approximates 10% irrespective of the specific drug (1,5,9,10). This is referred to as Hy’s Law after the late Hy Zimmerman, who noted that mortality from drug-induced hepatocellular jaundice ranged from 10% to 50%. Hy Zimmerman also noted that in most cases at risk for fatal outcome, aside from jaundice alanine aminotransferase (ALT) and aspartate aminotransferase were between 8 and 100! upper limit of normal (ULN) and alkaline phosphatase (Alk. Ptase) !3!ULN. Over the years the validity of Hy’s Law has held up in specific examples (Table 2) and is further verified in recent registries (9–11). This type of reaction is accompanied by systemic symptoms, jaundice, markedly elevated serum transaminases, ALT!ULN/Alk. Ptase.!ULNR5, and in the more severe cases, coagulopathy and encephalopathy indicative of acute (fulminant) liver failure. It is noteworthy that the height of the transaminases does not reliably predict severity except perhaps in the case of acute intrinsic toxins, e.g., acetaminophen. Cholestatic disease, although not usually life threatening, presents with jaundice, disproportionate increased Alk. Ptase, ALT!ULN/Alk. Ptase.!ULN%2, and pruritus; cholestatic reactions tend to resolve very slowly (i.e., months vs. weeks for hepatitis) The author of this chapter has relationships with the following corporations: Abbott, Adams Respiratory Therapy, Allergan, Amgen, Astra Zeneca, Avera, BG Medicine, Biogen, Boehringer/Ingelhem, Cadence, Daiichi Sankyo, DOV, Elan, Enanta, Encysive, GSK, Gtx, Incyte, ISIS, Janssen, Johnson & Johnson, Maxygen, Merck, Millenium, Ono, Pfizer, Rigel, Roche, Sankyo, TAP, Threshold, Teva, and Wyeth.

2

Kaplowitz

TABLE 1 Spectrum of Hepatic Manifestations of Drug-Induced Liver Disease Acute hepatitis Chronic hepatitisa Acute cholestasis Mixed hepatitis/cholestasis or atypical hepatitis Chronic cholestasisa Non-alcoholic steatohepatitis Fibrosis/cirrhosis Microvesicular fatty liver Veno-occlusive disease Peliosis hepatitis Adenoma and hepatocellular carcinoma a

Acetaminophen, isoniazid, troglitazone, bromfenac Nitrofurantoin, methyldopa, diclofenac, minocycline, dantrolene Amoxicillin clavulanic acid, erythromycins, sulindac, chlorpromazine, angiotensin-converting enzyme inhibitors Phenytoin, sulfonamides Chlorpromazine, numerous others on rare occasion Amiodarone, tamoxifen Methotrexate Valproic acid, nucleoside reverse transcriptase inhibitors Busulfan, cyclophosphamide Azathioprine, hormones Hormones

Drugs that cause chronic disease more frequently cause acute disease.

and on rare occasion lead to vanishing bile duct disease and biliary cirrhosis (12,13). Mixed injury patterns with intermediate ALT/Alk. Ptase. can resemble atypical hepatitis or granulomatous hepatitis. Individual drugs tend to exhibit a consistent pattern or clinicopathological signature of the reaction (Table 1) with characteristic latency and clinical presentation. However, some drugs may show several patterns: e.g., nimesulide can cause a short-latency, hypersensitivity-mediated cholestatic injury and a delayed idiosyncratic acute hepatitis-like reaction (14). Thus, although one pattern may predominate, crossover to other patterns is not unusual. Drug-induced liver disease can be predictable (high incidence and dose-related) or unpredictable (low incidence and may or may not be dose-related). Unpredictable reactions, also referred to as idiosyncratic, can be viewed as either immune-mediated hypersensitivity or nonimmune reactions. Most potent predictable hepatotoxins are recognized in the animal testing or clinical phase of drug development. Those that slip through are almost always unpredictable. Latency between the initiation of therapy and the onset of liver disease is a component of the signature of reactions to specific drugs and provides some clues as to the pathogenesis. Early onset within a few days (particularly if no previous exposure) is strong evidence for direct toxicity of the drug or its metabolite, which is characteristic of predictable reactions; acetaminophen overdose is an example (15). Unpredictable reactions manifested as overt or symptomatic disease usually occur with intermediate (one to eight weeks) or long latency (up to 12 months). Intermediate latency is characteristic of hypersensitivity reactions, but can be seen with nonimmune idiosyncrasy as well. The hypersensitivity reactions tend to be associated with fever, rash, and eosinophilia and a rapid positive rechallenge (5,8). Hepatotoxicity of sulindac (16), phenytoin (17), and amoxicillin–clavulanic acid (18) are typical examples. Most cases of cholestatic liver injury and chronic hepatitis caused by drugs are of the hypersensitivity type. It is important to recognize that these reactions may occur up to three to four weeks after a one to two week course of medication (e.g., amoxicillin–clavulanic acid). In contrast, the long-latency type of idiosyncratic reaction is characteristically not associated with features of hypersensitivity and the response to rechallenge is variable and delayed. Thus, one assumes that these events reflect TABLE 2 Drugs that Cause Hepatocellular Jaundice and Confirm Hy’s Law 1978 Iproniazid Isoniazid Phenytoin Halothane Cinchophen Dantrolene Nitrofurantoin

Later Phenylbutazone Ketoconazole Ticrynafen Valproic acid Enflurane Pemoline Labetalol Diclofenac Sulindac

1978 refers to first edition of Zimmerman’s textbook.

Probable Bromfenac Troglitazone Trovafloxacin Nefazodone

3

Drug-Induced Liver Disease

TABLE 3 Drugs Associated with Idiosyncratic Hepatitis Nonallergic Acarbose Benoxaprofenb Bosentan Bromfenacb Dantrolene Diclofenac Disulfiram Felbamate Flutamideb Isoniazid Isotretinoin Ketoconazole Labetalol a b

Allergic Leflunomide Nefazodoneb Nevirapine Pemoline Pyrazinamide Terbinafinea Tolcapone Troglitazoneb Trovafloxacinb Valproic acid Zafirlukast Zileuton

Allopurinol Diclofenac Dihydralazine Halothane Methyldopa Minocycline Nevirapine Nitrofurantoin Phenytoin Propylthiouracil ACE inhibitorsa Augmentinwa Phenothiazinesa

Erythromycinsa Sulfonamidesa Sulindaca Tricyclicsa

Cholestatic/mixed. Withdrawn from the market.

some type of late-onset change in the metabolism of the drug or the response to injury (repair or regeneration). Drugs associated with variable, long latency include isoniazid (19) and troglitazone (20). This type of idiosyncratic reaction is extremely challenging with respect to understanding the pathogenesis and predicting the problem in individual cases. Table 3 provides a list of drugs that are associated with idiosyncratic allergic and nonallergic reactions. A few can cause either allergic or nonallergic reactions, e.g., diclofenac and nevirapine. Low-frequency unpredictable reactions, either immune-mediated or not, often occur on a background, higher rate of mild, asymptomatic, and usually transient liver injury, which is detected as abnormal biochemical tests, particularly serum ALT. Generally, the biochemical abnormality defined as ALTO3!ULN may occur 10 to 20 times more frequently than overt disease. In almost all instances, the ALT returns to normal despite continued drug use. Thus, in the majority of patients with increased ALT some type of adaptation or “tolerance” occurs and in the minority progression to overt, severe injury occurs, which may reflect a failure to adapt. This issue is further complicated by the uncertain explanation for the very long latency in some of the idiosyncratic reactions. It should be emphasized that acute or chronic hepatitis induced by drugs subsides upon discontinuation of the drug without long-term sequelae with rare exception. A few reported cases of autoimmune hepatitis triggered by hypersensitivity drug reactions have continued on without the drug, but it is questionable as to whether this was drug-induced liver disease or underlying autoimmune chronic hepatitis. Scarring may persist after severe subacute or chronic injury but is of little consequence after removal of the drug cholestalic reactions are cholestatic reactions are not infrequently associated with loss of interlobular bile ducts. However, the development of cirrhosis or effects on longevity are exceedingly rare. PATHOGENESIS Hepatotoxicity of drugs can be principally metabolism-dependent, parent drug-dependent, or a combination of both (Fig. 1). Metabolism takes place largely in the liver, which accounts for its susceptibility to drug-induced injury (8). The metabolites may be electrophilic chemicals or free radicals that deplete glutathione (GSH), covalently bind to proteins, lipids, or nucleic acids, or induce lipid peroxidation. The consequences include hepatocellular necrosis, apoptosis, or sensitization to cytokines or inflammatory mediators produced by nonparenchymal cells. Alternatively, the reactive metabolites may covalently bind to or alter liver proteins such as cytochrome P450s (CYPs) leading to sensitization and immune-mediated injury. The immune phenomena nevertheless are metabolism dependent. Thus, the rare occurrence of immunemediated liver disease is often superimposed on a higher frequency of mild injury (abnormal ALT) suggesting that the drug has a mild toxic potential (e.g., phenytoin or halothane) but in rare individuals this toxic potential leads to more severe toxicity initiated by metabolism steps,

4

Kaplowitz

Haptenization Immune response Metabolite ” Covalent binding anger “D GSH depletion Reactive O2

Drug Mitochondria DNA Toxicity (mild)

Adaptation

Inflammatory/ toxic mediators

Recovery

Repair

Overt liver injury

FIGURE 1 Pathogenesis of drug-induced liver diseases. Upstream events in the hepatocytes affect viability of individual cells but sensitize to downstream processes leading to clinically overt organ damage. The latter involves a balance of effects of cytokines, chemokines, and inflammatory mediators, mainly produced by nonparenchymal cells and the effects on repair processes a such as regeneration.

but also heavily influenced by genetic and/or environmental factors that determine either an immune response or idiosyncratic reaction. Genetic polymorphisms of enzymes involving drug activation or detoxification have been implicated in the susceptibility to hypersensitivity reactions to sulfonamides (21,22), anticonvulsants (17,23), and tacrine (24). Presumably genetic polymorphisms of either major histocompatibility complex (MHC) I-dependent antigen presentation in hepatocytes or MHC II-dependent antigen presentation in macrophages, which have scavenged necrotic or apoptotic hepatocytes directly killed by the drug, may further contribute to determine the rare occurrence of these hypersensitivity reactions (25) which most often have an incidence of 1:1000 or less. Parent drug-dependent toxicity occurs as a result of the properties of the parent drug (or metabolite) to accumulate in organelles [weak bases such as amiodarone accumulate in mitochondria (26)], undergo nonspecific redox cycling (quinones cycle electrons from NADPH to O2 generating (OK 2 )), or specifically inhibit enzymes or transporters (nucleoside reverse transcriptase inhibitors block mitochondrial DNA polymerase (27)) or cyclosporin A inhibits canalicular transporters (28). In these cases, if the parent drug’s chemical properties account for direct toxicity, factors that enhance its availability (decreased metabolism or export) may increase susceptibility. Regardless of whether toxicity within a target liver cell (e.g., hepatocyte, sinusoidal endothelial cell, or bile duct cell) is parent drug- or metabolite-dependent, the ultimate severity of the liver disease in vivo may depend greatly on the subsequent downstream participation of toxic mediators released from various cell types and the recruitment of inflammatory cells as well as intracellular and tissue repair and regenerative responses. The toxic mediators include chemicals, such as NO and reactive oxygen metabolites, and the balance of cytokines that promote injury [e.g., tumor necrosis factor-a (TNFa), Interleukin-1 (IL-1), Interferon-g (IFNg), Interleukin-12 (IL-12), Interleukin-18 (IL-18)), or prevent injury (IL-4, IL-10, IL-13, monocyte chemotactic protein-1 (MCP-1)]. Thus, toxin may somewhat injure hepatocytes but then sensitize to the effects of an imbalance in injurious versus protective cytokines (Fig. 2). For example, the toxicity of carbon tetrachloride (CCl4) is abrogated in vivo by neutralizing TNF (29); the toxicity of acetaminophen is markedly enhanced in MCP-1 chemokine receptor Protection IL-4 IL-10 IL-13 MCP-1 Toxin

Injury IL-12 TNFα IFNγ IL-12

TNFα Sensitize hepatocytes IFNγ T(–) MCP-1 IL-10, 13

Lethal

FIGURE 2 Role of cytokine balance in determining susceptibility to toxins. Drugs or metabolites may directly injure hepatocytes to a minor extent, but may markedly sensitize to the lethal effects of TNFa and IFNg. The latter are modulated by cytokines that promote or inhibit their production or actions. Abbreviations: TNFa, tumor necrosis factor a; IFNg, interferon g.

5

Drug-Induced Liver Disease

Hepatocyte injury

Toxin HGF

Antiapoptosis

Regeneration

Acute liver Pro- or anti- failure apoptosis

IL-6

FIGURE 3 Role of cytokines and regeneration in toxin-induced liver disease. TNF activates NF-kB in nonparenchymal cells, leading to IL-6 production, and in hepatocytes, or with the regenerative response, worsening of the overt liver injury.

TNFα

LPS

Kupffer cell activation

knockouts associated with an enhanced TNFa response (30) and may be abrogated by inactivating Kupffer cells (31), although this is controversial. Similarly IL-10 null mice are sensitized to acetaminophen (32), whereas natural killer/natural killer cell with T cell receptor cell depletion, knockout of IFNg, and Fas/Fas ligand deficiency protect against acetaminophen (33). Thus, the direct and indirect influence of toxins on the production and balance of mediators, and genetic polymorphisms in these responses may play a major role in unmasking the overt toxic potential of a drug culminating in overt idiosyncratic toxicity. Another important factor that contributes to the extent of liver injury is the capacity of the liver to regenerate (Fig. 3). Thus, for example, TNFa will promote regeneration by acting on Kupffer cells (autocrine) to release IL-6, which will trigger, along with hepatocyte growth factor, regeneration. Interference with IL-6 (knockout) worsens CCl4 injury, and conversely, exogenous IL-6 treatment diminishes liver injury in wild-type mice (34). TNF also acts on hepatocytes through NF-kB signaling to promote survival gene transcription. If the toxin interferes with the latter pathway, TNFa-induced apoptosis may occur.

RISK FACTORS Regardless of whether hepatotoxicity is predictable (frequent) or unpredictable (rare), hypersensitivity-mediated or idiosyncratic, metabolism dependent or parent drug-dependent, the interplay of genetic and environmental risk factors influences susceptibility (Fig. 4) (35). Age, gender, concomitant drugs, and underlying diseases (e.g., hepatitis C virus, hepatitis

Toxic potential of drug - Reactive metabolite - Acylglucuronide - Mitochondrial effects

Genetic factors - Drug metabolism - Detoxification - Transport - Others

Environmental factors - Other drugs - Ethanol - Age - Underlying disease

FIGURE 4 Risk of hepatotoxicity. The ultimate development of hepatotoxicity is determined by the interplay of the toxic potential of the drug or its metabolites and the susceptibility of the host as determined by genetic and environmental factors, both of which influence gene expression.

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Kaplowitz

TABLE 4 Risk Factors for Drug-Induced Hepatotoxicity Drug Methotrexate Isoniazid Acetaminophen Valproate Diclofenac Anticonvulsants Sulfonamides

Factors Chronic alcohol, obesity, diabetes, chronic hepatitis, psoriasis HBV, HCV, HIV, alcohol, older age, female, slow acetylator, rifampin, pyrazinamide Chronic alcohol, fasting, isoniazid Young age, anticonvulsants, genetic defects of mitochondrial b-oxidation and respiratory chain enzymes Female, osteoarthritis Genetic defect in detoxification HIV, slow acetylator, genetic defect in defense

Abbreviations: HBV, hepatitis B virus; HCV, hepatitis C virus.

B virus, HIV) have been most frequently identified. Table 4 lists examples of drugs and associated risk factors. With the advent of new technologies in genomics and proteomics, one can anticipate that new insights into the mechanisms of susceptibility and liver injury from drugs will be forthcoming (36). Some of the genetic factors to consider are listed in Table 5. Polymorphisms (CYPs, cytokines, MHC, etc.) and rare heterozygous mutations (b-oxidation, bile salt export pump) will need to be assessed.

DIAGNOSIS Establishing a diagnosis of drug-induced liver disease in an individual case is mainly based upon circumstantial evidence aided by the signature type of reactions (if known) with respect to latency and clinical characteristics as well as exclusion of other more plausible alternative causes. Additional information can be gained from the response to removal of the drug—rapid improvement in cytotoxic reactions and slow improvement in cholestatic reactions. A rechallenge with recrudescence of liver abnormalities is the most definitive evidence, but hardly ever justified and not always positive in idiosyncratic cases. A practical approach is to consider the diagnosis probable/possible if the signature latency and pattern of disease fit and other causes are excluded (viral hepatitis, ischemic hepatitis, biliary disease). The remaining cases are unlikely or unrelated depending on the completeness of the workup and the strength of the evidence in favor of an alternative diagnosis. This ad hoc approach is equivalent to diagnosing as yes, no, or maybe. The presence of autoantibodies to specific forms of CYP has been associated with hypersensitivity reactions to certain drugs (25,37,38). Although uncertain but intriguing significance with respect to pathophysiology, their presence may be helpful in the diagnosis of drug-induced liver disease in these special cases (Table 6). However, testing for these autoantibodies is mainly a research tool at present. Furthermore, sensitivity and specificity of the presence of these autoantibodies is uncertain and autoantibodies to certain CYPs have been TABLE 5 Possible Genetic Determinants of Risk 1. Drug metabolism (e.g., CYP polymorphisms) 2. Detoxification (e.g., GSH-related, epoxide hydrase) 3. Apoptosis and survival genes 4. Signal transduction (kinases and phosphatases) 5. MHC I and II 6. Cytokines/chemokines and receptors 7. Inflammatory mediators (Cox, NOS, etc.) 8. Regeneration/repair 9. Transporters (BSEP, MRP2, etc.) 10. Mitochondrial b-oxidation and respiratory chain 11. Structural integrity (e.g., cytokeratin 8/18) Abbreviations: Cox, cyclooxygenase; NOS, nitric oxide synthase; BSEP, bile salt export pump; MRP2, multidrug resistance associated protein family; MHC, major histocompatibility complex; CYP, cytochrome P450s; GSH, glutathione.

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Drug-Induced Liver Disease

TABLE 6 Autoantibodies in Drug-Induced Liver Disease Autoantibody target a

CYP 2C9 CYP 1A2b CYP 3Ab CYP 2E1 mEH

Drug Tienilic acid Dihydralazine Anticonvulsants Halothane Germander

a

Also referred to as anli-LKM2 autoantibody. Also referred to as anti-LM autoantibody. Abbreviations: CYP, cytochrome P450; mEH, microsomal epoxide hydrolase.

b

observed in patients taking drugs without evidence of hepatotoxicity, limiting their diagnostic value. Several groups have attempted to generate quantitative systems designed to generate a numerical score that reflects the probability of a drug as the cause for liver disease (39–42). The Roussel-Uclaf casuality assessment method scoring system appears to be the most accurate (43,44) and puts numerical weight on the factors discussed above (Table 7) to generate a composite score that reflects the probability that liver injury is drug-induced. The advantage is that this system is less subjective than the ad hoc approach. This type of scoring system performs well when validated against well-documented cases of drug-induced liver disease. Specialists, the pharmaceutical industry, and regulatory bodies should be encouraged to use this scale. It also would be reasonable to apply the scoring system to individual case reports submitted to medical journals. Although it is not perfect and may not discriminate between multiple concurrently used candidate toxins, it does provide consistency and focuses the attention of the evaluator on most of the critical parameters that need to be considered in estimating the probability of causality. DRUG DEVELOPMENT Drug development involves a preclinical and clinical phase. Preclinical assessment is centered on animal testing using very high doses. Although animal testing is probably very reliable in screening out potent, predictable toxins, it is far less reliable in identifying a propensity for unpredictable toxicity. Experience has suggested that there are numerous false positives and false negatives. A better understanding of the mechanisms of unpredictable hepatotoxicity may eventually lead to the establishment of appropriate animal models that recapitulate the factors that determine susceptibility in humans. This will most likely be achieved with the use of transgenic and knockout mice to set up conditions that mimic human susceptibility. At present, some suggestion of hepatotoxicity at high doses in animals may at least warrant more careful or extensive assessment in the clinical phases of drug development. During phases I to III of drug testing the likelihood of encountering overt hepatotoxicity (i.e., jaundice and high transaminases) depends on the frequency of the reaction. Most idiosyncratic reactions occur in 1 in 1000 or more individuals and acute liver failure in 1 in 10,000 or more. Typically in clinical development, drugs are tested in 1500 to 2500 individuals. Exclusion of an overt reaction with 95% confidence, if the true incidence is 1:1000, would require 3000 treated patients, assuming all were exposed for the appropriate duration (e.g., six to nine months). This is usually not attained in clinical trials. Therefore, if one is not fortunate TABLE 7 Causality Assessment 1. 2. 3. 4. 5. 6. 7.

Latency Rate of resolution (dechallenge) Risk factors (age, alcohol, pregnancy) Exclusion of other causes (viral hepatitis, ischemia, biliary tract disease, alcohol) Concomitant drugs Track record (PDR, case reports) Rechallenge

Abbreviation: PDR, Physicians Desk Reference.

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Kaplowitz

enough to identify overt hepatitis in one or two cases in the study population, the appearance of lesser signals needs to be the focus for scrutiny. It is very rare indeed to identify acute liver failure from idiosyncratic hepatotoxins in drug development. The rule of threes indicates that to identify (not miss) acute liver failure with 95% confidence that has a true incidence of 1 in 10,000 would require 30,000 study patients. Since the probability of identifying overt or life-threatening liver injury in clinical trials is so low, one must focus on the incidence of asymptomatic ALT and bilirubin elevations (45). The most sensitive parameter appears to be the incidence of ALTO3!ULN in drug-treated versus placebo control-treated patients. Depending on the study population, the incidence of 3!ALT in controls may vary from 0.1% to nearly 1.0%. Thus, an incidence of 2% to 3% or greater in the drug-treated patients would be unequivocal cause for closer scrutiny. Although this is a sensitive indicator, it is not entirely specific since there are drugs, e.g., statins, tacrine, aspirin, etc., that are associated with an increased incidence of 3!ALT, but have proved relatively safe in postmarketing experience (?false-positive signal). More specificity is gained by examining the height of the transaminases. An ALT increase of eightfold or greater is a more specific signal since this rarely occurs in controls. Even more specific is conjugated hyperbilirubinemia (R1.5-fold) associated with elevated ALT. The experience with troglitazone exemplifies the issue of identification of a signal. In a cohort of 2500 study patients, ALTO3!ULN occurred in nearly 2% (vs. !1% in controls); ALTO8!ULN occurred in 0.6% (vs. none of controls); and two cases with overt jaundice were observed (46). Thus, all the criteria for a hepatic signal were present at the time of approval of the drug. A similar premarketing experience was observed with bromfenac (47), which was also withdrawn postmarketing. A critical issue is what is the appropriate regulatory response to the occurrence of a signal? In some cases, particularly when the drug is not crucial, approval is denied. If the drug is critical, warnings and education of physicians and patients are very important and there may be justification for recommending monitoring ALT (see below) and/or restrictions on the use of the drug. Two recent examples are bosentan and ximelagatran, which had unequivocal signals for idiosyncratic hepatotoxicity, perhaps stronger than troglitazone (Table 8). Bosentan was approved with monitoring because the disease being treated is fatal, i.e., pulmonary hypertension, and no other efficacious treatment is available, and therefore a favorable risk-benefit analysis. Ximelagatran, a thrombin inhibitor, was not approved because several cases of very severe hepatotoxicity were seen and alternative anticoagulants are available. A number of antituberculous, HIV and cancer drugs have remained on the market because benefit outweighs risks. POSTMARKETING MONITORING The background incidence of drug-induced mild, reversible liver injury provides the rationale for monitoring or surveillance. From this background of mild injury a minority of individuals will emerge with overt disease. Thus, by stopping the medications at the first sign of mild injury one should prevent serious consequences. Although this seems a logical approach, a number of problems must be considered. First, the approach applies only to delayed reactions.

TABLE 8 Comparison of Idiosyncratic Hepatotoxins in Clinical Trials Troglitazone nZ ALTO3!ULN ALTO10!ULN ALTO3! and bili O2! ALTO8! and bili O3! Fatal acute liver failure

Bosentan

2500 1.8% 0.6%

658 12 14% 2 7%

0.08% 0

0.3% 0

a 3 cases (one confounded by HBV). Abbreviations: ALT, alanine aminotransferase; ULN, upper limit of normal; HBV, hepatitis B virus.

Ximelagatran 6948 7.8% 1.9% 0.5% 0.1% 0.01 0.04%a

Drug-Induced Liver Disease

9

Hypersensitivity reactions occur relatively early and evolve rapidly so educating patients about symptoms is crucial in early cessation of the offending drug. Second, one is sacrificing potentially very important therapy to a much larger number of patients than would actually develop overt disease; third, compliance with such approaches is known to be very poor; fourth, the rate of development of overt disease from the first appearance of elevated ALT needs to be gradual for monthly monitoring to be efficacious in preventing life-threatening disease. Testing more frequently than monthly is not practical, although the future development of a fingerstick ALT test that could be applied in a fashion similar to monitoring glucose might change this by improving compliance and allowing more frequent monitoring. In any case, monthly monitoring for delayed idiosyncratic reactions is the best approach available, but the efficacy of the approach is assumed and not proven. Furthermore, this should not substitute for the need to educate patients about symptoms of hepatotoxicity, such as fever, rash, malaise, fatigue, anorexia, gastrointestinal complaints, abdominal pain, dark urine; jaundice, pruritus, etc., and the need to report them to the physicians to ensure expeditious cessation of offending agents. Despite monthly monitoring, some adverse events may appear rapidly in the few weeks after a normal test. Ultimately, the most difficult challenge to the application of monitoring is cost-effectiveness—monthly monitoring is expensive and one needs to weigh this quantitatively against the morbidity and mortality of adverse liver events. There is no clear answer to the question of what incidence of serious adverse idiosyncratic hepatitis warrants monitoring, how frequently it should be performed, and for how long. Furthermore, the postmarketing occurrence of adverse events must be weighed against the benefit of the drug. Risk/benefit assessment is ill defined but ultimately becomes the crucial factor in recommending monitoring ALT versus removal of the drug from the market. In the case of the nonsteroidal anti-inflammatory drug, bromfenac, continued use of the drug in the face of infrequent, delayed idiosyncratic severe hepatotoxicity (47) could not be justified since many alternative treatments were available. In the case of troglitazone, the decision to withdraw was delayed and more complicated owing to the important and unique therapeutic properties of the drug in managing a serious medical condition, albeit with benefits that would not be evident for many years (i.e., the effect of long-term control of blood sugar on complications of diabetes). It was decided that the implementation of monthly monitoring would likely protect the users and the drug was continued. Although this strategy may have worked to some extent, the issue of compliance with monitoring and the possibility of occasional “rapid risers” meant that the population could not be completely protected. At the same time, several new drugs in this class were approved and after a year of postmarketing experience with the alternative new agents, it was concluded that these agents were much less likely to induce severe hepatotoxicity, leading to the withdrawal of troglitazone. A major issue in postmarketing surveillance is the frequency of serious adverse events compared to the background in the general population. Reliable data on the occurrence of hospitalization for idiopathic hepatitis and acute liver failure are very limited. However, several databases (Medicaid, HMO) suggest that hospitalization for cryptic acute hepatitis occurs in about 1:50,000 to 1:100,000 adult individuals in the general population each year (48–50). Acute liver failure is estimated to occur in up to 3000 individuals annually in the United States. About 10% to 20% of these cases are idiopathic with a resulting annual incidence of one to two cases per million individuals in the population. Thus, when a new drug is marketed and more than a few cases of unexplained acute liver failure are reported, concern should be raised. In the example of troglitazone, at least 30 to 40 cases of acute liver failure were reported to the FDA in the first year on the market from a population of about one million taking the drug. This was a much higher rate than predicted. Such a postmarketing signal may be less apparent when far fewer individuals are exposed. However, the current MedWatch system for reporting adverse events, with all its attendant problems regarding poor compliance and accurate causality assessment, has been reasonably successful in rapidly identifying problems with numerous drugs leading to withdrawal or severe restrictions of use.

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CONCLUSIONS The liver is a particular target for drugs because of its role in clearing and metabolizing chemicals. The parent drug or more frequently the metabolites may either affect critical functions, or sensitize to the effects of cytokines or inflammatory cells, or elicit an immune response. This often occurs in an unpredictable fashion, implying that environmental and genetic factors alter the susceptibility to these adverse events. A wide range of liver diseases can occur as adverse events but the individual drug tends to induce a characteristic signature reaction with respect to latency and clinicopathological manifestations. Hepatotoxicity from drugs poses a major challenge in drug development and postmarketing surveillance. The future identification of the pathogenesis of idiosyncratic reactions represents the major challenge in this field and will likely advance rapidly with the application of methods of toxicogenomics and pharmacogenomics in the preclinical and clinical arenas. REFERENCES 1. Zimmerman H. Drug Hepatotoxicity. 2nd ed. Philadelphia, PA: Lippincott, 1999. 2. Ostapowicz G, Fontana RB, Larson AM, et al. Etiology and outcome of acute liver failure in the USA: preliminary results of a prospective multi-center study (abstr). Hepatology 1999; 30(4):221A. 3. Shakil A, Kramer D, Mazariegos G, et al. Acute liver failure: clinical features, outcome analysis, and applicability of prognostic criteria. Liver Transplant 2000; 16:163–9. 4. Larsen A, Polson J, Fontana R, et al. Acetaminophen-induced acute liver failure: results of a U.S. Multicenter, prospective study. Hepatology 2005; 42:1364–72. 5. Zimmerman H. Drug-induced liver disease. In: Schiff E, Sorrell M, Maddrey W, eds. Schiff’s Diseases of the Liver. 8th ed. Philadelphia, PA: Lippincott-Raven, 1999:973–1064. 6. Sgro C, Clinard F, Ouazir K, et al. Incidence of drug-induced hepatic injuries: a French populationbased study. Hepatology 2002; 36:451–5. 7. Meier Y, Cavallaro M, Roos M, et al. Incidence of drug-induced liver injury in medical inpatients. Eur J Clin Pharmacol 2005; 61:135–43. 8. Kaplowitz N. Drug metabolism and hepatotoxicity. In: Kaplowitz N, ed. Liver and Biliary Diseases. 2nd ed. Baltimore, MD: Williams & Wilkins, 1996:103–20. 9. Bjornsson E, Olsson R. Outcome and prognostic markers in severe drug-induced liver disease. Hepatology 2005; 42:481–9. 10. Andrade R, Lucena M, Fernandez M, et al. Drug-induced liver injury: an analysis of 461 incidences submitted to the Spanish registry over a 10-year period. Gastroenterology 2005; 129:512–21. 11. Lewis JH. “Hy’s law”, the “Rezulin rule” and other predictors of severe drug-induced hepatotoxicity: putting risk-benefit perspective. Pharmacoepidemiol Drug Saf 2006; 15:221–9. 12. Desmet VJ. Vanishing bile duct syndrome in drug-induced liver disease. J Hepatol 1997; 26:31–5. 13. Degott C, Feldmann G, Larrey D, et al. Drug-induced prolonged cholestasis in adults: a histological semiquantitative study demonstrating progressive ductopenia. Hepatology 1992; 15:244–51. 14. Van Steenberen W, Peeters P, DeBondt J, et al. Nimesulide-induced acute hepatitis: evidence from six cases. J Hepatol 1998; 29:135–41. 15. Pham T-V, Lu S, Kaplowitz N. Acetaminophen hepatotoxicity. In: Taylor MB, ed. Gastrointestinal Emergencies. 2nd ed Baltimore, MD: Williams & Wilkins, 1997:371–88. 16. Tarazi E, Harter JG, Zimmerman HJ, et al. Sulindac-associated hepatic injury. Analysis of 91 cases reported to the food and drug administration. Gastroenterology 1993; 104:569–74. 17. Shear N, Spielberg S. Anticonvulsant hypersensitivity syndrome: in vitro assessment of risk. J Clin Invest 1988; 82:1826–32. 18. Larrey D, Vial T, Micaleff A, et al. Hepatitis associated with amoxycillin-clavulanic acid combination. Report of 15 cases. Gut 1992; 33:368–71. 19. Thompson N, Caplin M, Hamilton M, et al. Anti-tuberculosis medication and the liver: dangers and recommendations in management. Eur Respir J 1995; 8:1384–8. 20. Murphy E, Davern T, Shakil O, et al. Troglitazone-induced fulminant hepatic failure. Dig Dis Sci 2000; 45:549–53. 21. Rieder M, Uetrecht J, Shear N, et al. Diagnosis of sulfonamide hypersensitivity reactions by in vitro “rechallenge” with hydroxylamine metabolites. Ann Intern Med 1999; 110:286–9. 22. Rieder M, Shear N, Kanee A, et al. Prominence of slow acetylator phenotype among patients with sulfonamide hypersensitivity reactions. Clin Pharmacol Ther 1991; 49:13–7. 23. Gennis M, Vemusi R, Burns E, et al. Familial occurrence of hypersensitivity to phenytoin. Am J Med 1991; 91:631–4. 24. Becquemont L, Lecoeur S, Simon T, et al. Glutathione S-transferase q genetic polymorphism might influence tacrine in Alzheimer’s patients. Pharmacogenetics 1997; 7:251–3.

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25. Robin M, LeRoy M, Descatoire V, Pessayre D. Plasma membrane cytochromes P450 as neoantigens and autoimmune targets in drug-induced hepatitis. J Hepatol 1997; 26(1):23–30. 26. Berson A, DeBeco V, Letteron P, et al. Steatohepatitis-inducing drugs cause mitochondrial dysfunction and lipid peroxidation in rat hepatocytes. Gastroenterology 1998; 114:764–74. 27. Brinkman K, Hofstede H, Burger D, et al. Adverse effects of reverse transcriptase inhibitors: mitochondrial toxicity as common pathway. AIDS 1998; 12:1735–44. 28. Kowdley K, Keefe E. Hepatotoxicity of transplant immunosuppressive agents. Gastroenterol Clin North Am 1995; 24:991–1001. 29. Czaja M, Xu J, Alt E. Prevention of carbon tetrachloride-induced rat liver injury by soluble tumor necrosis factor receptor. Gastroenterology 1995; 108:1849–54. 30. Hogaboam C, Bone-Larson C, Steinhauser M, et al. Exaggerated hepatic injury due to acetaminophen challenge in mice lacking C–C chemokine receptor 2. Am J Pathol 2000; 156:1245–52. 31. Laskin D, Gardner C, Price V, Jollow D. Modulation of macrophage functioning abrogates the acute hepatotoxicity of acetaminophen. Hepatology 1995; 21:1045–50. 32. Bourdi M, Masubuchi Y, Reilly TP, et al. Protection against acetaminophen induced liver injury and lethality by interleukin 10: role of inducible nitric oxide synthase. Hepatology 2002; 35:289–98. 33. Liu Z-X, Govindarajan S, Kaplowitz N. Innate immune system plays a critical role in determining the progression and severity of acetaminophen hepatotoxicity. Gastroenterology 2004; 127:1760–74. 34. Cressman P, Greenbaum L, DeAngelis R, et al. Liver failure and defective hepatocyte regeneration in interleukin-6-deficient mice. Science 1996; 274:1379–83. 35. DeLeve L, Kaplowitz N. Prevention and therapy of drug-induced hepatic injury. In: Wolfe M, ed. Therapy of Digestive Disorders Philadelphia, PA: WB Saunders/Harcourt Brace & Company, 2000:334–48. 36. Nuwayser E, Bittner M, Trent J, et al. Microarrays and toxicology: the advent of toxicogenomics. Mol Carcinog 1999; 24:153–9. 37. Beaune P, Lecoeur S. Immunotoxicology of the liver: adverse reactions to drugs. J Hepatol 1997; 26(Suppl. 2):37–42. 38. Neuberger J. Immune mechanisms in drag hepatotoxicity. Clin Liver Dis 1998; 2:471–82. 39. Maria V, Victorino R. Development and validation of a clinical scale for the diagnosis of drug-induced hepatitis. Hepatology 1997; 26:664–9. 40. Benichou C. Criteria of drug induced liver disorders: report of an international consensus meeting. J Hepatol 1990; 11:272–6. 41. Danan G, Benichou C. Causality assessment of adverse reactions to drugs I. A novel method based on the conclusions of international consensus meetings: application to drug-induced liver injuries. J Clin Epidemiol 1993; 46:1323–30. 42. Benichou C, Danan G, Flahault A. Causality assessment of adverse reactions to drugs II. An original model or validation of drug causality assessment methods: case reports with positive rechallenge. J Clin Epidemiol 1993; 46:1331–6. 43. Lucena M, Camargo R, Andrade R, et al. Comparison of two clinical scales for causality assessment in hepatotoxicity. Hepatology 2001; 33:123–30. 44. Kaplowitz N. Causality assessment versus guilt by association in drug hepatotoxicity. Hepatology 2001; 33:308–10. 45. Kaplowitz N. Idiosyncratic drug hepatotoxicity. Nat Rev Drug Discov 2005; 4:489–99. 46. Watkins P, Whitcomb R. Hepatic dysfunction associated with troglitazone. N Engl J Med 1998; 338:916–7. 47. Moses P, Schroeder B, Alkhatib O, et al. Severe hepatotoxicity associated with bromfenac sodium. Am J Gastroenterol 1999; 94:1393–6. 48. Walker A, Cavanaugh R. The occurrence of new hepatic disorders in a defined population. Post Mark Surveill 1992; 6:107–17. 49. Carson J, Strom B, Duff A, et al. Safety of nonsteroidal anti-inflammatory drugs with respect to acute liver disease. Arch Intern Med 1993; 153:1331–6. 50. Dun M-S, Walker A, Kronlund K. Descriptive epidemiology of acute liver enzyme abnormalities in the general population of central Massachusetts. Pharmacoepidemiol Drug Saf 1999; 8:275–83.

2

Cytochrome P450 Activation of Toxins and Hepatotoxicity F. Peter Guengerich

Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee, U.S.A.

INTRODUCTION The history of bioactivation of chemicals predates cytochrome P450 (P450) research, beginning with structure-activity studies of carcinogens by Fieser (1) and biochemical experiments by Millers (2,3). The enzymatic activation of inert chemicals by oxidation or other processes can generate electrophilic products that react with nucleophilic sites on DNA and proteins. The concept was extended to drugs and other chemicals and can provide an explanation for toxicity, following the classic studies of the Gillette and Brodie laboratories with acetaminophen and bromobenzene in the 1970s (4,5). In the case of carcinogenesis, causality can be demonstrated at least to the extent that mutagenicity can be demonstrated with defined DNA lesions (6). As discussed later, the evidence linking toxicities with covalent binding is considerable but must still be considered correlative (7,8). P450 research began with these and other studies in steroid metabolism (9,10) and microbial biochemistry (11). Much of the field is still centered in these areas. Drug metabolism is one area in which basic knowledge about P450 has had tremendous practical impact. Some of the key findings in the development of the field were the demonstration that the hemoprotein P450 is the component of the system that functions as the terminal oxidase [i.e., actually reacts O2 with the substrate (12)], the separation and reconstitution of the components of the microsomal system (13), the discovery that P450 is inducible (14,15), the demonstration of multiple P450s (16), and the purification (17), sequence analysis (18), and crystallization (19) of mammalian P450s. P450 ENZYMES The human genome contains 57 P450s, and these are named systematically based on sequence similarity (20,21). The sequence of a P450 does not provide direct insight into catalytic function, however. One approach to categorizing the human P450s is shown in Table 1. Some assignments are equivocal (e.g., 1B1 and 27A1), but the P450s involved in the metabolism of steroids, eicosanoids, and fat-soluble vitamins are clearly important in development and normal physiology. The P450s in the “xenobiotics” column are not considered critical but these are the enzymes involved in drug metabolism (22). In considering metabolism of all commercial drugs, w75% is attributed to P450s (Fig. 1A). Within this group, w95% is catalyzed by five P450s (Fig. 1B). Thus, the determination of which P450s are involved in the metabolism of a drug can usually be done quickly, and this information is expected for new drug candidates by the U.S. Food and Drug Administration (FDA). Determining which human P450s are involved in bioactivation and detoxication reactions can also be readily established in in vitro systems (25). Methods also exist to put The author of this chapter has relationships with the following corporations. Stock holdings: Abbot Laboratories, Glaxo SmithKline, and Orchid Biosciences. Consulting: Merck Research Laboratories, Schering-Plough Research Institute, Targacept, Biogen-Idec ProCor, Otsuka Pharmaceuticals, BioPure, and ThermoFisher Scientific.

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Guengerich

TABLE 1 Classification of Human P450s Based on Major Substrate Class Sterols

Xenobiotics

Fatty acids

1A1 1A2 2A6 2A13 2B6 2C8 2C9 2C18 2C19 2D6 2E1 2F1 3A4 3A5 3A7

2J2 4A11 4B1 4F12

1B1 7A1 7B1 8B1 11A1 11B1 11B2 17A1 19A1 21A2 27A1 39A1 46A1 51A1

Eicosanoids 4F2 4F3 4F8 5A1 8A1

Vitamins 2R1 24A1 26A1 26B1 26C1 27B1

Unknown 2A7 2S1 2U1 2W1 3A43 4A22 4F11 4F22 4V2 4X1 4Z1 20A1 27C1

Source: From Refs. 22, 23.

in vitro findings into the context of human in vivo situations, although this is obviously more difficult in the case of bioactivation. Humans vary considerably in terms of amounts of each P450 and catalytic activity (Fig. 2), and some of the reasons will be discussed in the next section. This variability can be the basis of unexpected pharmacokinetics (Fig. 3). Ideally, the same pharmacokinetic pattern (Fig. 3) would be observed in all individuals, with the increase and decrease in plasma levels (and presumably levels of drug and metabolites in each tissue) being the same. However, an individual with a deficit of the P450 needed to metabolize the drug would show a response such as that shown in Figure 3 after receiving the same doses. Alternatively, unexpectedly rapid metabolism and low levels of the drug could result in therapeutic effectiveness. Further, if a P450 converts the drug to a toxic metabolite, the patient could be at higher risk. At this point, it is useful to consider differences between experimental animal models and humans. As discussed later, pharmacodynamic differences are often seen and these can influence drug efficacy and also toxicology. The choice of an appropriate test species is also important in considerations of metabolism and pharmacokinetics. The P450s in humans and laboratory mammals have similarity but are not identical, even when they have the same P450 name. As an example, the case of P450 1A2 is presented, where the rat and human enzymes have very similar activities toward the model substrate 7-methoxyresorufin, but human P450 1A2 has 10-fold higher catalytic efficiency than rat P450 1A2 in the activation of some FMO

UGT

NAT

1A1

MAO

1A2

Esterases

2B6

2C9

3A4(+5) P450 2D6

(A)

(B)

2C19

2E1

FIGURE 1 (A) Contribution of individual enzymes in (human) drug metabolism. (B) Contribution of individual P450 enzymes in drug metabolism. Abbreviations: UGT, UDP-glucuronosyl transferase; FMO, flavin-containing monooxygenase; NAT, N-acetyltransferase; MAO, monoamine oxidase. Source: Adapted from Ref. 24.

15

Cytochrome P450 Activation of Toxins

(A)

Nifedipine oxidation

0.2

10

5

8 6 4 2

0

(B)

0

39 98 100 107 108 109 110 112 115 116 118 123 126 129 130 132 133 134

0.4

P450 3A4

P450 2E1

39 98 100 107 108 109 110 112 115 116 118 123 126 129 130 132 133 134

chlorzoxazone 6-hydroxylation

P450 1A2

0

10

15

39 98 100 107 108 109 110 112 115 116 118 123 126 129 130 132 133 134

7-Ethoxyresorutin O-deethylation

0.6

(C)

FIGURE 2 Variation of activities of three P450s in human liver samples. A set of 18 organ donor samples was used to prepare liver microsomes and the indicated catalytic activities were measured (units of nmol product/formed/nmol total P450) (numbers indicate sample numbers). (A) P450 1A2, (B) P450 2E1, and (C) P450 3A4. Source: Adapted from Ref. 22.

heterocyclic amines to genotoxic products (26). One of the first exercises usually done with a new drug candidate is in vitro comparisons of metabolism to determine which animal species most resembles humans. The issue of metabolites that are unique to humans and not seen in toxicology test species is a matter of current concern with pharmaceutical companies and the FDA (27). Another point is that the FDA expects to have toxicology test animals exposed to multiples of both the parent drug and major human metabolites, and the choice of a species to use for testing can be a problem if metabolism is very different. Regulation of P450s Variation in P450 activities can be due to several factors. One issue is genetic polymorphism that control expression and/or activity, which are treated in chapter 11. The other issues are enzyme induction and inhibition, which underlie many of the drug–drug interactions attributed to pharmacokinetics. P450 Inhibition Many examples of P450 inhibition are known and contribute to drug interaction problems. Because of this, pharmaceutical companies screen new drug candidates for inhibition in vitro. At an early stage, the throughput can be at a high volume. The results are used (1) to select

Extensive Metabolizer (EM, normal)

C

Plasma level of drug

p,max

AUC

Poor Metabolizer (PM)

Time (arrows show repeated doses)

FIGURE 3 Effect of variability of a P450 (or other enzyme) on pharmacokinetics of a drug. Both the EM and PM patients received the same dose of the drug at each time interval (indicated with the arrow). The drug accumulates in the PM patient. Abbreviations: EM, extensive metabolizer; PM, poor metabolizer. Source: Adapted from Ref. 22.

16

Guengerich

among a series of candidates for those that should be less likely to cause problems and (2) to rationally decide which in vivo interaction studies will be most important. On the basis of the mechanism, P450 inhibition may be divided into two categories: (1) The first is competitive inhibition, the competition of two chemicals for a site on the same P450. Although several complex models are possible (28), the analysis is relatively straightforward and the information can be applied directly to pharmacokinetic equations. The inhibition is reversible. (2) The second type is mechanism-based, or “suicide,” inhibition, which is irreversible. In the course of catalytic action on the inhibitor (which is actually a substrate), at least a fraction of the reaction cycles results in inactivation of the enzyme (29). This process has the effect of lowering the concentration of the P450. Examples include components of oral contraceptives (30,31) and components of grapefruit juice (32). P450 Induction New drug candidates are often screened with in vitro induction assays in many pharmaceutical companies. Two major issues are of concern. The first is that a rough correlation among compounds exists between the inducibility of some of the P450s and liver tumors found in rodent cancer bioassays. This pattern is seen with P450s in the 1A, 2B, and 4A subfamilies. However, many exceptions to the trend exist, and the relevance of the rodent tumors to human cancer is questionable, in light of the successful use of omeprazole (P450 1A inducer) (33), the lack of tumors in individuals on long-term barbiturate therapy (P450 2B inducer) (34), and the limited responses to peroxisome proliferators (P450 4A inducer) in humans (35). The point should also be made that the P450 induction is not causal (of tumors) in the rodent models. The other reason to study induction is the potential for drug–drug interactions. Induction of a P450 by a drug can lead to changes in pharmacokinetics as a function of time, if the drug is metabolized by the induced P450. In addition, induction of a P450 by one drug can lead to increased metabolism of another. Classic examples are the early work of Remmer (14) and the loss of efficacy of oral contraceptives after use of barbiturates, rifampicin, or St. John’s wort (30,36). Although several mechanisms of P450 induction are possible and have been characterized in experimental systems, the most common mode of induction involves transcriptional regulation. A generalized scheme is shown in Figure 4. In general, a ligand is bound to a cytosolic receptor, which then forms a heterodimer with another protein in the nucleus and

R' L +

L R

R

L R

R'

(movement to nucleus?)

Coactiv L R DNA

R'

P450 gene

L R

R'

P450 gene

DNA

RNA pol

(increased access to promoter, start site)

Increased transcription

FIGURE 4 General scheme of P450 induction. A ligand L binds to a receptor R. The LR complex then interacts with another protein, R 0 , to yield a heterodimer. In the nucleus, this complex binds to a specific enhancer sequence, possibly with the assistance of a co-activator, and changes the chromatin structure to facilitate transcription by RNA polymerase. Source: Adapted from Ref. 22.

Cytochrome P450 Activation of Toxins

17

binds to a specific DNA sequence upstream of the P450-coding region. The DNA region is what is called an “enhancer” element. In some cases, a co-activator protein is also bound to the complex. The net effect of the binding of these proteins is to rearrange the structure of the gene, in the chromatin, to open the DNA promoter region, increasing the access for RNA polymerase and the rate of transcription of the P450 gene. Brief descriptions of the major characterized systems are provided here. The point should be made that some genes respond to multiple regulatory systems. The family 1 P450s are regulated by the aryl hydrocarbon receptor (AhR). A variety of polycyclic hydrocarbons and related materials are ligands, including some compounds in cigarette smoke. Binding of a ligand to the AhR involves the release of AhR from heat shock protein 90 and dimer formation with the AhR nuclear transferase protein (37). This heterodimer binds to xenobiotic regulatory element sites. This response explains the interactions of certain drugs with cigarette smoking, ingestion of charbroiled food, and use of certain drugs (e.g., omeprazole). The P450 2B subfamily genes are inducible by barbiturates, related drugs, and a variety of certain other chemicals (e.g., the pesticide DDTand the herbal medicine St. John’s wort). Induction involves the dimerization of the constitutive androgen receptor (CAR) with one of the retinoid X receptor (RXR) forms and interaction with an element far upstream of the start site (38). This system is still enigmatic in the initial response. Some artificial ligands do bind to CAR [e.g., “TCBOPOP” (39)] but apparently barbiturates and many other typical inducers do not (40). P450 3A subfamily enzymes respond to a variety of compounds, including steroids, macrolide antibiotics, and a variety of drugs. These inducers are ligands for the pregnane X receptor (PXR) which, after ligand binding, dimerizes with an RXR protein and then binds to a defined element on the P450 gene and, with the assistance of hepatic nuclear factor (HNF)-4a and possibly other co-activators (41,42), enhances transcription. The last case presented is that of the P450 4A subfamily enzymes [in humans, this consists of only P450 4A11 (and possibly 4A22), but experimental animals have multiple 4A genes]. The ligands, which can be drugs, fatty acids, or other chemicals (35), bind to the peroxisome proliferator–activated receptor a (PPARa). Liganded PPARa dimerizes with an RXR and the complex interacts with an element on the DNA. This dimer also involves the transcription of other genes and leads to the proliferation of peroxisomes, up to 25% of the cell volume, in rodents. The point should be made that CAR, PXR, PPAR, and RXR are all members of the so-called steroid receptor superfamily, which also includes the steroid hormone receptors, glucocorticoid receptor, vitamin D receptor, and retinoic acid receptor (43). The dimerization with RXR is important in that the interaction and the response are functions of the particular form of RXR involved and also its (retinoid) ligand. As mentioned earlier, several of these systems involve co-activators, e.g., HNF-4a in the case of P450 3A4, so use of cell lines with the appropriate factors is critical in mimicking the in vivo response (and also explaining the basis of tissue specificity). The point should also be made that other receptors in this family, e.g., liver X recptor and farnesoid X receptor, regulate the steroidogenic P450s (44), and some of these systems may be involved in “cross talk” with the regulatory components of the xenobioticmetabolizing P450s. Cell-based models can be used to test compounds for the ability to induce P450s, either by measuring P450 induction directly or using a reporter-based system, provided that the cells have the appropriate factors. Another consideration in using experimental animals to predict induction responses is that some species differences exist among the receptors. For instance, the response of PXR in rats, rabbits, and humans varies considerably with the ligands rifampicin and pregnenolone 16a-carbonitrile (41), and the results can be understood in the context of a few amino acid substitutions (45).

CONTEXTS OF TOXICITY One way to consider drug toxicity is classification with regard to general mechanism. Thus, it is possible to divide toxicities into five categories [Table 2; (46); see also chap. 5].

18

Guengerich

TABLE 2 Contexts of Drug Toxicity On-target Hypersensitivity and immune reactions Off-target Bioactivation to reactive intermediates Idiosyncratic Source: From Ref. 46.

The first of the five contexts, on-target or mechanism-based toxicity, is relatively straightforward to deal with. An example is the statins. Most of the toxicities can be explained by the intended action of the parent drug on 3-hydroxymethylglutaryl-CoA reductase, but in a different cell (e.g., muscle). The toxicity can be reduced by lowering the dose or, in this case, by administration of mevalonate, the product of the targeted enzyme. The second context is due to immune reactions resulting from modification of a protein by the parent drug. An example is penicillins and related b-lactams. This process is part of a “hapten” hypothesis (47). To expand this, there is a “danger” hypothesis (48), basically that the drug–macromolecule complex generates a “danger” signal that ultimately results in antibody/ T-cell responses (47). A further twist on this is the “p-i concept” of “direct pharmacological interaction of drugs with immune receptors” (47,49) (see chap. 7). The third case is off-target pharmacology. In this case, the parent drug interacts with additional targets that were not anticipated and yields unexpected pharmacology, which can result in toxicity. A relevant example is terfenadine, an antihistamine that was recalled in 1995 (Fig. 5). In this case, P450 inhibition is also an issue. Normally, terfenadine is rapidly oxidized by P450 3A4 to fexofenadine (50). Both terfenadine and fexofenadine bind to the H1 receptor and have antihistaminic activity. In most individuals, the oxidation is rapid and no terfenadine is found in plasma. However, individuals who use P450 3A4 inhibitors concurrently (e.g., erythromycin, ketoconazole) have lowered P450 3A4 activity and terfenadine accumulates.

Erythromycin Ketoconazole H+ N

CH3

CH3 Terfenadine

CH3

X P450 3A4

CH2OH

+ H+ N

Dehydrogenases or P450

CH3

H+ N

CH3 CH3

H+ N

CH3 –

CO 2 CH3

Fexofenadine

CH3 CH3

Arrhythmia, QT intervals Antagonists of H1 receptor (antihistamines)

Zwitterionic; does not cross blood/brain barrier

FIGURE 5 Terfenadine as an example of off-target toxicity and a role for metabolism. Terfenadine is extensively oxidized in three steps to a carboxylic acid (fexofenadine). Both the terfenadine and the product fexofenadine affect the target, the H1 receptor, and have antihistaminic activity. Terfenadine is normally not found in plasma following oral ingestion because of rapid metabolism. However, inhibitors of P450 3A4 block oxidation and cause terfenadine to accumulate. Terfenadine has off-target pharmacology and interacts with hERG receptors to produce cardiac problems; in some cases, abnormal QT intervals and fatal arrhythmias have been attributed to the increased terfenadine load. Abbreviation: hERG, human ether-ago-go related gene. Source: From Ref. 50.

19

Cytochrome P450 Activation of Toxins

O

O

CH3

HN

HN

CH3

O HN

O – SO3

OH P450s (2E1/1A2/3A4)

O HN

CH3

O gluc

Acetaminophen

CH3

HN

+ O

OH CH3

O

OH

N

CH3

SG GSH

OH

Protein O Iminoquinone

O HN

CH3

Cys-protein OH

FIGURE 6 Metabolism of acetaminophen and relevance to toxicity. At low doses, the metabolism of acetaminophen is largely through conjugation. Some oxidation does occur but GSH conjugation renders this inactive. At higher doses, the three conjugation pathways are not effective and larger amounts of the protein conjugates are formed and apparently contribute to toxicity. See chapter 18 for more details.

Terfenadine (but not fexofenadine) also binds to the human ether-ago-go related gene (hERG) receptor and inhibits ion channels, resulting in abnormal QT intervals and arrhythmias. Terfenadine, the first non-sedating antihistamine, was recalled and replaced with fexofenadine. The fourth context of toxicity is bioactivation, which will be discussed later. An example shown is that of acetaminophen (Fig. 6), where some oxidation to a reactive iminoquinone is always detected but can be tolerated, until the dose of the drug exceeds the ability of the conjugation systems to clear the compound. The concept of bioactivation, already presented, is generally associated with the concept of reaction of the products with proteins or other cellular macromolecules to cause damage (51), except in cases in which the products are themselves dangerous due to altered pharmacology (vide infra) or cause oxidative stress. Thus, this context has some similarity to the second one (Table 2), except that an immune system component is usually not invoked. Historically, the concept was what can be called the “critical protein hypothesis” (7), where ablation of the function of an individual protein was responsible for cell toxicity; i.e., that protein acts as a “master switch” for the cell. However, a more accepted current view is that cells have very complex networks and that disruption of those signaling pathways (46) or any of several of the systems involved in energy production (7) may lead to toxicity, with effects being cell and tissue specific (see also chaps. 4 and 5). The final context in this discussion is idiosyncratic, which is rare (!1/104 patients), a serious problem, and not understood (by definition). Dihydralazine is usually regarded as an example of an idiosyncratic drug issue, although closer analysis reveals aspects of metabolic activation, covalent binding, and possibly immunological components involved in the response (Fig. 7). BIOACTIVATION BY P450s AND OTHER ENZYMES Basic Aspects As indicated earlier in this chapter, bioactivation of drugs is one context of toxicity of drugs and other chemicals. As indicated, the phenomenon has been known since the 1930s and

20

Guengerich

HN

NH2 N N

HN

HN P450

HN

HN

Dihydralazine

NH2

N=O N N

N N

1A2

NH2

NHOH

HN

P450 1A2 adducts

NH2 Anti-LKM antibodies (recognize unmodified P450 1A2) Cause/ effect? Hepatitis

FIGURE 7 Possible mechanisms for dihydralazine-induced hepatitis. Dihydralazine is oxidized by P450 1A2 and the hydroxylamine and/or nitroso product form protein adducts. LKM antibodies are found in patients. Whether these antibodies are causal in hepatitis is not established. See also chapter 7. Abbreviation: LKM, liver kidney microsomal. Source: From Ref. 52.

extensively applied to drugs since the early 1970s. Although bioactivation may seem complex, the fundamentals are quite basic, even if the interpretation may not be straightforward. Seven principles are used to summarize this area (8): 1. The reactions are described by two basic types of chemistry. (a) The first is the reaction of an electrophile (E; formed from the drug) and a nucleophile (NucZprotein, DNA, or other component in the cell) EdC CNucdK/E

2.

3.

4. 5. 6.

Nuc

or (b) Free radical propagation, usually resulting from an odd-electron enzymatic process or lipid peroxidation. Radical processes and oxidative stress from reactive oxygen (or nitrogen) species have not been addressed extensively here, but in some cases are probably very important (53). How much reactive oxygen species contribute to the overall hepatoxicity of drugs is unclear (see chap. 3). The first product or the most obvious reactant may not be the one that reacts with the nucleophile. For instance, in some cases, an epoxide may be generated by a P450 but it will undergo a rearrangement to an acyl halide or other product that participates in the reaction (54–56). The stability (and also the reactivity) of reactive products may vary considerably (57). A short-lived product, e.g., aflatoxin B1 (AFB1) exo-8,9-epoxide (t1/2Zone second at neutral pH) (58) can be formed in the endoplasmic reticulum but still migrates to the cell nucleus to react with DNA very efficiently (Fig. 8). Other reactive products may have half-lives on the order of minutes to hours. In contrast to the shorter t1/2 products, these can often migrate from one cell to another and even one tissue to another. In vitro systems are good models for elucidating details but ultimately all covalent binding results must be considered in the context of in vivo situations. In in vitro settings, some factors may be overemphasized or biased. The dose is an issue or, in many cases, the issue, going back 500 years to an axiom of toxicology from Paracelsus (60). Covalent binding can be an index of toxicity and often correlates (4,61). Some pharmaceutical companies are using data from in vitro and in vivo covalent binding studies to at least stratify candidates for development (62). However, many exceptions are known, i.e., nontoxic compounds show covalent binding and toxic compounds do not bind. Thus, caution is advised in interpretation.

O

OCH3

O O H

DNA

GS

OCH3

O

O O

OCH3

1.5 ×

106

H 2O (H+)

M–1 min–1

HO

H

O

OCH3

O

AFB1 GSH conjugate

O H O

OH

O

42 min–1 + 1.3 × 105 exo–8.9– epoxide AFB1 M–1 min–1 + GST M1 1.2 × 10−5 M–1 min–1 (H component)

O 1.8 × 105 M–1 min–1 H O

P450 3A4

O

O

AFB1 Guanine–N 7 adduct

O H O

H

O

O

O O

O OCH3 AFB1 dihydrodiol

O

OH

(H–) 0.12 min–1 (at pH 7)

OH–

HO

O

O

HO

O

O

OCH3

O

O

O

OCH3

O

OCH3

O

1.8 x 106 M–1 min–1

O

+

O

Protein

2.4 × 102 M–1 min–1 (BSA) to 2.4 × 103 M–1 min–1 (N 2–AcLys)

AFB1 monoaldehydes

HO

OH

HO

OH

O

OCH3

AFB1 dialdehyde

HO

OH

AFAR AKR 7A2,7A3

O

1.2 × 105 M–1 min–1 O

HO

O

O

AFB1 Lys adduct

Lys N

O

FIGURE 8 Metabolism of AFB1 and relevance to toxicity. The major human enzymes involved in each reaction are included and rates have been determined. First-order rates are listed in units of reciprocal time. Units of minK1 MK1 indicate either second-order rate constants (for chemical reactions) or catalytic efficiency (kcat/Km) for enzymatic reactions measured using the most relevant purified recombinant human enzymes. The exo-8,9-epoxide plays a central role in this process and appears to be the only product that reacts with DNA. The dialdehyde reacts with proteins (BSA). The dehydrogenase AFAR is now classified as an AKR. Several other oxidation products (AFQ, AFM, endo epoxide) are not shown, which are all less toxic than AFB1. Abbreviations: AFAR, aflatoxin aldehyde reductase; AFB1, aflatoxin B1; AFQ, aflatoxin Q1; AFM, aflatoxin M1; AKR, aldo-keto reductase; BSA, bovine serum albumin. Source: From Ref. 59.

AFB1

O H O

H

O

Gua–N 7

OH

Cytochrome P450 Activation of Toxins

21

22

Guengerich

7. Numerous other factors influence the relevance of covalent binding to toxicity. Among these are receptor-medicated events (i.e., with the parent drug itself), the disruption of particular signaling events by the reactive products, the ability of the cells to repair damage, the intervention of immunological processes, and cell proliferation (see also chaps. 4, 5, and 11). Examples of P450-Dependent Bioactivation Acetaminophen This is a classic example of P450 bioactivation (4) (see also chap. 18). It is also a very practical issue because, as pointed out elsewhere in this book, this is one of the most commonly used drugs and overdoses are a leading cause of liver failure today (see also chap. 19). At low doses, the conjugating enzymes allow only a small amount of acetaminophen to be oxidized (P450 2E1, 3A4, 1A2) to the reactive iminoquinone, a reactive “Michael acceptor” (Fig. 6). With higher doses and in situations in which the cofactors needed for the various conjugations are depleted (e.g., GSH, UDP-glucuronic acid, 3 0 -phosphoadenosine 5 0 -phosphosulfate, as in nutritionally challenged states), more acetaminophen is activated and reacts with proteins. Which proteins are the most critical targets has been considered extensively (7,63) but is still not clear (see chap. 18). The process is somewhat more complex in terms of other processes not shown in Figure 6. The iminoquinone is reduced back to acetaminophen by NADPH-P450 reductase, hydrolysis occurs to yield benzoquinone, and peroxidases can also be involved in the activation (64,65). Aflatoxin B1 AFB1 is not a drug but is a mycotoxin of considerable significance regarding human exposure, in that it is one of the most potent (and prevalent) hepatocarcinogens known (66). P450 oxidations of AFB1 produce several products, all of which are less dangerous except one, the exo-8,9-epoxide (Fig. 8). All of the genotoxicity can be explained in terms of this epoxide, specifically the exo isomer (67). Even though the t1/2 of the epoxide is one second under physiological conditions (58), the compound can migrate from the endoplasmic reticulum to the cell nucleus to bind efficiently to DNA (68). Alternatively, the epoxide can be enzymatically conjugated with GSH (69) or (nonenzymatically) hydrolyze to a dihydrodiol. The dihydrodiol is unstable and undergoes reversible ring opening to a dialdehyde species (58). The dialdehyde reacts with protein lysines (but not with DNA) (59), but enzymatic reduction of the dialdehyde occurs and prevents the covalent binding (70). Measurements of AFB1–lysine adducts in human serum albumin have been used to assess AFB1 exposure and biologically relevant exposure (71). Trichloroethylene Although trichloroethylene (TCE) was used formerly as an anesthetic, the major concerns are from industrial and environmental exposure to this solvent. Oxidation of TCE by P450s, especially P450 2E1 (72), yields two primary products (Fig. 9). One is chloral (hydrate), formed via rearrangement of an enzyme–substrate intermediate (Fig. 9) (74). (Chloral is a known sedative, although this is probably not an issue in the pharmacology of TCE.) The other major oxidation product is TCE oxide, which has a t1/2 of 12 seconds under physiological conditions (55,74). Labeling experiments with 18O and 2H (55) support a mechanism for reaction with lysines that does not involve a direct reaction by the epoxide but instead by an acyl halide rearrangement product (Fig. 10). TCE oxide [and its rearrangement product(s)] does not react well with DNA (75). The reactions with protein lysine groups yield stable amides, both N6-formyl lysine and N6dichloroacetyl lysine (55). Modern electrospray mass spectrometry methods can be used to quantify the number and amount of the two adducts formed on an oligopeptide or small protein in vitro (55). In the course of such analysis, it was found that some of the adducts are unstable at basic or neutral pH (75). These appear to be adducts formed with serine, cysteine, and tyrosine (75). The t1/2 for loss under physiological conditions is wone hour. When model

23

Cytochrome P450 Activation of Toxins

Cl FeO3+

+

Cl

Cl

H

Cl

FeO2+

Cl Cl

H

O H

+ Cl

FeIII

Cl Cl Cl

H

FeIV

H

Cl

HO Cl

O

Cl Cl

Cl Cl H

O

Cl O

Cl

Cl

Cl

H Cl N N N N Heme (porphyrin) adduct

FIGURE 9 Oxidation of TCE by P450. The activated form of P450 is indicated as the peroxyferryl complex, FeO3C. Reaction with TCE yields a one-electron oxidation product and then a carbocation intermediate, which can rearrange to chloral or react with heme. The epoxide is unstable (t1/2Z12 seconds) and undergoes rapid reaction (Fig.10) but does not form chloral. Abbreviation: TCE, trichloroethylene. Source: From Refs. 73, 74.

enzymes with these three residues critical for catalytic activity were partially inactivated with TCE oxide, the catalytic activity slowly returned with a t1/2 of wone hour, suggesting the loss of the acylating entity and “recovery” of the enzymes (75). Such unstable protein adducts would not have been detected using more traditional, slower analytical methods, and the existence of such a phenomenon raises several questions. On one hand, the transient nature of the adducts might argue that a given amount of protein damage is not as serious as first thought from consideration of the level of binding. Alternatively, one can take the other view that the extra burden on an enzyme involved in cell regulation may be serious, if the regulatory system is distorted for even a short period of time. Troglitazone This compound was the first in the thiazolidinedione group of PPAR activators marketed to treat diabetes (see chap. 28). However, the drug was withdrawn in 2000 due to hepatotoxicity, with the incidence of liver transplant or fatality due to liver failure being 1/104 (47). The FDA withdrew the drug following 560 reports of hepatotoxicity. X O

X

H

X

a O

O

H b aX NH2-Lys b

O

O

H

NH-Lys

− O

+ Cl H Cl

X

O

NH-Lys H

O X NH2-Lys

X

O

X

NH-Lys

H

H

O NH-Lys

FIGURE 10 Reactions of TCE oxide (Fig. 9) with lysine residues in proteins. Evidence for the proposed pathways was obtained using 18O and 2H labeling studies. The acyl halide intermediates are the species that react with the protein, not the epoxide itself. Abbreviation: TCE, trichloroethylene. Source: From Ref. 55.

24

Guengerich

Although the exact cause of the hepatotoxicity has not been firmly established, the covalent binding of troglitazone is considerable in model systems. P450 3A4 is involved in the pathways that can lead to electrophilic products (Fig. 11). One pathway (Fig. 11A) involves the formation of a quinone, a potent Michael acceptor capable of reacting with nucleophilic groups in proteins. The other pathway involves oxidation of the thiazolidinone ring (Fig. 11B). Although troglitazone was withdrawn from the market, several other “glitazones” with the thiazolidinone structure are now marketed. If the oxidation of that ring is the cause of covalent binding (and covalent binding is related to toxicity) then one might expect these to also be toxic. They appear not to be, but part of the reason may be that the doses used (!10 mg QD) are more than an order of magnitude less than used with troglitazone.

RELATIVE CONTRIBUTION OF METABOLISM TO DRUG TOXICITY The contexts of drug toxicity have been discussed earlier. How does metabolism fit into this picture, i.e., in what fraction of the cases are metabolites really involved in toxicity of drugs? Two aspects can be considered. One is metabolism of drugs to stable products. The process of metabolism almost always occurs to some extent, and the products generally have attenuated activity toward the same target (as the parent drug). In some cases, the product may show enhanced biological activity at the same site, and this is really a “pro-drug” phenomenon. [An example is the oxidation of the antihypertensive losartan to a carboxylic acid by P450 2C9 (79)]. Recently, the FDA has expressed concern about safety assessment of drug metabolites that are unique to humans (compared to experimental animals) [FDA/CDER Draft: Guidance for Safety Testing of Drug Metabolites, 2005]. However, few drugs appear to be transformed to products that have pharmacology totally different from the parent drug (46), even if the possibility cannot be completely excluded. Examples from the non-pharmaceutical world include the oxidation of the anesthetic/solvent TCE to the sedative chloral (vide supra) and the oxidation of the pesticide methoxychlor to an estrogen (80). The other issue is metabolism of drugs to reactive products. Indeed, all of the four examples cited by FDA/CDER in the 2005 Draft on Guidance for Safety Testing of Drug Metabolites (vide supra) involve reactive products, which cannot usually be tested in the usual manner because they are unlikely to reach the target site. Reactive products are an interesting topic and have a tendency to reach the open literature because of their interesting chemistry. However, what fraction of drug problems really involves bioactivation? One way of looking at the issue involves consideration of acetaminophen itself, probably the most widely used drug today in the United States. (see chap. 19). Nearly, one-half of druginduced liver injury can be related to drugs, and about one-half of these cases are due to overdoses of acetaminophen. The statement can be accurately made that all acetaminopheninduced injury is the result of bioactivation, even if unknown other events are also important in determining the course of injury (7,81,82). Another way of analyzing the roles of metabolism and covalent binding is the analysis of Walgren et al. (83). Of six drugs that have been withdrawn from the market (in a given recent time period), five are known to have reactive metabolites (benzoxaprofen, iproniazid, nefazodone, tienilic acid, and troglitazone). Of 15 drugs with “Black Box” warnings, eight have reactive metabolites (dacarbazine, dantrolene, felbamate, flutamide, isoniazid, ketoconazole, tolcapone, and valproic acid). In considering the above two sets of compounds, 62% have reactive metabolites. Further, at least 14 drugs known to produce reactive metabolites have warnings about hepatotoxicity (acetaminophen, carbamazepine, clozapine, diclofenac, disulfiram, halothane, leflunomide, methyldopa, rifampicin, tacrin, tamoxifen, terbinafine, ticlopidine, and zileuton). Another 14 drugs with reactive metabolites were never approved in the United States (alpidem, amineptine, amodiaquine, cinchophen, glufenine, ibufenac, isoxanine, niperotiden, perhexiline, pirprofen, and tibroquinol) (83). Looking at the amassed literature, one might conclude that bioactivation is a prominent aspect of drug toxicity. Indeed, several pharmaceutical companies now conduct early in vitro screens to stratify compounds to avoid those most likely to show reactive products (62). However, the above discussion may present a biased view of the reasons why new drug

Troglitazone

O

O

(B)

N H

O

O

N H

O– S+

O

O

O

· O

S

S

O

NH2

SG

GSH, H2O –CO2

O

N=C=O

OH

O

O

GSH

OH O

CH2

O

S O

OH

O

GSH

SG

O

O

HO

O

–O

O

O

O

O

–H2O

O

OH

OH

S+

S

OH

GSH

O

SG

O

SG

SG

S

NH2 SG

O

FIGURE 11 Activation of troglitazone by P450 3A4. Pathway (A) results in the formation of a benzoquinone. Pathway (B) involves oxidation of the thiazolidinedione ring. Source: From Refs. 47, 76 78.

HO

O

S

(A)

·

O

Cytochrome P450 Activation of Toxins

25

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Guengerich

candidates fail. First, when reactive metabolites are found, there is almost universally no demonstrated mechanism of causality with disease, as of yet. Another issue is that the true cause of toxicity of many failed drug candidates is never investigated, and there is a strong possibility that many show off-target pharmacology as the context of toxicity (vide supra). Unfortunately, a complete analysis of the causes of candidate failure may never be available because of the expense of analyzing mechanisms for large numbers of compounds that are not viable as leads. One final important point should be made. Although many of the toxicities, particularly those considered idiosyncratic, are not considered to be dose-dependent, there is a clear inverse correlation between the dose and the proclivity for toxicity. Most of the drugs that have been withdrawn from the market were used at high doses (O100 mg/day) (83) and few problems have been seen at doses on the order of 10 mg/day (47). This information might be considered as evidence for roles of bioactivation and either protein modification or oxidative stress, in that there is a hint of a threshold. The relationship is not absolute and one can consider the case of ceruvistatin, which was used at sub-milligram per day doses and withdrawn due to muscle and kidney problems. However, this is probably a case of on-target toxicity, in that the effect is due to the intended action of the drug (albeit in a different tissue) (84). Thus, the conclusion is that drug toxicity is the result of several factors, including the dose, the usage (chronic use is more of a problem than temporary use), the activation to reactive metabolites and existence of detoxication pathways, and the inherent on- and off-target pharmacology. More problems arise when a single pathway is dominant in the metabolism steps and large variations in the population can have major impacts (as opposed to situations where several enzymes contribute to detoxication and the result is a dampening of the effects of changes in any single system) (7,85).

APPROACHES TO SAFETY PREDICTION OF NEW DRUGS Toxicity, particularly hepatotoxicity, has become one of the most serious concerns in the development of new drugs. A major problem is the development of toxicity problems late in the process of clinical trials, after considerable investment in a drug candidate. One issue is the prediction of chemical groups that make drugs more likely to be activated to reactive products (86). This issue has been considered by medicinal chemists, and a number of entities are now either avoided completely or, if any assays suggest toxicity in early screening, changed to reduce toxicity while maintaining efficacy (47,86). Although some chemical entities are clearly more prone to cause problems than others (e.g., thiophenes), this approach cannot completely avoid problems in that even a simple phenyl ring is only from one to three steps away from a reactive product. One view of the drug development process is shown in Figure 12. A generation ago the loop cycled only between chemistry (drug design and synthesis) and biology (efficacy and selectivity testing). At that time, most of the screening was with animals (in vivo). Today, the field involves more in vitro approaches, particularly with systems of human origin. A separate assay loop is involved, with “developability screening” and predictions. Many of the desirable properties of a drug candidate are related to P450s, transporters, and detoxication processes. The overall goal is to select the most desirable candidates before proceeding to the more complex (and expensive) safety studies and clinical trials. In many respects, the prediction of pharmacokinetics has been considerably improved with better understanding of the P450s, conjugating enzymes, and transporters. However, early prediction of toxicity is more difficult (7,46). The basic problem is that the key events in most types of toxicity remain poorly understood (see chap. 5). Genotoxicity is an exception, to a large extent, in that simple mutations can be scored quickly. Some of the issues in the biology of cell toxicity have been introduced elsewhere (see chap. 5). Several newer approaches are being examined in both industrial and academic settings (46) (see chap. 15).

27

Cytochrome P450 Activation of Toxins

Target Lead compounds

Design & synthesis

Efficacy & selectivity testing

Candidate

"Developability screening" & predictions

Toxicology pharmaceutics ADME: Pharmacokinetics Pharmacodynamics Cytochrome P450 inhibition Cytochrome P450 induction Permeability Transporter interactions Instrinsic clearance Reaction phenotyping Reactive metabolites

Detailed physicochemical, ADME, & safety workup

Clinical trials

FIGURE 12 A typical strategy used in drug development. A target receptor or enzyme is established on the basis of knowledge about the biology for the disease of interest. Lead compounds are developed, usually by screening procedures (sometimes by rational design). These candidates enter a series of reiterative development loops. Efficacy and selectivity testing involve pharmacological assays based on the target; developing screening and predictions involve the aspects shown at the right; design and synthesis involve more chemistry. The goal of the process is to develop candidates that are less likely to fail in the detailed later studies and the clinical trials. Thanks are extended to W. G. Humphreys (Bristol-Myers Squibb) for the concept. Abbreviation: ADME, absorption, distribution, metabolism, and excretion. Source: Adapted from Ref. 87.

1. One approach is in silico prediction. The difficulty is that few targets directly related to toxicity have been identified and structures of even fewer are available, so modeling of receptor–ligand interactions is possible in only a few cases (e.g., possibly Ah receptor). Thus, most approaches are based on quantitation–structure relationships, mostly involving structural entities and crude toxicity parameters. 2. The second approach is transcriptonomics, or mRNA profiling. A number of experiments have been done in this arena (88,89), with the general goal of finding early changes in patterns that will be predictive of toxicity. Because the extent of changes in gene expression is extensive (one can easily find changes in expression of 10% of the genes due to treatment in many experiments), the analysis is not trivial and enlightened new approaches to the informatics are necessary. Another complication is that in vivo changes are probably much more valuable than work done in cell cultures; extrapolation to humans is still an issue. 3. The third approach is proteomics. Briefly, four aspects can be considered. The first is changes in the levels of expression of individual genes, similar to the approach used in transcriptome profiling. The same problems arise as in the consideration of mRNA profiles. Another aspect is the analysis of changes in posttranslational events, such as phosphorylation (redox changes are generally even more difficult). The third approach is to map drug modification of individual proteins (90). To date the modification of a single protein has not clearly defined a role in toxicity, but as increased information becomes available more insight may be possible. The fourth approach, like the second and third of these, uses mass spectrometry approaches and involves the use of the techniques to profile patterns of localization of proteins within tissues (91,92).

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4. Metabonomics (or sometime “metabolomics”) is the analysis of changes in the profiles of low Mr compounds, usually using blood or urine samples. The methods are usually NMR spectroscopy and mass spectrometry (93). The point is not to look at the drug or its metabolites but instead at changes in the profiles of endogenous compounds. These are not necessarily identified, but the goal is to find changes in patterns that can be related to toxicity and serve as early predictors, e.g., with pattern sorting using principal components analysis and related approaches. 5. Another approach is the use of covalent binding data. The point is to stratify lead candidates to find those that produce less binding. Two major approaches are used. One is in vitro (cell-free) systems searching for conjugates of the drug with GSH or a surrogate thiol (62,94). This aspect can be done without radiolabeled material. Quantitation of in vivo (animal) results is done subsequently, with radiolabeled drug. A rough decision cutoff value of 50 pmol adduct/mg protein has been proposed (62), but obviously this value would be a function of dose, time, etc. The overall plan in these strategies is to find better and earlier biomarkers that can be used effectively in the development process and even in clinical trials and marketed drugs. A critical issue with candidates found in animals is extrapolation to humans. Another lingering problem is the idiosyncratic toxicities. With time, more of these will be classified into other “contexts of toxicity” (vide supra). However, the problem of eliminating all possible adverse reactions in large clinical populations without a mechanism or an animal model is still a formidable task. CONCLUSIONS Metabolism is an important issue in drug efficacy and toxicity. The P450s are the most important enzymes in these processes, but other enzymes have roles, including transporters. The regulation of P450s is understood largely in terms of transcription and classic receptor– ligand systems. Metabolism by P450s and other systems clearly has a role in the toxicity of many drugs, generally either deactivating the drug or activating it to a reactive form. There are several contexts of drug toxicity, and exactly what the relative contributions are to overall injury is not clear. However, a sizeable function of toxicity is somewhat related to reactive products, although even when these compounds are produced they appear to require other factors for toxicity. A number of newer approaches to toxicity prediction are being considered, but the validation of biomarkers of toxicity is still an important issue. REFERENCES 1. Fieser LF. Carcinogenic activity, structure and chemical reactivity of polynuclear hydrocarbons. Am J Cancer 1938; 34:37–124. 2. Miller EC, Miller JA. The presence and significance of bound amino azodyes in the livers of rats fed p-dimethylaminoazobenzene. Cancer Res 1947; 7:468–80. 3. Mueller GC, Miller JA. The metabolism of 4-dimethylaminoazobenzene by rat liver homogenates. J Biol Chem 1948; 176(11):535–44. 4. Jollow DJ, Mitchell JR, Potter WZ, et al. Acetaminophen-induced hepatic necrosis. II. Role of covalent binding in vivo. J Pharmacol Exp Ther 1973; 187(1):195–202. 5. Zampaglione N, Jollow DJ, Mitchell JR, et al. Role of detoxifying enzymes in bromobenzene-induced liver necrosis. J Pharmacol Exp Ther 1973; 187(1):218–27. 6. Basu AK, Essigmann JM. Site-specifically modified oligodeoxynucleotides as probes for the structural and biological effects of DNA-damaging agents. Chem Res Toxicol 1988; 1(1):1–18. 7. Park BK, Kitteringham NR, Maggs JL, et al. The role of metabolic activation in drug-induced hepatotoxicity. Annu Rev Pharmacol Toxicol 2005; 45(1):177–202. 8. Guengerich FP. Principles of covalent binding of reactive metabolites and examples of activation of bis-electrophiles by conjugation. Arch Biochem Biophys 2005; 433(2):369–78. 9. Dorfman R, Cook JW, Hamilton JB. Conversion by the human of the testis hormone, testosterone, into the urinary androgen, androsterone. J Biol Chem 1939; 130:285–95. 10. Ryan KJ. Conversion of androstenedione to estrone by placental microsomes. Biochim Biophys Acta 1958; 27(3):658–9.

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Antioxidant Defense in Liver Injury: Oxidant Stress, Antioxidant Defense, and Liver Injury Hartmut Jaeschke

Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas, U.S.A.

INTRODUCTION The liver, the body’s largest solid organ, performs a substantial number of vital metabolic functions and is the main organ for drug and xenobiotic metabolism. These functions require an extensive aerobic metabolism to generate sufficient quantities of ATP in mitochondria. However, this metabolic activity causes a continuous formation of reactive oxygen species (ROS). In addition, drug metabolism and cell injury with inflammation can dramatically increase the oxidant stress burden for each individual cell and the organ. This review will focus on the general discussion of reactive oxygen and peroxynitrite formation, description of antioxidant systems in different cellular and vascular compartments, and analysis of potential adverse consequences of excessive oxidant stress in the liver. REACTIVE OXYGEN AND NITROGEN INTERMEDIATES Molecular oxygen (3O2) can be reduced by one-electron steps to superoxide ðOK 2 Þ, hydrogen peroxide (H2O2), the hydroxyl radical (OH%), and then to water (Fig. 1). Superoxide is not very stable and dismutates rapidly to form hydrogen peroxide and singlet oxygen (1O2), another ROS. However, in the presence of nitric oxide (%NO), superoxide reacts preferably with NO to generate peroxynitrite (ONOOK). The rate of peroxynitrite formation depends on the concentrations of both NO and superoxide (first-order kinetics) and this reaction is near diffusion controlled (1,2). With the ubiquitous presence of carbon dioxide (CO2)/bicarbonate in vivo, peroxynitrite reacts rapidly with CO2 to form reactive intermediates, which are highly effective oxidizing and nitrating species (1). In addition, peroxynitrite can be protonated to form peroxynitrous acid (ONOOH), which is a powerful oxidant. Hydrogen peroxide can be reductively cleaved to the extremely reactive hydroxyl radical in the presence of transition metals (Fenton reaction). However, if phagocytes release myeloperoxidase (MPO), then hypochlorous acid (HOCl), another potent oxidant, is generated. In addition to the described primary reactive intermediates (Fig. 1), a number of secondary radicals can also be formed, e.g., alkyl (R%), peroxy (ROO%), and alkoxy (RO%) radicals. In general, the secondary radicals are less reactive and more selective in their target. Formation and steady-state concentrations of any of these described reactive oxygen and nitrogen species in vivo are dependent on a number of factors including the formation rates of the precursors, detoxification reactions, pH, and availability of transition metals. INTRACELLULAR AND VASCULAR SOURCES OF OXIDANTS Mitochondria Superoxide and hydrogen peroxide are the main initial ROS generated in all liver cell types and the vascular space. A major continuous intracellular source of superoxide formation is the electron transport chain of mitochondria (3). Approximately 2% of total oxygen utilized in a cell is reduced to superoxide (3). NADH dehydrogenase (complex I) and ubiquinone–cytochrome b

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OCI– H2O

MPO 3

O2

e

•

O2

–

e • – O2

– CI

H2O2 1

Fe

O2

e 2+

OH Fe

•

e

H2O

3+

•

NO

ONOO – ONOOH

CO2

•

NO2CO3

•

–

– NO2 + CO3

•

•

FIGURE 1 Reactive oxygen species generated by oneelectron reduction steps of molecular oxygen (3O2). During the spontaneous dismutation of superoxide ðOK 2 Þ, singlet oxygen (1O2) is formed. If nitric oxide (†NO) is present, superoxide reacts with NO and forms peroxynitrite (ONOOK) and peroxynitrous acid (ONOOH). Peroxynitrite reacts with carbon dioxide to generate nitrating species such as the (†NO2) radical. Neutrophils release the enzyme myeloperoxidase (MPO) to form hypochlorite (OClK).

complex (complex III) release superoxide even under physiological conditions. The highest mitochondrial superoxide formation is observed during slow resting state 4 respiration, i.e., when the components of the respiratory chain are mainly in the reduced form (3,4). Mitochondrial superoxide can increase substantially when mitochondria are damaged. When superoxide is released from the electron transport chain, it can combine with NO to form peroxynitrite. It was postulated that mitochondria contain a nitric oxide synthase (NOS) (5). However, the existence of a mitochondrial NOS was questioned (6); nevertheless, peroxynitrite formation in mitochondria can occur when NO from an extramitochondrial source diffuses into mitochondria (6). In addition to the electron transport chain of the inner mitochondrial membrane, monoamine oxidases, which are located in the outer membrane, generate substantial amounts of hydrogen peroxide during the oxidative deamination of biogenic amines (7). Because of the localization, monoamine oxidases contribute to the oxidant stress in mitochondria and in the cytosol. A mitochondrial oxidant stress has been demonstrated in connection with mitochondrial dysfunction during hypoxia–reoxygenation (8), chemical hypoxia (9), extracellular oxidant stress (10), and the toxicity of ethanol (11) and bile acids (12). In the case of acetaminophen hepatotoxicity, reactive oxygen (13) and peroxynitrite formation (14) has been localized to the mitochondria. Microsomes During phase I metabolism of xenobiotics, the microsomal P450 enzyme system can release activated oxygen intermediates. The formation of hydrogen peroxide and superoxide has been documented in isolated microsomes (15). However, during in vivo drug metabolism there is little evidence for increased oxidant stress (16,17), suggesting less leakage of ROS from cytochrome P450 enzymes in the intact cell than in isolated microsomes. On the other hand, cytochrome P450 2E1 is considered a major source of ROS during metabolism of ethanol in hepatocytes (18). In addition, drug metabolism can lead to secondary oxidant stress in the liver, e.g., injury to mitochondria (13) and mobilization of transition metals (19). A severe oxidant stress can be generated by metabolism of redox-cycling agents such as diquat (20), paraquat (21), and menadione (22). These compounds are reduced by P450 reductase to a radical species, which can reduce oxygen to superoxide thereby regenerating the parent compound. Redoxcycling agents can undergo numerous cycles before they are excreted and create an enormous oxidant stress and cause severe liver damage (20). Peroxisomes These cell organelles contain a number of oxidases, e.g., fatty acyl-CoA oxidase, amino acid oxidase, and urate oxidase, which generate hydrogen peroxide as a regular product. Owing to the very high levels of catalase in peroxisomes, the adverse effects are limited under physiological conditions. However, high fat diet and drugs that are peroxisome proliferators cause an increase in fatty acyl-CoA oxidase and potentiate the oxidant stress in this cell organelle (23,24).

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Cytosol Xanthine dehydrogenase is a major enzyme in the cytosol of all liver cells. Although the specific activity of this enzyme is the same in all three cell types, hepatocytes contain more than 85% of the total enzyme activity in the liver (25). Prolonged periods of ischemia (26) and certain drug toxicities (13) can cause a proteolytic cleavage of the enzyme, resulting in the loss of the capability to bind the cofactor NADC. Instead, the enzyme acts as an oxidase by using molecular oxygen as electron acceptor, which leads to formation of superoxide and hydrogen peroxide in the cytosol. Some time ago, xanthine oxidase (XO) was considered the main intracellular source of reactive oxygen formation during ischemia–reperfusion. However, it is questionable if XO can actually generate a quantitatively relevant oxidant stress in hepatocytes (27). The restricted availability of the substrate, hypoxanthine or xanthine, may be the limiting factor for the duration and extent of XO-mediated reactive oxygen formation (8). It was suggested that XO might be a relevant source of reactive oxygen in Kupffer cells (25) and, after release by hepatocytes and binding to endothelial cells, a major source of oxidant stress in vascular lining cells (28). Vascular Oxidant Stress Kupffer cell activation and hepatic neutrophil recruitment can occur during drug metabolism (29–32), ischemia–reperfusion (33–35), endotoxemia (36,37), alcoholic hepatitis (38), and obstructive cholestasis (39,40), and may contribute to liver injury. A number of inflammatory mediators activate and prime Kupffer cells and neutrophils for enhanced superoxide formation including activated complement factors (41), TNF-a (42), and platelet-activating factor (43). Kupffer cells are in a fixed position within the sinusoidal lining. Superoxide, generated by NADPH oxidase in Kupffer cells, is released into the sinusoidal lumen and the space of Disse. Because of the close proximity to other cells, Kupffer cell-derived reactive oxygen can directly cause cell injury, which can be inhibited by vascular antioxidants (44). In contrast to Kupffer cells, neutrophils adherent to vascular endothelial cells release cytotoxic mediators only when excessively stimulated. However, this is rarely the case under realistic pathophysiological conditions in vivo. Injury occurs mainly after chemotactic stimulation, transmigration, and adherence of the neutrophil to hepatocytes (45,46). These processes require a number of adhesion molecules including b2 integrins and intercellular adhesion molecule-1 (ICAM-1) (47). Upregulation of the b2 integrin Mac-1 (CD11b/CD18) and the adhesion through this receptor (48) is critical for neutrophil-induced reactive oxygen formation. In support of this hypothesis, enhanced Mac-1 expression was shown in every model where neutrophils contribute to liver injury (39,49–53). In addition, antibodies against Mac-1 attenuated the postischemic oxidant stress by neutrophils and protected against neutrophil-induced liver injury (36,50). Furthermore, the neutrophil-induced oxidant stress and injury were attenuated in CD18 gene knockout mice after obstructive cholestasis (39) or a-naphthyl-isothionate treatment (54). The role of reactive oxygen in the mechanism of neutrophil-mediated cell killing remains controversial. Coculture systems showed that activated neutrophils damage hepatocytes by protease release and not oxidant stress over a time frame of 15 hour in vitro (55,56). However, recent findings suggest that reactive oxygen generated by transmigrated and adherent neutrophils causes not only an oxidant stress in the vasculature but also intracellularly (37,39,57). The appearance of chlorotyrosine protein adducts (37,39,40) and HOCl-modified protein epitopes (58) during neutrophil cytotoxicity clearly documents the presence of neutrophil-derived oxidants (HOCl) in the target cell. In addition, the higher susceptibility of glutathione peroxidase (GPx) gene knockout mice to neutrophil cytotoxicity indicates that neutrophils can kill hepatocytes by reactive oxygen, a process that requires not more than one hour after neutrophil attack (57). How can we explain the drastic differences between results of in vivo experiments and the coculture system in vitro? The most likely explanation for the opposite results is in the role of hepatocytes. In vivo, hepatocytes are exposed to the same inflammatory mediators as neutrophils resulting in the upregulation of adhesion molecules such as ICAM-1 (40,49,59) as well as the formation and release of CXC chemokines (60,61).

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Chemokines and ICAM-1 are important for neutrophil chemotaxis and adherence to hepatocytes (60), which is the final activating step for neutrophil degranulation and long-lasting adherence-dependent reactive oxygen formation. In contrast, all coculture experiments were done with control hepatocytes. Since neutrophils do not attack healthy cells (37,46), they do not firmly adhere in vitro and the cytotoxicity is dependent on the excessive stimulation with inflammatory mediators. Although reactive oxygen may be generated, it may not be long enough to cause toxicity and not in close proximity to hepatocytes. Therefore, the cytotoxicity in vitro is mainly caused by slow proteolytic digestion and not by the rapid targeted killing by reactive oxygen (45,46).

PATHOPHYSIOLOGICAL CONSEQUENCES OF OXIDANT STRESS Lipid Peroxidation Oxidant stress in the liver can cause lipid peroxidation (LPO), which is still a frequently hypothesized mechanism of cell injury. However, on a quantitative basis, the magnitude of LPO in vivo is mostly insufficient to directly cause cell death (62). Excessive intracellular superoxide formation alone does not kill hepatocytes by LPO even after depletion of glutathione (GSH) (63). Extensive hepatic LPO in vivo was only observed when, in addition to reactive oxygen formation, the cellular antioxidants, e.g., vitamin E and GSH, were depleted (64,65), high levels of polyunsaturated fatty acids were present in membranes (65), and iron was mobilized from intracellular stores (19). If some of these factors come together, massive LPO with severe cell injury ensues. However, under most realistic pathophysiological conditions, LPO is minimal and is not likely to be directly responsible for cell damage. Does this mean LPO is not important? LPO products are potent chemotactic factors for neutrophils and can modulate superoxide formation (66). In addition, LPO products may enhance chemokine formation (67). These observations may explain the role of LPO products in maintaining an inflammatory response beyond the initial mediator formation (68). Furthermore, LPO products were shown to promote induction of collagen gene expression in activated stellate cells and contribute to fibrosis (69). Thus, LPO products can be important signaling molecules under certain pathophysiological conditions. Peroxynitrite Formation The reaction of peroxynitrite with carbon dioxide yields nitrating species which react preferentially with tyrosine (70). During acetaminophen hepatotoxicity, a mitochondrial oxidant stress (13) and NO derived at least in part from the inducible NOS (71) cause peroxynitrite formation in mitochondria (14). Aided by the massive depletion of cytosolic and mitochondrial GSH, peroxynitrite proved to be a critical mediator in the mechanism of cell injury (71,72) and trapping of it did not only strongly attenuate cell death but also promoted cell cycle activation and regeneration (73). Although the detailed mechanisms are not completely understood, peroxynitrite appears to be involved in the mitochondrial membrane permeability transition (MPT) pore opening (74). The MPT leads to breakdown of the mitochondrial membrane potential and ATP depletion (75) and the release of intermembrane proteins such as endonuclease G and apoptosis-inducing factor, which translocate to the nucleus and cause DNA fragmentation (14,76). These events are essential for the development of acetaminopheninduced necrosis (77). Nitrotyrosine residues were also detected in the liver during hepatic ischemia–reperfusion injury (78,79) and during alcohol-induced liver injury (80), but the pathophysiological relevance is less clear (81). Since ischemic stress promotes vasoconstriction in the hepatic vasculature (82), NO formation in the liver is also critical for maintenance of liver blood flow (83,84). In many situations, peroxynitrite formation, especially in the presence of scavengers such as GSH and NADH, is less damaging than prolonged vasoconstriction and ischemia (84,85).

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Reactive Oxygen and Cell Death Reactive oxygen can cause hepatocellular necrosis without gross cell damage by LPO. The mechanism of this necrotic cell death is linked to the opening of the MPT pore, which causes mitochondrial uncoupling and loss of the membrane potential (86). A significant oxidant stress causes oxidation of mitochondrial pyridine nucleotides and the formation of ROS in mitochondria, both of which increase mitochondrial free Ca2C(10). The MPT can be induced by an increase in mitochondrial Ca2C directly (87) or through the activation of mitochondrial serine proteases (calpains) (88). Cytosolic calpains can induce membrane blebbing by degrading cytoskeletal proteins (89). These events lead to rapid necrotic cell death of hepatocytes within one hour (10,86). Reactive oxygen can also induce cell death through apoptosis (90–94). Generation of superoxide with menadione triggers caspase-mediated apoptotic cell death dependent on activation of the MAPkinase JNK in both hepatocyte cell lines (92) and primary cultured hepatocytes (22). Induction of ERK1/2 attenuated superoxide-induced apoptosis (22,92). In contrast to superoxide, higher levels of H2O2 induce necrotic cell death in hepatocytes (22). The reason for the lack of apoptosis after H2O2 exposure may be the oxidative inhibition of caspases by high concentrations of H2O2 (95). This mechanism may be responsible for the delayed apoptosis of activated neutrophils at an inflammatory site (96). In addition, since only H2O2 and HOCl generated by neutrophils can actually cross cell membranes and induce an intracellular oxidant stress in target cells, this could explain why neutrophils cause necrotic cell death and not apoptosis (37,97). Reactive Oxygen and Gene Transcription The activation of many transcription factors including nuclear factor-kappa B (NF-kB) and activating protein-1 (AP-1) can be induced or modulated by ROS (98,99). Despite extensive experimental data, the molecular mechanism of the redox sensitivity of these transcription factors is still not completely clear (100). However, a number of these transcription factors have thiols, which are critical for their activity, and are known to be regulated at least in part by thioredoxin (Trx) (98,99,101). Pro-inflammatory cytokines (e.g., TNF-a, interleukin (IL)-1), chemokines (e.g., IL-8), adhesion molecules (e.g., ICAM-1, VCAM-1 and E-selectin), and stress genes (e.g., heme oxygenase-1 (HO-1)) are regulated by these redox-sensitive transcription factors. Therefore, reactive oxygen can significantly enhance an inflammatory response thereby indirectly contributing to cell damage. In the liver, TNF-a formation can be modulated by oxidant stress. Antioxidants inhibited endotoxin-induced NF-kB activation and the formation of TNF mRNA and protein in isolated Kupffer cells (102). In support of these results, TNF-a formation in vivo could be prevented by the radical scavenger dimethyl sulfoxide (103) and endotoxin-induced TNF-a generation was three times higher in GPx knockout mice (57). Furthermore, increased nonheme iron concentrations and increased reactive oxygen formation in hepatic macrophages isolated from alcohol-treated animals responded to endotoxin exposure with higher NF-kB activation and elevated transcription of cytokines and chemokines (104). Together these data clearly indicate that gene transcription in Kupffer cells can be modulated by reactive oxygen. However, not only pro-inflammatory genes, but also genes which protect against oxidant stress can be activated by ROS (101). In general, these genes have an antioxidant response element (ARE) within their promoter. AREs are regulated by the transcription factor NF-E2-related factor 2 (Nrf2), which translocates from the cytosol into the nucleus. Nrf2 is normally associated with the cytoplasmic inhibitor, Kelchlike ECH-associated protein 1 (Keap1) in the cytosol. When thiols of Keap1 are oxidized, Nrf2 is released and can enter the nucleus (105). Nrf2 activation occurs after acetaminophen treatment and limits liver cell injury possibly through upregulation of HO-1 and other protective genes (106,107). Induction of HO-1 in hepatocytes during hemorrhagic shock and resuscitation are dependent on the activation of AP-1 (108). Both the activation of AP-1 and the induction of HO-1 could be inhibited by antioxidants (109). HO-1 generates the antioxidant biliverdin and the vasodilator carbon monoxide, both of which may contribute to the hepatoprotective effect of HO-1 induction (109,110).

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Stellate cells regulate sinusoidal blood flow and are, after transformation, the major source of extracellular matrix protein formation leading to fibrosis. There is considerable evidence that reactive oxygen and LPO can stimulate fibrogenesis (69,111). ROS induce or modulate transforming growth factor b1-induced collagen a1 (I) gene expression in vivo (112,113). Furthermore, reactive oxygen can stimulate chemokine transcription in stellate cells (114) thereby enhancing the inflammatory response. Although there is extensive evidence that ROS can directly cause cell death or contribute to modulation of transcription factor activation and gene expression in liver cells, it should be kept in mind that much of the data on ROS toxicity and redox regulation generated in cultured cells have to be interpreted with caution. Gene expression profiles of primary cultured hepatocytes change rapidly after isolation and are considerably different from the intact liver in vivo (115). Since liver cells in culture are in a state of oxidant stress due to hyperoxia (21% oxygen), adaptive changes in response to these artificial culture conditions may modulate the susceptibility to oxidant stress and trigger redox regulation of signaling pathways, which may have limited relevance in vivo (116). ANTIOXIDANT DEFENSE SYSTEMS The continuous formation of reactive oxygen and reactive nitrogen species during physiological functions of liver cells and the potential for a substantially increased oxidant stress under many pathophysiological conditions (Fig. 1) require an effective defense system against these reactive intermediates. Because of the variety of oxygen and nitrogen metabolites formed and their different localization and reactivity, a sophisticated, multilevel network of antioxidant enzymes and small molecules is operative in every liver cell (Fig. 2.). Enzymatic Defense Mechanisms Superoxide is removed by superoxide dismutases (SODs) in the major cellular compartments (117). Cu2C/Zn2C-SOD (SOD1) is mainly located in the cytosol and nuclear matrix whereas Mn3C-SOD (SOD2) is present in the mitochondria (118). Superoxide first reduces the redoxactive metal (Cu2C or Mn3C) which yields molecular oxygen; a second superoxide molecule is then reduced to hydrogen peroxide by the metal ion. The reaction of superoxide with SOD is diffusion limited. The high intracellular SOD levels (approx. 10 mM) keep the steady-state levels of superoxide in the range of 1 to 10 pM (2,4). Since superoxide is not a very toxic molecule by itself and the spontaneous dismutation has the same reaction products, then why is it beneficial to have these high levels of SOD? In addition to the fact that SOD catalyzed dismutation avoids the formation of singlet oxygen (Fig. 1), the main reason for the importance of SOD might be to limit peroxynitrite generation (1,2). Using the rate constants for the reaction of superoxide with SOD (2.4!109/M/sec) and NO (2!1010/M/sec), the rate of disappearance for superoxide is

2H2O Catalase

·– O2

Superoxide dismutases

·

O2, A H2O2, AH2

H2O2

Ferritin Fe2+

NO 2 GSH

GSSG

ONOO – H+

ONOO H

Glutathione peroxidase 2 GSH

GSSG

·

OH

Vitamin E

Fe3+ Glutathione peroxidase

2H2O

NO2– +H2O

LPO

FIGURE 2 Cellular antioxidant defense mechanisms include enzymes for the rapid metabolism of reactive oxygen and nitrogen species, binding proteins for transition metals (e.g., ferritin), and chain-breaking antioxidants (e.g., vitamin E). Abbreviations: GSH, glutathione; GSSG, glutathione disulfide; LPO, lipid peroxidation.

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20,000/sec with SOD (10 mM) and 200/sec with NO (estimated physiological concentrations: 10 nM) (1). Thus, under physiological conditions, SOD prevents peroxynitrite formation. However, if the NO concentrations are increased (10 mM), e.g., during an inflammatory response, the reaction with NO increases to 40,000/sec (1). This indicates that SOD cannot prevent intracellular peroxynitrite formation under inflammatory conditions. This situation is even more critical in the extracellular space where SOD levels are much lower. Hydrogen peroxide (H2O2) formed by SOD is degraded by catalase, GPx, or peroxiredoxin (Prx). Most of the catalase enzyme activity is located in peroxisomes. Mammalian catalase is a hemeprotein which reduces hydrogen peroxide to water by utilizing electrons from either hydrogen peroxide (catalase reaction) or from other small molecules such as ethanol or methanol (peroxidase reaction) (119). Catalase is inducible in the liver by caloric restriction, phenobarbital, and hypolipidemic drugs, e.g., clofibrate (119). The main function of catalase is to metabolize hydrogen peroxide generated by oxidases in peroxisomes. Only under extreme conditions will any relevant amount of hydrogen peroxide escape peroxisomes or will significant amounts of cytosolic hydrogen peroxide be detoxified by catalase (120). Cellular glutathione peroxidase (cGPx-1), one of the four known selenium-dependent GPxs, is ubiquitously expressed (121). The other three forms include GPx-2 (gastrointestinal GPx), GPx-3 (plasma GPx), and GPx-4 (phospholipid hydroperoxide GPx) (121). cGPx-1 is located in the cytosol (75%) and mitochondria (25%). The enzyme contains selenium in the form of selenocysteine, which is critical for the catalytic function (121). cGPx-1 can reduce peroxides, including hydrogen peroxide and organic peroxides (121). Although there is evidence in vitro that cGPx-1 can also reduce peroxynitrite (122), the peroxynitrite-dependent injury after acetaminophen overdose was not enhanced in GPx-1 gene knockout mice (72). In contrast to its low specificity for peroxide substrates, the enzyme requires GSH as cofactor. Glutathione disulfide (GSSG) is rapidly reduced back to GSH by glutathione reductase and NADPH. Since the reductase is the rate-limiting step of this cycle, GSSG accumulates to some degree and can be excreted into bile and plasma (16). The removal of GSSG either by the reductase (O95%) or export from hepatocytes (!5%) protects protein sulfhydryl groups from oxidation by high levels of GSSG. Mitochondria take up and release GSH but are not able to export GSSG (123). Consequently, reduction of GSSG within the mitochondria is the only option to prevent GSSG accumulation. As in the case of acetaminophen toxicity, this cannot always be avoided (13). A member of the glutathione-S-transferase family (GST-B) was identified as the enzyme responsible for the GPx activity in selenium-deficient mice (124). GST-B, which is inducible by selenium deficiency (125), can only metabolize organic hydroperoxide but not hydrogen peroxide (126). However, despite this adaptation, selenium-deficient animals are more susceptible to reactive oxygen-induced liver injury (127). Another enzyme, GPx-4, which selectively uses lipid hydroperoxides as substrates, was identified (128). This enzyme is located in the mitochondria, nuclei, and microsomes and is involved in the metabolism of peroxidized lipids and therefore inhibits the propagation of LPO (129). Trxs and Prxs In mammalian cells, thioredoxin-1 (Trx-1) is located intracellularly in the cytosol, but is also found in the nucleus, and extracellularly in plasma (101). Trx-2 is detected primarily in mitochondria. Trx contains redox-active dithiols which can be oxidized to the corresponding disulfide (Fig. 3). The oxidized form of Trx-1 and -2 is reduced by the respective thioredoxin reductase-1 (TR1) and -2 (TR2) (101). Multiple functions of these enzymes have been reported, which are mainly complementary to the GSH/GPx system (101). One important function of Trx is to reverse oxidative changes in proteins including reduction of protein disulfides (Fig. 3). This function does not only restore protein function, but can also regulate the activity of certain transcription factors. Reduction of critical thiol groups by Trx increases the DNA binding of NFkB and AP-1 after nuclear translocation (130). Trx can function as antioxidant by reducing hydrogen peroxide and peroxynitrite (101,131). However, this requires the combined activity of Trx and peroxiredoxins (Prxs) (Fig. 4) (131). Mammalian cells express at least six Prx isoforms (Prx I–VI) (131). Prx I, II, and VI are located in the cytosol, Prx III and V reside in the mitochondria, and Prx IV is present in the extracellular space. Common to all Prx family

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Trx

Pr-SH

S

Pr-S-S-Pr

S

TR

TR

SeH SH

FAD

TR

TR

Trx SH SH

Se

S

NADPH

NADP

FADH2

+

FIGURE 3 Reduction of protein disulfides by thioredoxin (Trx). Trx is regenerated by thioredoxin reductase, (TR) which uses electrons derived from NADPH. Source: Adapted from Ref. 101.

members is a conserved cysteine residue near the N-terminal, which is the primary cysteine oxidized by hydrogen peroxide. In this reaction, the sulfhydryl group (cys-SH) is oxidized to the sulfenic acid (cys-SOH) as intermediate, which is then reduced by a second cysteine residue to the disulfide (Prx-S–S-Prx; Fig. 4). This disulfide can be reduced by Trx. Due to the high concentration of Prx in the cytosol (0.1–0.8% of soluble proteins), the Prx/Trx system is even more effective than GPx/GSH system in removing low levels of hydrogen peroxide (131,132). However, under certain conditions, Prx-SOH can be further oxidized to the sulfinic acid form (Prx-SO2H), which can no longer be reduced by Trx (131). Prx can be recovered through reduction by another enzyme, sulfiredoxin (Fig. 4) (133). However, this reaction is slow. Thus, hyperoxidation results in a temporary inactivation of Prx, which may allow accumulation of hydrogen peroxide for signal transduction purposes (132). Low Molecular Weight Antioxidants Ascorbate (vitamin C), a-tocopherol (vitamin E), and GSH are examples of low molecular weight antioxidants. a-Tocopherol is the most effective chain-breaking compound in biological membranes (134) However, as for most antioxidants, the intracellular concentrations of a-tocopherol are not high enough to be a relevant hydroxyl radical scavenger. However, it effectively reduces peroxyl radicals (ROO%), one of the less reactive secondary radicals, to the lipid hydroperoxide which can then be metabolized by GPx-4 (134). Thus, a-tocopherol prevents the propagation of the radical chain by avoiding the formation of new alkyl radicals. The a-tocopherol radical can be reduced by ascorbate and thiols such as GSH (135). Ascorbate is regenerated in the aqueous phase by a GSH-dependent dehydroascorbate reductase or a ATP R-SH ADP R-S-S-R

Prx

Sulfiredoxin

SO2H Sulfinic acid

H2O2 H2O2

Prx SH

Prx

Prx-SH

Prx

SOH

S

Sulfenic acid

S Prx

Trx S

S Trx SH SH

FIGURE 4 Detoxification of hydrogen peroxide (H2O2) by peroxiredoxin (Prx). During the catalytic cycle, the sulfenic acid is reduced by Prx-SH and thioredoxin (Trx). However, excess H2O2 may further oxidize Prx to the sulfinic acid, which is inactive. Sulfiredoxin can regenerate the active enzyme in a multistep process. Source: Adapted from Ref. 131.

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NADH-dependent semidehydroascorbate reductase (134). Thus, low molecular weight antioxidants act together to interrupt radical chain reactions and to divert radicals away from sensitive areas, e.g., hydrophobic membranes, to the aqueous phase (136). The critical importance of these compounds as defense systems to protect membranes has been shown in acetaminophen hepatotoxicity. Despite the substantial mitochondrial oxidant stress and peroxynitrite formation (13,14,72), LPO is not a relevant mechanism of cell injury for this drug in vivo (137). However, in animals fed with vitamin E-deficient diet, LPO becomes the predominant injury mechanism with complete destruction of the liver within 1 to 4 hour after administration of acetaminophen (64). GSH is the most important water-soluble antioxidant (138). It is used as a cofactor for GPxs and GSTs and is important for the maintenance of protein sulfhydryl groups. GSH is also an effective scavenger of peroxynitrite and HOCl. The key functional group of GSH is the cysteine sulfhydryl moiety, which is less susceptible to autoxidation than the same moiety in the isolated amino acid. GSH is synthesized intracellularly by two ATP-dependent enzymes: g-glutamylcysteine synthetase and glutathione synthetase. Due to its protease resistant g-glutamyl bond, GSH cannot be degraded by intracellular proteases. Thus, for the cellular turnover, GSH has to be exported from the cell and is degraded by g-glutamyltranspeptidase, which is present on the surface of epithelial cells in the kidney, lung, and intestine and also in the biliary tract. Approximately 90% of the GSH in plasma is supplied by the sinusoidal GSH release of hepatocytes (139). Cellular export proteins for GSH include members of the multidrug resistance-associated proteins (Mrps), which are members of the ATP-binding cassette superfamily, and organic anion-transporting polypeptides (Oatps) (140). Mrps use ATP and Oatps use an electrochemical gradient as the driving force (140). Mrp2 appears to be the main carrier for GSH in the canalicular membrane. Oatp1 and 2 contribute to the export across the sinusoidal membrane (140). Mrp4, located at the basolateral membrane, is responsible for cotransport of GSH with bile acids (141). In addition to the biliary export of GSH, Mrp2 is also the main carrier for GSSG and GSH conjugates (142,143). Much less is known about a sinusoidal GSSG transport. However, functional studies showed a release of GSSG into the sinusoids in the isolated perfused liver (63,144) as well as in vivo (145). Quantitative estimations of GSSG formation and release even during severe oxidant stress indicated that 1% to 5% of all GSSG formed is exported and 95% to 99% is reduced by glutathione reductase (63). Approximately 80% of the exported GSSG is released into bile and 20% into the sinusoid (144). Thus, the biliary GSSG efflux is the most sensitive marker of intracellular oxidant stress. GSH is not only present in the cytosol, but also in other cellular compartments including mitochondria (15% of total cellular GSH) (146). However, GSH is not synthesized in mitochondria but has to be taken up from the cytosol. The carriers involved include the dicarboxylate and 2-oxoglutarate carriers in the inner mitochondrial membrane (147). The transport of GSH into the mitochondrial matrix, which occurs against an electrochemical gradient, is facilitated by the release of phosphate (dicarboxylate carrier) or oxoglutarate (oxoglutarate carrier) (147). A depletion of mitochondrial GSH impairs the detoxification mechanisms in this cell organelle and can lead to increased oxidative injury, loss of mitochondrial function, and cell death (146–149). Reactive oxygen escaping from mitochondria can induce NF-kB activation and promote gene transcription (149). On the other hand, any GSSG formed in the mitochondria through the activity of GPx cannot be exported into the cytosol (122); GSSG has to be reduced or it will accumulate as has been shown during acetaminophen toxicity (13). Metal-Binding Proteins Free radical processes such as LPO are dependent on the availability of redox-active transition metals. Therefore, another defense strategy is to keep metal ions such as Fe2C/Fe3C or CuC tightly bound to transport or storage proteins. Metal-binding proteins include ferritin, transferrin, and lactoferrin for iron, ceruloplasmin for copper, and metallothionein for other metals (150). Because of its large number of cysteines, metallothionein may also act directly or indirectly as antioxidant (151).

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ANTIOXIDANT DEFENSE IN NONPARENCHYMAL CELLS The previous sections described various antioxidant strategies in the liver, i.e., hepatocytes. However, a limited number of studies suggest that similar systems are operative in nonparenchymal cells (152,153). In general, activities of SODs and selenium-dependent GPx are similar in nonparenchymal cells and in hepatocytes (152). Likewise, the GSH contents on a nmol/mg cellular protein basis are very similar in all cell types. However, due to the much smaller cell size of nonparenchymal cells compared to hepatocytes, the total GSH content in nonparenchymal cells represents less than 5% of the liver GSH content (152). Thus, the detoxification capacity for ROS is only a fraction of hepatocytes. Interestingly, Kupffer and endothelial cells adapt differently to an inflammatory stimulus. During endotoxemia, Kupffer cells modulate pro-oxidant pathways resulting in increased superoxide formation (154). In contrast, endothelial cells upregulate SOD and GPx activities, the glucose transporter GLUT1, and the key enzymes of the carbohydrate metabolism (153,154). This response supports the detoxification potential for reactive oxygen in endothelial cells and helps to maintain the integrity of the vascular lining cells. ANTIOXIDANT DEFENSE IN THE VASCULAR SPACE Inflammatory cells can release reactive oxygen and nitrogen species into the vascular space and generate a substantial oxidant stress. Plasma antioxidants include albumin, transferrin, lactoferrin, ceruloplasmin, haptoglobin, urate, ascorbate, vitamin E, bilirubin, and extracellular SOD and GPx activities (reviewed in Ref. 155). However, the main problems of plasma antioxidant systems are the low concentrations of the scavengers and the low activities of the enzymes, which makes them much less effective than their intracellular counterparts. The exceptions are the metal transport proteins which bind metals with high affinity and, therefore, virtually eliminate free iron from plasma (155). Extracellular Cu2C/Zn2C-SOD (EC-SOD, SOD3) (156) and extracellular selenium-dependent GPx (eGPx) (121,157) are proteins distinct from the cellular enzymes. However, their biological relevance is not clear. EC-SOD can bind to surface proteoglycans on the endothelial cell surface (158). It can be speculated that this SOD may protect endothelial cells from a vascular oxidant stress induced by phagocytes and may limit peroxynitrite formation. Plasma eGPx is dependent on the cofactor GSH. In contrast to the mM Km value of the enzyme (157), plasma GSH concentrations are generally in the 5 to 200 mM range (139,159). Thus, it would be expected that eGPx is not very effective in removing peroxides from plasma. A more liver-specific antioxidant defense system has recently been recognized (160,161). GSH can be oxidized in the vasculature during ischemia–reperfusion (34,160) and endotoxemia (159), reflecting a Kupffer cell-induced oxidant stress. In this situation, the plasma GSH levels are substantially increased (100–200 mM in the hepatic vein) due to enhanced release of GSH from hepatocytes (159). These elevated levels, which may even be higher in the space of Disse, are sufficient to make GSH a fairly effective trapping agent for ROS. In support of this conclusion, depletion of plasma GSH concentrations aggravated inflammatory liver injury (160) and an increase above baseline levels protected against injury (161,162). The oxidation of GSH occurred in the vascular space (34) and was not enzymatically catalyzed (160). In vitro studies showed that only H2O2 reacts with GSH and forms GSSG (161). Other relevant oxidants, i.e., hypochlorite and peroxynitrite, are trapped by GSH but generate mainly higher oxidation states such as sulfenic or sulfinic acid and very little GSSG (72). These data are supported by in vivo experiments which showed that NOS inhibitors increased plasma GSH and GSSG levels during hepatic inflammation (85,86). In contrast, NO donors decreased GSH and GSSG concentrations in plasma (86). This suggests that when NO and superoxide are generated in the vascular space, at least some of these molecules react to form peroxynitrite which can be trapped by GSH. Thus, GSH appears to be an important scavenger of reactive oxygen and nitrogen species in the vascular space under inflammatory conditions. Another important antioxidant system in plasma is selenoprotein P (163). The plasma concentrations of this protein are in the range of 25 to 30 mg/mL. The protein contains 7 to 10 selenocysteine and 17 cysteine residues in each molecule (163). Thus, it should be able to

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function as a scavenger for reactive oxygen and peroxynitrite. Since many different organs can produce and release this protein, it was postulated that selenoprotein P acts as an antioxidant in the interstitial space (163). The importance of this selenoprotein P was demonstrated in experiments where the redox-cycling agent diquat or GSH depletion with phorone induced LPO and liver injury in selenium-deficient animals (164,165). Treatment of these animals with a dose of selenium, which was sufficient to restore selenoprotein P levels in plasma but did not affect the low levels of GPx in plasma or liver tissue, prevented LPO and liver injury (164,165). These observations suggest a significant role of selenoprotein P as antioxidant in plasma. REFERENCES 1. Squadrito GL, Pryor WA. Oxidative chemistry of nitric oxide: the role of superoxide, peroxynitrite and carbon dioxide. Free Radic Biol Med 1998; 25:392–403. 2. Koppenol WH. The basic chemistry of nitrogen monoxide and peroxynitrite. Free Radic Biol Med 1998; 25:385–91. 3. Turrens JF. Mitochondrial formation of reactive oxygen species. J Physiol 2003; 552:335–44. 4. Cadenas E, Davies KJA. Mitochondrial free radical generation, oxidative stress and aging. Free Radic Biol Med 2000; 29:222–30. 5. Tatoyan A, Giulivi C. Purification and characterization of a nitric-oxide synthase from rat liver mitochondria. J Biol Chem 1998; 273:11044–8. 6. Tay YM, Lim KS, Sheu FS, et al. Do mitochondria make nitric oxide? no? Free Radic Res 2004; 38(6):591–9. 7. Hauptmann N, Grimsby J, Shih JC, et al. The metabolism of tyramine by monoamine oxidase A/B causes oxidative damage to mitochondrial DNA. Arch Biochem Biophys 1996; 335:295–304. 8. Jaeschke H, Mitchell JR. Mitochondria and xanthine oxidase both generate reactive oxygen species after hypoxic damage in isolated perfused rat liver. Biochem Biophys Res Commun 1989; 160:140–7. 9. Gores GJ, Flarsheim CE, Dawson TL, et al. Swelling, reductive stress, and cell death during chemical hypoxia in hepatocytes. Am J Physiol 1989; 257:C347–54. 10. Nieminen AL, Byrne AM, Herman B, et al. Mitochondrial permeability transition in hepatocytes induced by t-BuOOH—NAD(P)H and reactive oxygen species. Am J Physiol 1997; 272:C1286–94. 11. Kukielka E, Dicker E, Cederbaum AI. Increased production of reactive oxygen species by rat liver mitochondria after chronic ethanol treatment. Arch Biochem Biophys 1994; 309:377–86. 12. Sokol RJ, Winklhofer-Roob BM, Devereaux MW, et al. Generation of hydroperoxides in isolated rat hepatocytes and hepatic mitochondria exposed to hydrophobic bile acids. Gastroenterology 1995; 109:1249–56. 13. Jaeschke H. Glutathione disulfide formation and oxidant stress during acetaminophen-induced hepatotoxicity in mice in vivo: the protective effect of allopurinol. J Pharmacol Exp Ther 1990; 255:935–41. 14. Cover C, Mansouri A, Knight TR, et al. Peroxynitrite-induced mitochondrial and endonucleasemediated nuclear DNA damage in acetaminophen hepatotoxicity. J Pharmacol Exp Ther 2005; 315(2):879–87. 15. Kuthan H, Ullrich V. Oxidase and oxygenase function of the microsomal cytochrome P-450 monooxygenase system. Eur J Biochem 1982; 126:583–8. 16. Lauterburg BH, Smith CV, Hughes H, et al. Biliary excretion of glutathione and glutathione disulfide in the rat. J Clin Invest 1984; 73:124–33. 17. Bajt ML, Knight TR, Lemasters JJ, et al. Acetaminophen-induced oxidant stress and cell injury in cultured mouse hepatocytes: protection by N-acetyl cysteine. Toxicol Sci 2004; 80(2):343–9. 18. Wu D, Cederbaum AI. Oxidative stress mediated toxicity exerted by ethanol-inducible CYP2E1. Toxicol Appl Pharmacol 2005; 207(Suppl. 2):70–6. 19. Jaeschke H, Kleinwaechter C, Wendel A. NADH-dependent reductive stress and ferritin-bound iron in allyl alcohol-induced lipid peroxidation in vivo: the protective effect of vitamin E. Chem Biol Interact 1992; 81:57–68. 20. Smith CV, Hughes H, Lauterburg BH, et al. Oxidant stress and hepatic necrosis in rats treated with diquat. J Pharmacol Exp Ther 1985; 235:172–7. 21. Brigelius R, Anwer MS. Increased biliary GSSG-secretion and loss of hepatic glutathione in isolated perfused rat liver after paraquat treatment. Res Commun Chem Pathol Pharmacol 1981; 31:493–502. 22. Conde de la Rosa L, Schoemaker MH, Vrenken TE, et al. Superoxide anions and hydrogen peroxide induce hepatocyte death by different mechanisms: involvement of JNK and ERK MAP kinases. J Hepatol 2006; 44(5):918–29. 23. Lazarow PB, De Duve C. A fatty acyl-CoA oxidizing system in rat liver peroxisomes: enhancement by clofibrate, a hypolipidemic drug. Proc Natl Acad Sci USA 1976; 73:2043–6.

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139. Lauterburg BH, Adams JD, Mitchell JR. Hepatic glutathione homeostasis in the rat: efflux accounts for glutathione turnover. Hepatology 1984; 4:586–90. 140. Ballatori N, Hammond CL, Cunningham JB, et al. Molecular mechanisms of reduced glutathione transport: role of the MRP/CFTR/ABCC and OATP/SLC21A families of membrane proteins. Toxicol Appl Pharmacol 2005; 204(3):238–55. 141. Rius M, Hummel-Eisenbeiss J, Hofmann AF, et al. Substrate specificity of human ABCC4 (MRP4)mediated cotransport of bile acids and reduced glutathione. Am J Physiol Gastrointest Liver Physiol 2006; 290(4):G640–9. 142. Chen C, Hennig GE, Manautou JE. Hepatobiliary excretion of acetaminophen glutathione conjugate and its derivatives in transport-deficient (TR-) hyperbilirubinemic rats. Drug Metab Dispos 2003; 31(6):798–804. 143. Muller M, Jansen PL. Molecular aspects of hepatobiliary transport. Am J Physiol 1997; 272(6 Pt 1):G1285–303. 144. Jaeschke H. Glutathione disulfide as index of oxidant stress in rat liver during hypoxia. Am J Physiol 1990; 258:G499–505. 145. Adams JD, Lauterburg BH, Mitchell JR. Plasma glutathione and glutathione disulfide in the rat: regulation and response to oxidative stress. J Pharmacol Exp Ther 1983; 227:749–54. 146. Fernandez-Checa JC, Kaplowitz N. Hepatic mitochondrial glutathione: transport and role in disease and toxicity. Toxicol Appl Pharmacol 2005; 204(3):263–73. 147. Lash LH. Mitochondrial glutathione transport: physiological, pathological and toxicological implications. Chem Biol Interact 2006; 163:54–7. 148. Colell A, Garcia-Ruiz C, Miranda M, et al. Selective glutathione depletion of mitochondria by ethanol sensitizes hepatocytes to tumor necrosis factor. Gastroenterology 1998; 115:1541–51. 149. Garcia-Ruiz C, Colell A, Morales A, et al. Role of oxidative stress generated from the mitochondrial electron transport chain and mitochondrial glutathione status in loss of mitochondrial function and activation of transcription factor nuclear factor-kappa B: studies with isolated mitochondria and rat hepatocytes. Mol Pharmacol 1995; 48:825–34. 150. Jaeschke H. In: Sipes IG, McQueen CA, Gandolfi AJ, eds. Antioxidant Defense Mechanisms. Comprehensive Toxicology, Vol. IX Oxford: Elsevier, 1997:181–97. 151. Hidalgo J, Campmany L, Borras M, et al. Metallothionein response to stress in rats: role of free radical scavenging. Am J Physiol 1988; 255:E518–24. 152. DeLeve LD. Glutathione defense in non-parenchymal cells. Semin Liver Dis 1998; 18:403–13. 153. Spolarics Z. Endotoxemia, pentose cycle, and the oxidant/antioxidant balance in the hepatic sinusoid. J Leukoc Biol 1998; 63:534–41. 154. Spolarics Z, Wu JX. Role of glutathione and catalase in H2O2 detoxification in LPS-activated hepatic endothelial and Kupffer cells. Am J Physiol 1997; 273:G1304–11. 155. Halliwell B, Gutteridge JMC. The antioxidants of human extracellular fluids. Arch Biochem Biophys 1990; 280:1–8. 156. Zelko IN, Mariani TJ, Folz RJ. Superoxide dismutase multigene family: a comparison of the CuZnSOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, evolution, and expression. Free Radic Biol Med 2002; 33(3):337–49. 157. Takahashi K, Avissar N, Whitin J, et al. Purification and characterization of human plasma glutathione peroxidase: a selenoglycoprotein distinct from the known cellular enzyme. Arch Biochem Biophys 1987; 256:677–86. 158. Karlsson K, Lindahl U, Marklund SL. Binding of human extracellular superoxide dismutase C to sulphated glycosaminoglycans. Biochem J 1988; 256:29–33. 159. Jaeschke H. Enhanced sinusoidal glutathione efflux during endotoxin-induced oxidant stress in vivo. Am J Physiol 1992; 263:G60–8. 160. Jaeschke H. Vascular oxidant stress and hepatic ischemia/reperfusion injury. Free Radic Res Commun 1991; 12–13:737–43. 161. Liu P, Fisher MA, Farhood A, et al. Beneficial effect of extracellular glutathione against reactive oxygen-mediated reperfusion injury in the liver. Circ Shock 1994; 43:64–70. 162. Bilzer M, Baron A, Schauer R, et al. Glutathione treatment protects the rat liver against injury after warm ischemia and Kupffer cell activation. Digestion 2002; 66(1):49–57. 163. Burk RF, Hill KE. Selenoprotein P: an extracellular protein with unique physical characteristics and a role in selenium homeostasis. Annu Rev Nutr 2005; 25:215–35. 164. Burk RF, Hill KE, Awad JA, et al. Pathogenesis of diquat-induced liver necrosis in selenium-deficient rats: assessment of the roles of lipid peroxidation and selenoprotein P. Hepatology 1995; 21:561–9. 165. Burk RF, Hill KE, Awad JA, et al. Liver and kidney necrosis in selenium-deficient rat depleted of glutathione. Lab Invest 1995; 72:723–30.

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Hepatotoxicity Due to Mitochondrial Injury Dominique Pessayre, Bernard Fromenty, Abdellah Mansouri, and Alain Berson quipe Mitochondries, INSERM, U773, Centre de Recherche BiomØdicale Bichat Beaujon, FacultØ de MØdecine Xavier Bichat, UniversitØ Paris 7 Denis Diderot, Paris, France

INTRODUCTION There are three main mechanisms for drug-induced liver injury. The most frequent mechanism is the formation of reactive drug metabolites that can be directly toxic or can cause immune reactions (1,2). The other is drug-induced impairment of mitochondrial function, which may decrease fat oxidation (thus causing steatosis) and/or energy production (thus causing cell dysfunction or cell death) (3–7). A third mechanism involves the opening of the mitochondrial permeability transition (MPT) pore, causing either necrosis or apoptosis. Although a few parent drugs can trigger this transition (8,9), it is more commonly caused by reactive metabolites, which can lead to MPT either through direct toxicity or through immune reactions (8,9). Thus, even when liver injury is initially due to the formation of reactive metabolites, mitochondrial damage plays a major role in the final mechanisms of cell death (8,9). Therefore, most forms of drug-induced liver lesions initially or secondarily involve mitochondrial injury (9). ORIGIN AND STRUCTURE OF MITOCHONDRIA Some 1.5 to 2 billion years ago, a precursor of present day eukaryotes engaged in a parasitic/symbiotic partnership with a wild bacterium (10–12). This precursor allowed the bacterium to reside and divide within its cytoplasm. In exchange, the bacterium used the emerging oxygen atmosphere to completely degrade fuels into CO2 and water, thus providing the host with considerable energy (11,12). Like their bacterial ancestors, mitochondria have two membranes. A circular outer membrane surrounds the intermembrane space, while an inner membrane with inner folds (the mitochondrial cristae) limits the mitochondrial matrix (12). Although most of the initial bacterial genes have been transferred to the nucleus of the host, mitochondria have retained a small genome, located in the matrix (12). Each cell contains many copies of this circular, double-stranded genome, as there are several copies of mitochondrial DNA (mtDNA) in a single mitochondrion and many mitochondria per cell (10). This mtDNA encodes 13 polypeptides of the respiratory chain, while nuclear DNA encodes the remaining respiratory polypeptides and all other mitochondrial proteins. These proteins are synthesized in the cytoplasm (usually with a mitochondrial targeting presequence) and are then imported into the mitochondrial membranes or the matrix, where the presequence is cleaved (10). The authors of this chapter have relationships with the following corporations: Dominique Pessayre has been a consultant for Abbott, Actelion, Astellas, Astra Zeneca, Aventis, Bayer, Ciba-Geigy, DuPont, EliLilly, Fournier Laboratories, Glaxo SmithKline, Helsinn Healthcare, Hoechst-Marion-Roussel, HoffmanLaRoche, Novartis, Parke-Davis, Pfizer, Rhone Poulenc-Rorer, Roussel-UCLAF, Sanofi-Synthelabo, Servier Laboratories, Solvay, TROPHOS and Wyeth. Bernard Fromenty has been a consultant for Aventis and Bristol Myers Squibb. The laboratory of Dominique Pessayre and Bernard Fromenty at INSERM has benefited from research grants from Astra Zeneca, Beaufour, Bristol-Myers-Squibb, Cassenne Laboratories, Ciba Geigy, Helsinn Healthcare, Hoffmann-LaRoche, Knoll, Roger Bellon, Sanofi-Synthelabo, Schering-Plough, Servier Laboratories, and Sigma-Tau.

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Respiratory chain polypeptides are located in the inner membrane, except for cytochrome c (cyt. c), which is targeted to the intermembrane space, but is mostly associated with cardiolipin on the inner membrane (13). Although the initial shortening of very-long and long-chain fatty acids is mediated by enzymes located in the inner membrane, most of the other enzymes involved in fatty acid b-oxidation and the tricarboxylic acid cycle are located in the mitochondrial matrix (10). ROLES OF MITOCHONDRIA Fat Oxidation and Energy Production Mitochondria play a major role in fat oxidation and energy production. The entry of long-chain fatty acids into mitochondria is modulated by carnitine palmitoyltransferase I (CPT I, Fig. 1). This outer membrane enzyme is inhibited by malonyl-CoA, which is the first step in the synthesis of fatty acids (14). This inhibition normally ensures a reciprocal relationship between fatty acid synthesis and the mitochondrial uptake, and oxidation of fatty acids (14). Once fatty acids are taken up by mitochondria, they are targeted towards oxidation (14). Fatty acyl-CoAs (FA-CoAs) are split by b-oxidation cycles into acetyl-CoA subunits, which can either form ketone bodies or can, like other fuels, be completely degraded to CO2 by the tricarboxylic acid cycle (Fig. 1). The NADH and FADH2 that are generated by both b-oxidation and the tricarboxylic acid cycle are then re-oxidized by the mitochondrial respiratory chain (12). This regenerates the NADC and FAD necessary for other cycles of fuel oxidation (Fig. 1), and also initiates the process of energy production (3). NADH and FADH2 transfer their electrons to the first complexes of the respiratory chain (12). Although a fraction of these electrons react with oxygen to form reactive oxygen species

Uptake CPT I

Synthesis FFA FA-CoA

CYTOSOL TG

VLDL

Secretion

MITOCHONDRIA O2

H2O

e–

Cyt. c oxidase

NAD+, FAD

β-Oxidation

MATRIX

Tricarboxylic acid cycle

e–

NADH, FADH2 ADP CO2

INTERMEMBRANE SPACE

H+

ATP ANT

O2 O2– H+ ATP synthase

FIGURE 1 Fat metabolism and energy production in hepatocytes and mitochondria. FFA are either synthesized in the liver or transferred from the intestine or adipose tissue. Long-chain FFA form FA-CoA thioesters, which either enter mitochondria, a step regulated by CPT I, or are esterified into triglycerides that are stored in the cytoplasm or secreted as VLDLs. Inside mitochondria, the FA-CoA thioesters are cut and oxidized by the b-oxidation and tricarboxylic acid cycles, generating NADH and FADH2 that transfer electrons to the respiratory chain. Although part of these electrons react with oxygen to form the superoxide anion radical and other ROS, most electrons migrate up to cyt. c oxidase, where they safely combine with oxygen and protons to form water. The transfer of electrons along the respiratory chain is associated with the extrusion of protons from the mitochondrial matrix into the intermembrane space. The reentry of protons in the matrix through ATP synthase transforms ADP into ATP, which is then extruded by the ANT, in exchange for cytosolic ADP. Abbreviations: FA-CoA, fatty acyl-CoA; CPT I, carnitine palmitoyltransferase I; VLDLs, very lowdensity lipoproteins; cyt. c, cytochrome c; ANT, adenine nucleotide translocator; TG, triglycerides; FFA, free fatty acids.

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(ROS) as discussed further on, most electrons migrate all the way along the respiratory chain, up to cyt. c oxidase, where four electrons react in quick succession with oxygen and protons to safely form water, without any release of ROS (Fig. 1). During this transfer of electrons along the respiratory chain, protons are extruded from the mitochondrial matrix into the intermembrane space (Fig. 1) (12). This creates a large electrochemical potential across the inner membrane whose potential energy is then used to generate ATP. When ADP is high, protons reenter the matrix through the F0 portion of ATP synthase, causing the rotation of a molecular rotor within the F 1 portion of ATP synthase and the conversion of ADP into ATP (Fig. 1). The adenine nucleotide translocator (ANT) then extrudes the formed ATP, in exchange for cytosolic ADP (12). Thus, mitochondria turn fat and other fuels into CO2 and water, providing most of the cell’s ATP. However, the price of this oxidative phosphorylation is a high formation rate of ROS (12). Mitochondrial ROS Formation A small fraction of the electrons transferred to complexes I and III of the respiratory chain directly react with oxygen to form the superoxide anion radical (Fig. 1) and other ROS, including peroxynitrite (12). Mitochondria are the main source of ROS in the cell (15). Although mitochondria actively repair ROS-mediated mtDNA lesions (16), mtDNA is very sensitive to ROS-induced damage, due to its proximity to the inner membrane (the main cellular source of ROS) and the absence of protective histones. ROS oxidize mtDNA bases, and these modifications can cause errors during either mtDNA replication (17) or repair (18), leading to point mutations. ROS also cause mtDNA strand breaks, which can cause mtDNA deletions (19). The mitochondrial theory of aging suggests that the accumulation of these mtDNA lesions eventually decreases mtDNA-encoded polypeptide synthesis and the transfer of electrons along the respiratory chain (15). Whenever the flow of electrons is blocked at some point along the respiratory chain, then the respiratory chain components, which are located upstream to the block, become overly reduced and directly transfer their electrons to O2, thus increasing the basal formation of ROS (20). This increased ROS formation further increases ROS-induced damage to mtDNA, causing more impairment of respiration and more ROS formation (10). This vicious circle could explain why mtDNA deletions (21), and some point mutations (22), which are uncommon before the age of 40 years, exponentially accumulate during old age. The high mitochondrial formation rate of ROS, and the oxidative damage it causes to mtDNA and nuclear DNA, is probably one important mechanism of aging (10,23). Another role of mitochondria is to modulate cell death. Cell Survival and Cell Death The partnership with parasitic bacteria may have been initially rather dangerous, because wild bacteria tend to proliferate when they are well fed. The problem was solved when the transcription and replication of the bacterial/mitochondrial genome were placed under the control of nuclear genes, thus transforming wild bacteria into tame mitochondria (24). Before this could occur, however, the host may have found ways to partially invalidate the proliferating bacteria, while bacteria may have disabled any mutant host that took advantage of this control mechanism. The present day sequel to this double warfare is a shared decisionmaking process, in which both symbiotic partners play a role in regulating cell death (25). Mitochondria are involved in this decision through the regulation of the permeability of their outer membrane and also through either closure or opening of a pore in the inner membrane, called the MPT pore (Fig. 2) (9,25). Pore closure allows the cell to survive, while pore opening causes cell death (Fig. 2) (9). Pore opening allows massive reentry of protons through the inner membrane, causing collapse of the mitochondrial membrane potential and interrupting mitochondrial ATP synthesis. If the pore opens quickly in all mitochondria, severe ATP depletion prevents apoptosis (an energy-requiring process) and causes necrosis (Fig. 2) (26). Pore opening also causes matrix expansion, rupture of the spherical outer membrane,

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Normal mitochondria

MPT in all mitochondria

MPT in some mitochondria

MITO

ATP

MPT H+

No ATP

MITO

ATP

MITO

ATP

MPT H+

No ATP

MPT H+

No ATP

Cytochrome c Caspases

LIFE

NECROSIS

APOPTOSIS

FIGURE 2 Regulation of life and death by the MPT pore. Pore closure allows cell survival, whereas pore opening causes cell death. MPT causes the reentry of protons into the mitochondrial matrix, thus bypassing ATP synthase and preventing ATP formation. Pore opening also causes matrix expansion, outer membrane rupture and release of mitochondrial cyt. c from the intermembrane space into the cytosol. If the pore opens in all mitochondria, decreased ATP synthesis causes ATP depletion and necrosis. If the pore only opens in some mitochondria, unaffected mitochondria keep synthesizing ATP (thus avoiding necrosis), while permeabilized mitochondria release cytochrome c, which activates caspases, causing apoptosis. Abbreviations: MPT, mitochondrial permeability transition; cyt. c, cytochrome c; MITO, mitochondria.

herniation of the inner membrane and matrix through the outer membrane gap, and cyt. c release from the intermembrane space into the cytosol (27,28). If the pore only opens in some mitochondria, unaffected mitochondria keep synthesizing ATP, while permeabilized mitochondria release cyt. c, which activates caspases in the cytosol, causing apoptosis (Fig. 2) (9,27,28). CONSEQUENCES OF IMPAIRED MITOCHONDRIAL FUNCTION Primary Impairment of Fatty Acid Oxidation Primary impairment of fatty acid oxidation causes hepatic steatosis (Fig. 3) (3). Let us recall that two main pathways can remove fat from the liver. First, the mitochondrial b-oxidation and tricarboxylic acid cycles can degrade fatty acids into CO2 and water. Second, triglycerides (TG) can be secreted as very low-density lipoprotein (VLDL). In the lumen of the endoplasmic reticulum (ER), microsomal triglyceride transfer protein (MTP) lipidates apolipoprotein B (Apo B) into triglyceride-rich VLDL particles, which then follow vesicular flow to the plasma membrane to be secreted. Drugs can cause steatosis by impairing mitochondrial b-oxidation (3), by inhibiting MTP activity and thus hepatic VLDL secretion (29), or, quite frequently, by concomitantly inhibiting both MTP activity and mitochondrial b-oxidation (30). Mild inhibition of mitochondrial b-oxidation is not enough; severe impairment is required to trigger steatosis (3). In this case, the free fatty acids (FFA) which are taken up by the liver or synthesized within the liver are insufficiently oxidized by the deficient mitochondria and are instead esterified into TG, which accumulate within the cytoplasm of hepatocytes, thus causing hepatic steatosis (Fig. 3) (3). Acute impairments of fatty acid b-oxidation typically cause microvesicular steatosis (3). In this peculiar form of steatosis, numerous tiny lipid vesicles leave the nucleus in the center of the cell and give the hepatocyte a “foamy”, “spongiocytic” appearance. However, when b-oxidation is more chronically impaired, mixed forms of steatosis can also occur.

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Microvesicular steatosis

CYTOSOL Lipid peroxidation Fat vesicle

FFA

TG

FA-CoA

Time Macrovacuolar steatosis

Toxic to mitochondria

MITOCHONDRIA Fatty acid β-oxidation CO2

NAD+ FAD

O2 e–

NADH FADH2

+

H

MATRIX

ADP

H2O Respiration

e– H+

INTERMEMBRANE SPACE

Steatosis

Gluconeogenesis

ATP

Glucose

Cell dysfunction

FIGURE 3 Primary impairment of fatty acid oxidation and its consequences. The impairment of FA-CoA b-oxidation in mitochondria increases FFA, and TG, which accumulate as small lipid vesicles that can progressively coalesce into larger vacuoles. The impairment of fat oxidation deprives the cell of an important source of energy during fasting. It also inhibits gluconeogenesis so that hypoglycemia may occur. In extrahepatic organs, this limits pyruvate oxidation as an alternative source of energy. Finally, the toxic effects of lipid peroxidation products, FFA and FA-CoA on mitochondrial further decrease energy formation, thus causing cell dysfunction. Abbreviations: FFA, free fatty acids; FA-CoA, fatty acyl-CoA; TG, triglycerides.

Some hepatocytes are then filled with tiny lipid vesicles, while others exhibit large fat vacuoles or both small vesicles and larger vacuoles. These associations and transitions suggest that tiny lipid vesicles can progressively coalesce into larger vacuoles (Fig. 3). Indeed, prolonged causes of steatosis rather tend to cause macrovacuolar steatosis (3). In this other form of steatosis, hepatocytes are distended by a single, large vacuole of fat, displacing the nucleus to the periphery of the cell. Primary impairment of fatty acid oxidation also secondarily impairs mitochondrial energy production (3). Fatty acid oxidation represents the main cellular source of energy between meals, and subjects whose mitochondrial b-oxidation is impaired, do not tolerate fasting (31). The inhibition of b-oxidation secondarily inhibits hepatic gluconeogenesis (3). Fasting may trigger hypoglycemia in these patients (31), thus hampering energy production from this still oxidizable fuel. Furthermore, fasting also causes massive adipose tissue lipolysis, thus flooding the liver with FFA, which are not oxidized by the deficient mitochondria and therefore accumulate in hepatocytes (3). FFA and their dicarboxylic acid derivatives inhibit and uncouple mitochondrial respiration, further decreasing energy production (3). Furthermore, steatosis leads to lipid peroxidation whose reactive products damage the respiratory chain and mtDNA (10). All these effects may cause an energy deficit in cells (Fig. 3), which may cause cell dysfunction in different organs. These patients may develop mild liver failure (sometimes with renal failure and pancreatitis) and severe brain dysfunction, causing coma and death (31). Primary Impairment of Mitochondrial Respiration Impairment of mitochondrial respiration decreases energy formation and, depending on the severity of the deficit, can cause either cell dysfunction or cell death (Fig. 4) (3). Moderate impairment of respiration only causes cell dysfunction. Severe impairment of respiration can cause liver cell death, cholestasis, and fibrosis (32–34).

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CYTOSOL FA-CoA

TG

β-oxidation

NAD+ FAD

Pyruvate oxidation

NADH FADH2 Acetyl-CoA CO2

MATRIX

MITOCHONDRIA H2O

O2 e− e− H+

Respiration e−− e−

O2–

e

H+

ADP

O2

INTERMEMBRANE SPACE ATP

Steatosis

Lactic acidosis

Cell dysfunction Cell death

Oxidative stress mtDNA aging

FIGURE 4 Primary impairment of respiration and its consequences. The block in the flow of electrons in the respiratory chain decreases ATP formation, thus causing cell dysfunction or cell death. It also causes the accumulation of electrons in upstream respiratory chain complexes, thus increasing ROS formation, which can cause oxidative stress and aging of mtDNA. Finally, the block in electron flow also decreases the re-oxidation of NADH into NADC. A first consequence of the lack of NADC is to decrease the b-oxidation of fatty acids, thus causing steatosis. A second consequence is to decrease the oxidation of pyruvate by the pyruvate dehydrogenase complex. Pyruvate is not degraded and, due to the excess in NADH, it is increasingly reduced into lactate, which can trigger lactic acidosis. Abbreviation: mtDNA, mitochondrial DNA.

Impairment of respiration also blocks the transfer of electrons along the respiratory chain, causing over-reduction of the respiratory chain components located upstream, which react with oxygen to form the superoxide anion radical (Fig. 4) (20). The increased ROS formation could damage mtDNA and further impair respiration. ROS could also contribute to the appearance of necroinflammation and fibrosis, as discussed further on. Finally, severe impairment of respiration secondarily impairs mitochondrial b-oxidation (35). Normally, the NADH, which is formed by b-oxidation, is then re-oxidized by the mitochondrial respiratory chain, thus regenerating the NADC required for fatty acid b-oxidation. When respiration is severely impaired, NADC regeneration is insufficient to sustain b-oxidation (35). This secondarily impairs b-oxidation and causes microvesicular steatosis (Fig. 4) (3). The lack of NADC also inhibits the oxidation of pyruvate by the pyruvate dehydrogenase complex. Instead, due to high NADH levels, pyruvate is reduced into lactate, whose accumulation can trigger lactic acidosis (Fig. 4). Common Features Thus, primary impairment of b-oxidation causes both microvesicular steatosis and secondary energy deficiency (causing cell dysfunction), while primary impairment of respiration causes both energy deficiency (leading to cell dysfunction) and secondary impairment of b-oxidation (causing microvesicular steatosis). Thus, irrespective of the initial defect, drug-induced mitochondrial dysfunction may associate features of both steatosis and cell dysfunction. However, each of these features can predominate according to the initial mechanism.

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DIVERSE MECHANISMS IMPAIRING MITOCHONDRIAL FUNCTION Drugs may impair mitochondrial function by several mechanisms (3–7). They can degrade mtDNA, inhibit or terminate mtDNA replication, inhibit mtDNA transcription, impair the stability of mtDNA transcripts, inhibit the translation of mtDNA transcripts, inhibit or uncouple mitochondrial respiration, sequester CoA (which is required for fatty acid activation before b-oxidation), and/or directly inhibit b-oxidation enzymes, ATP synthase, or the ANT. Degradation of mtDNA Alcohol Ethanol abuse causes oxidative stress in the liver (36). The metabolism of ethanol first into acetaldehyde and then into acetate by alcohol dehydrogenase and aldehyde dehydrogenase is associated with the conversion of NADC into NADH (37). This conversion increases the NADH/NADC ratio and thus the NADPH/NADPC ratio, which can reduce ferric iron into ferrous iron, a potent generator of the hydroxyl radical (37). Other ROS sources are increased levels of the ROS-generating cytochrome P450 (CYP) 2E1 (38) and increased mitochondrial ROS formation in hepatocytes (39), as well as the activation of the NADPH oxidase of Kupffer cells (10). ROS cause oxidative damage to mitochondrial lipids (40), proteins (41), and DNA (41,42) in intoxicated animals. A single, high dose of ethanol causes extensive degradation and depletion of hepatic mtDNA within two hours in mice (42,43). Depletion of mtDNA also occurs in skeletal muscle, heart, and brain (43). Depletion of mtDNA can be prevented by 4-methylpyrazole, which blocks ethanol metabolism, or by melatonin, vitamin E, or ubiquinone, three antioxidant drugs (42,43). After a single alcoholic binge, damaged mtDNA molecules are quickly repaired or resynthesized de novo, and mtDNA depletion is only transient (42,43). In contrast, after four days of daily alcohol administration, the accumulation of non-repaired (bulky) DNA lesions on mtDNA inhibits the resynthesis of mtDNA, due to the limited number of intact mtDNA templates. As a consequence, mtDNA depletion lasts for several days after interruption of alcohol intoxication (44). The repetition of mtDNA strand breaks during chronic alcoholism can also cause mtDNA deletions. The prevalence of hepatic mtDNA deletions is increased in alcoholics with microvesicular steatosis, but not in patients with alcoholic hepatitis or cirrhosis (45,46). The latter conditions increase liver cell turnover (47,48), which could eliminate mutated mtDNA genomes if cells with a high proportion of mutated genomes fail to replicate and/or are progressively eliminated through apoptosis. When ethanol ingestion is stopped, alcoholinduced mtDNA deletions disappear quickly in white blood cells (49), which have a quick cell turnover. Alcohol abuse can cause diverse liver lesions. Macrovacuolar steatosis seems to be due to a combination of different mechanisms (50). These include an increased hepatic expression of sterol regulatory element-binding protein-1 causing increased hepatic fatty acid synthesis (51), decreased hepatic expression of MTP, which may sometimes limit hepatic lipoprotein secretion (52), and finally, impaired hepatic fatty acid oxidation (53). The latter effect is due to the excessive reduction of NADC into NADH during the metabolism of ethanol. The lack of NADC then slightly impairs mitochondrial b-oxidation and markedly inhibits the tricarboxylic acid cycle (53). Microvesicular steatosis may be due to a combination of this mild inhibition of b-oxidation, together with severe ROS-dependent damage to mitochondrial lipids, proteins, and DNA, thus further impairing mitochondrial function (46). Necroinflammation and fibrogenesis seem to be mediated by increased intestinal absorption of endotoxin, activation of toll-like receptor 4 on macrophages (54), induction of inducible nitric oxide synthase (55), increased formation of tumor necrosis factor-a (TNF-a), and finally increased apoptosis, which triggers transforming growth factor-b synthesis and collagen synthesis by stellate cells (56). Acetaminophen (Paracetamol) The inadvertent or deliberate intake of large doses of acetaminophen leads to an extensive formation of N-acetyl-p-benzoquinone-imine. This electrophilic metabolite depletes hepatic glutathione (GSH) and protein thiols, increases cell calcium, damages mitochondria, increases

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the formation of ROS including peroxynitrite, activates c-Jun N-terminal kinase (JNK), and causes MPT and liver cell necrosis, as discussed later on (see section MPT). Hepatic mtDNA was rapidly depleted after a paracetamol overdose in mice, whereas nuclear DNA, albeit partially fragmented, was not significantly depleted (57). The rapid depletion of mtDNA may be due to the DNA damage caused by peroxynitrite and other ROS (57). The absence of a rapid rebound in mtDNA levels may suggest impaired mtDNA resynthesis. Possible reasons could be the development of cell necrosis and the poisoning of topoisomerase II by N-acetyl-pbenzoquinone-imine (58). Impairment of mtDNA Replication by Drugs that Inhibit Topoisomerases and/or Bind to DNA The replication of mtDNA can be inhibited by drugs, which either intercalate between DNA bases or bind strongly to DNA grooves (59). Furthermore, intercalating drugs can inhibit and/or poison DNA topoisomerases, and these effects can also impair mtDNA replication (59). Topoisomerases play an important role in DNA replication and transcription (60). While type I topoisomerases act as monomers and only cut one strand of DNA, type II topoisomerases act as dimers or multimers and cut both strands of DNA. These enzymes cut the phosphodiester DNA backbone by forming a covalent bond between the liberated phosphorus of DNA and a tyrosine of the enzyme. Normally, this reaction is quickly reversible: topoisomerases transiently cut DNA, thus allowing DNA strand(s) to cross the gap, and then promptly reseal the DNA gap. Several antibacterial drugs (4-quinolones, novobiocine) or anticancer drugs (amsacrine, etoposide, anthracyclines, ellipticines, and actinomycins) are topoisomerase inhibitors (60). Although a few inhibitors inhibit the initial cutting of DNA by topoisomerases, most inhibitors instead inhibit mostly the resealing of DNA. These drugs, which are said to “poison” topoisomerases, increase the number of enzyme-bound DNA complexes (60). These complexes are called “cleavable complexes,” because detergents can destabilize the complexes so that the DNA ends are no longer held together, and the protein-linked DNA breaks are revealed. Both the inhibition and the “poisoning” of topoisomerases are deleterious to cells. The collision of a transcription complex or a replication fork against the accumulated topoisomerase-associated DNA breaks interrupts RNA or DNA synthesis, and can lead to real (non-topoisomerase– bound) double-strand breaks and to gene translocations, which can trigger apoptosis and/or cancer (61). Because mitochondria contain both a type I topoisomerase (62) and a bacterial-like type II topoisomerase (63), topoisomerase inhibitors or poisons can affect the replication of mtDNA. Quinolone Antibiotics The 4-quinolone antibiotics inhibit gyrase (a bacterial type II topoisomerase) and also the mitochondrial type II topoisomerase (64). Ciprofloxacin blocks the resealing of mtDNA breaks, causing accumulation of protein-linked double-strand mtDNA breaks (65). Ciprofloxacin and nalidixic acid progressively decrease mtDNA in cultured mammalian cells, impairing mitochondrial respiration and cell growth (64). 4-Quinolone antibiotics can cause cholestasis, steatosis, and necrosis in treated patients (66,67), and both trovafloxacin and alatrofloxacin were taken off the market because of an unacceptable risk of fulminant liver failure. However, it remains unknown whether mtDNA depletion actually occurs in humans or experimental animals treated with quinolone antibiotics. Alternative mechanisms for quinolone-induced hepatitis could include lysosomal membrane permeabilization and MPT (68), altered expression of mitochondrial proteins (69), and the occurrence of immune reactions in some patients (70). Tacrine and Tamoxifen The reversible, cholinesterase inhibitor, tacrine, has been used for the symptomatic treatment of Alzheimer’s disease. Monitoring of serum alanine aminotransferase (ALT) activity and tolerance-dependent, stepwise escalation of the doses were recommended, because the drug increased ALT activity in 50% of recipients, usually after about six weeks of treatment (71). The weak base tacrine is taken up by mitochondria, where it may cycle back and forth across the

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mitochondrial inner membrane, uncoupling respiration, and wasting energy without ATP production (72). First-pass metabolism in the liver ensures low exposure in other organs, explaining why the liver is selectively injured (72). The electrophoretic accumulation of tacrine within mitochondria also explains why this organelle is a selective target (72,73). Tacrine intercalates between mtDNA bases, poisons topoisomerases, and decreases the synthesis of mtDNA in mice (73). This leads to a progressive depletion of mtDNA in mice, eventually followed by the death of a few hepatocytes by necrosis or apoptosis (73). Tamoxifen is an antiestrogen, which is used in the treatment of advanced breast cancer, but can cause steatohepatitis, particularly in overweight women (74). This cationic amphiphilic drug accumulates electrophoretically within mitochondria, where it directly inhibits mitochondrial respiration and mitochondrial b-oxidation, thus causing steatosis (75). In addition, tamoxifen intercalates between DNA bases, and inhibits topoisomerases and mtDNA synthesis (75). Tamoxifen progressively depletes hepatic mtDNA in mice (75). Pentamidine Pentamidine is used in the prevention and treatment of Pneumocystis carinii infections. Although pentamidine inhibits the topoisomerases of P. carinii and trypanosomes, it has little effect on mammalian topoisomerases (59). However, pentamidine binds to the minor groove of duplex DNA and can deplete mtDNA in mammalian cells in vitro (59). Pentamide may also inhibit mitochondrial translation (76). Ethidium Bromide, Ditercalinium, Dequalinium, and 1-Methyl-4-Phenylpyridinium Ion Ethidium bromide, ditercalinium, and dequalinium are cationic drugs, which are concentrated electrophoretically in the mitochondrial matrix, where they intercalate between mtDNA bases, and inhibit mtDNA synthesis, to cause progressive depletion of mtDNA (77–79). 1-Methyl-4-phenylpyridinium ion (MPPC) is an oxidative metabolite of a mitochondrial toxin, which causes Parkinson’s disease in humans. This positively charged metabolite accumulates into mitochondria where it inhibits mtDNA synthesis and depletes mtDNA (80). Polyamine Analogs and Methylglyoxal bis(Guanine Hydrazone) The polycationic nature of polyamines leads to their strong interaction with the successive phosphate groups of the DNA backbone, on the major groove of DNA. This strong binding causes conformational changes in DNA (59). The polycationic nature of these compounds also causes their electrophoretic accumulation within the mitochondrial matrix (59). Methylglyoxal bis(guanine hydrazone) and several polyamine analogs, including N1,N12-bis(ethyl)spermine and N1,N8-bis(ethyl)spermidine, have been shown to progressively decrease mtDNA levels in cultured cell lines (81,82). Impairment of mtDNA Replication by Nucleoside Analogs Incorporated into mtDNA 2 0 ,3 0 -Dideoxynucleosides and Abacavir Several 2 0 ,3 0 -dideoxynucleosides are used in patients with the human immunodeficiency virus. These include 3 0 -azido-2 0 ,3 0 -dideoxythymidine (zidovudine, AZT), 2 0 ,3 0 -dideoxycytidine (zalcitabine, ddC), 2 0 ,3 0 -dideoxyinosine (didanosine, ddI), 2 0 ,3 0 -didehydro-3 0 -deoxythymidine (stavudine, d4T), and (K)-2 0 -deoxy-3 0 -thiacytidine (lamivudine, 3TC). A related molecule is abacavir, although this analog contains a cyclopentene–methanol moiety instead of a dideoxyribose moeity. The normal 5 0 -hydroxyl group found in deoxyribose is present in the sugar analog of these diverse molecules, allowing the formation of the triphosphate derivative and the incorporation of the analog into a nascent chain of DNA (Fig. 5). However, the normal 3 0 -hydroxyl group of deoxyribose is absent in these analogs. Once a single molecule of the analog has been incorporated, the DNA molecule lacks a 3 0 -hydroxyl group. No other nucleotide can be incorporated, interrupting DNA replication (Fig. 5) (83,84). The effects of these dideoxynucleosides depend on the ability of various polymerases to incorporate them into DNA. The human immunodeficiency virus reverse transcriptase can perform this incorporation, impairing reverse transcription of the viral RNA (83). In contrast, the

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Normal deoxyribose (5'-OH and 3'-OH)

5'OH

P

Dideoxyribose (5'-OH but no 3'-OH)

P

(No 3'OH-end)

mtDNA Interruption of mtDNA replication

mtDNA depletion Acquired mitochondrial cytopathy

FIGURE 5 Interruption of mtDNA replication by dideoxynucleosides. Once DNA polymerase g has incorporated a single molecule of dideoxynucleoside into a growing chain of mtDNA, the DNA now lacks a 3 0 hydroxyl group, and no other nucleotide can be incorporated, unless the analogue can be removed by the proofreading activity of polymerase g. Interruption of mtDNA replication can cause mtDNA depletion and an acquired mitochondrial cytopathy. Abbreviation: mtDNA, mitochondrial DNA.

DNA polymerases, which act in the nucleus, barely perform this incorporation, thus allowing the therapeutic use of these analogs (84). However, DNA polymerase g, which acts in mitochondria, also incorporates the dideoxynucleoside analogs into growing chains of mtDNA. This incorporation terminates mtDNA replication (Fig. 5) (85,86), unless the dideoxynucleotide analog is eventually removed by the proofreading, 3 0 -5 0 -exonuclease activity of polymerase g (87) However, polymerase g has a poor proofreading efficiency with some dideoxynucleotides, which are correctly base-paired, although they have an abnormal phospho-sugar analog backbone. Thus, the rate for the incorporation of these analogs into mtDNA is much faster than the rate for their removal (87). Furthermore, after one analog molecule has been eventually removed, and elongation has transiently resumed, another molecule of the analog is likely to be incorporated again further on. This new blocking molecule will have to be removed again, and so on. The end result is to considerably slow down the rate of efficient, complete mtDNA replication (87). Even in post-mitotic tissues, there is a constant turnover of mitochondria, requiring basal replication of mtDNA. When mtDNA replication is markedly slowed down, mtDNA levels may progressively decrease (Fig. 5). For reasons, which are not fully understood, different dideoxynucleosides tend to have differential effects on mtDNA in diverse organs. Although AZT can occasionally cause mtDNA depletion in the liver (88), it has been suggested that the so-called “D-drugs,” namely ddC, ddI, and d4T are more likely to cause hepatic mtDNA depletion than AZT, 3TC, or abacavir (89). In a patient with lactic acidosis after treatment with both ddI and d4T, all mitochondrial complexes were markedly decreased, except for complex II, which is only encoded by nuclear DNA (90). The impaired synthesis of mtDNA-encoded respiratory chain polypeptides can partially block the flow of electrons in the respiratory chain (91). Whenever the flow of electrons in the respiratory chain is partially blocked, the accumulation of electrons in complex I and complex III increases their reaction with oxygen, and the formation of the superoxide anion. In the case of AZT, this effect could be further aggravated by the inhibitory effect of this analog on the ANT (92). The lack of the ANT in knock out mice blocks the exchange of mitochondrial ATP for cytosolic ADP (20). This prevents the reentry of protons through ATP synthase and causes a high mitochondrial potential, which blocks the flow of electrons in the respiratory chain, and increases mitochondrial ROS formation, thus causing precocious appearance of mtDNA deletions (20). In mice, AZT increased peroxide formation by hepatic mitochondria and caused extensive oxidation of guanosine into 8-hydroxydeoxyguanosine in mouse liver mtDNA (93). Like AZT-treated mice, asymptomatic HIV-infected patients treated with AZT had higher urinary excretion of 8-hydroxydeoxyguanonise than untreated patients (94).

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As previously discussed, oxidative damage to mtDNA can cause both point mutations and deletions. In patients treated with diverse nucleoside reverse transcriptase inhibitors, heteroplasmic point mutations were shown to accumulate in the mtDNA of peripheral blood cells (95). Likewise, mtDNA deletions were more prevalent in the sperm of patients treated with various nucleoside analogs for 12 months or more than in those treated for lesser lengths of time (96). In one of these patients, multiple deletions were present after six months of treatment, whereas none had been found before treatment (96). In another patient treated with 3TC, d4T, and saquinavir, most of the hepatic mtDNA existed as six distinct deleted forms (97). This patient developed lactic acidosis, although hepatic mtDNA was not depleted (97). Thus, once mtDNA deletions and point mutations have developed, severe mitochondrial dysfunction may occur, even though tissue mtDNA level (which is the sum of normal and mutated mtDNA) may be normal. Tissues, whose mtDNA is decreased or abnormal, attempt to increase both mtDNA replication and transcription by inducing compensatory increases in mitochondrial biogenesis. In human volunteers treated for two weeks with either d4T/3TC or AZT/3TC, a decrease in mtDNA-encoded messenger RNAs was associated with an increased expression of peroxisome proliferator receptor g coactivator 1 (PGC1), nuclear respiratory factor 1, and mitochondrial transcription factor A in adipose tissue (98). All three factors are master regulators of mitochondrial biogenesis (99) and may help attenuate the adverse effects of nucleoside reverse transcriptase inhibitors. Indeed, an increase in the number of muscle mitochondria is observed in patients with AZT-induced myopathy (86). Mitochondrial proliferation can also occur in the liver (100). In one patient with d4T-induced lactic acidosis, extensive hepatic mitochondrial proliferation led to tightly packed mitochondria on electron microscopy, and to an aspect of oncocytic hepatocytes (cells with a pink granular cytoplasm) on light microscopy (100). Fialuridine and Ganciclovir Clinical trials testing the efficacy of fialuridine in patients with chronic hepatitis B were interrupted after several patients developed microvesicular steatosis and unmanageable lactic acidosis, sometimes with pancreatitis, neuropathy, or myopathy (101). This complication was unexpected because fialuridine possesses both a 5 0 -hydroxyl group and a 3 0 -hydroxyl group, so that the incorporation of a single molecule of fialuridine into DNA should not immediately terminate mtDNA replication. However, when several adjacent molecules of fialuridine are successively incorporated, further activity of DNA polymerase g is inhibited, decreasing mtDNA replication and mtDNA levels (102). Ganciclovir is mainly used in the treatment of cytomegalovirus infections (103). This nucleoside analog is converted into ganciclovir-monophosphate by viral kinases encoded by cytomegalovirus, varicella/zoster, and herpes simplex viruses (103). Cellular kinases then form ganciclovir-triphosphate, which can lead to the incorporation of ganciclovir nucleotides into growing chains of DNA (103). The acyclic, pseudo-sugar analog present in ganciclovir has two hydroxyl groups so that its incorporation does not terminate DNA replication. However, incorporated ganciclovir molecules may cause DNA helix perturbations, which may block the next DNA replication cycle, when the ganciclovir-modified DNA chain has to serve as a replication template (103). Ganciclovir is also incorporated in mtDNA and triggers mtDNA depletion, ultrastructural mitochondrial lesions and steatosis, together with apoptosis (104). Decreased Synthesis and Stability of mtDNA Transcripts in Cells Treated with Interferon-a Interferon-a is used in patients with chronic viral hepatitis B or C. Interferons induce 2 0 ,5 0 -oligoadenylate synthases, which synthesize 2 0 ,5 0 -oligoadenylates in the presence of double-stranded RNAs (105). The 2 0 ,5 0 -oligoadenylates activate RNase L, which, furthermore, is induced by interferons-a and -b (105). RNase L cleaves RNAs (105). This may cleave the nuclear DNA-encoded mRNA of mitochondrial transcription factor A (106), thus decreasing mtDNA transcription and the synthesis of mitochondrial mRNAs (107). Furthermore, RNase L is also present in mitochondria, where it degrades mitochondrial mRNAs (108). Thus, interferon-a decreases both the synthesis and also the stability of mitochondrial transcripts

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(107,108), which can eventually decrease mtDNA-encoded respiratory chain polypeptides and mitochondrial respiration in cultured cells (109). Although it is not known whether these in vitro effects also occur in interferon-treated patients, it is noteworthy that some adverse effects of interferon-a, such as minor blood dyscrasias, myalgias, paresthesias, convulsions or depression (110), or hepatic steatosis (111), resemble those observed in mild forms of inborn mitochondrial cytopathies. Decreased Translation of mtDNA Transcripts Erythromycins Erythromycins are amphiphilic cationic drugs, which accumulate in acidic compartments including lysosomes, where they can inhibit phospholipases to cause phospholipidosis (112). These antibiotics bind to the 50S ribosomal subunit of bacteria to inhibit the transfer of amino acids from the aminoacyl-tRNA to the peptide chain (113). Erythromycins can also inhibit mitochondrial protein synthesis, to some extent (114). They can cause megamitochondria (115) and can trigger sensorineural hearing loss (116). Finally, erythromycins are transformed into reactive metabolites, which may covalently bind to proteins, thus forming neoantigens, which may trigger immunization and hepatitis (117). It is unknown whether mitochondrial effects can contribute to erythromycin-induced cholestatic or mixed hepatitis (possibly by causing bile duct lesions and the release of immunizing neoantigens). Chloramphenicol and Thiamphenicol Chloramphenicol and thiamphenicol also bind to the 50S ribosomal subunit to inhibit protein synthesis in both bacteria and mitochondria (118). Mitochondrial dysfunction is probably involved in the reversible bone marrow suppression induced by chloramphenicol (119). However, it remains unknown whether other effects of chloramphenicol, including aplastic anemia and cholestatic hepatitis, are due to mitochondrial dysfunction and/or reactive metabolite formation (119). Sequestration of CoA or Carnitine and/or Direct Inhibition of b-Oxidation Aspirin Aspirin is quickly hydrolyzed into salicylic acid, which is activated into salicylyl-CoA on the outer mitochondrial membrane (120). Extensive salicylyl-CoA formation sequesters extramitochondrial CoA, leaving insufficient CoA to activate long-chain fatty acids and preventing their entry into mitochondria and b-oxidation (121). Even though lethal overdoses of aspirin frequently cause microvesicular steatosis (122), therapeutic doses do not, although they can trigger Reye’s syndrome in children with viral infections. Interferon-a, TNF-a, and nitric oxide are released during viral infections, and all impair mitochondrial function. As discussed above, interferon-a decreases the synthesis and stability of mitochondrial transcripts (107,108). Nitric oxide reversibly inhibits mitochondrial respiration (123) and may open the MPT pore (124). TNF-a also inhibits respiration and opens the MPT pore (8). Nevertheless, viral infections rarely cause Reye’s syndrome, suggesting that these endogenous substances do not impair mitochondrial function enough to trigger the disease. However, if children take aspirin during a viral illness, the added effects of salicylate on mitochondrial function may sufficiently impair mitochondrial function to trigger the syndrome in some children. The potentiating effect of aspirin is based on the following evidence. In the past, 93% of children with Reye’s syndrome had received aspirin during an acute viral illness (125), and children with Reye’s syndrome had received aspirin more frequently than those with similar viral diseases not followed by Reye’s syndrome (126). When aspirin use was advised against in feverish children, there was a corresponding decline in the use of aspirin and the incidence of Reye’s syndrome in the United States (127). The few residual cases of Reye’s syndrome now mainly occur in children with another potentiating factor, namely a latent genetic defect in mitochondrial b-oxidation enzymes (128).

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∆4-Ene-valproate

CYP

Mito

Valproate

Uncoupling Ca2+ Other stimuli

Mito

∆2, ∆4-Diene-valproyl-CoA

Valproyl-CoA

MPT

Inactivation of β-oxidation enzymes?

Sequestration of CoA

Cell death

Fatty acid β-oxidation

Pyruvate oxidation

FIGURE 6 Mitochondrial effects of valproate. Valproate is extensively transformed into valproyl-CoA in mitochondria, thus sequestering intramitochondrial CoA and impairing mitochondrial fatty acid b-oxidation and pyruvate oxidation. Valproate is also desaturated by CYP into D4-ene-valproate, which forms D4-ene-valproyl-CoA in mitochondria, and then D2,D4-diene-valproyl-CoA, an electrophilic metabolite, which may inactivate b-oxidation enzymes. Finally, valproic acid uncouples mitochondrial respiration, thus favoring MPT and cell death. Abbreviations: CYP, cytochrome P450; MPT, mitochondrial permeability transition; Mito, mitochondria.

Yet another effect of salicylate is to slightly uncouple mitochondrial respiration (121) and open the MPT pore (129), as discussed further on. The latter effect could contribute to Reye’s syndrome and could also be involved in the spotty liver cell death observed in patients receiving high therapeutic doses of aspirin (130). Valproic Acid Valproic acid is a branched-chain fatty acid used in several forms of seizures. Like natural short-chain fatty acids, valproic acid can enter mitochondria without undergoing previous activation. Inside the mitochondria, valproic acid is extensively transformed into valproyl-CoA (131). The sequestration of intramitochondrial CoA inhibits the b-oxidation of long-, medium-, and short-chain fatty acids (Fig. 6) (131,132). The lack of CoA may also inhibit pyruvate dehydrogenase which requires CoA as a necessary cofactor. This may explain why valproate markedly decreases mitochondrial respiration from pyruvate, although it has little effect on the respiration supported by malate and glutamate (133). Several other effects contribute to the mitochondrial toxicity of valproate (Fig. 6). CYPs 2C9 and 2A6 desaturate the outer carbons of valproate, forming 4-ene-valproate (134). This metabolite is activated into 4-ene-valproyl-CoA inside mitochondria (135,136). The first dehydrogenation step of the b-oxidation cycle then forms 2,4-diene-valproyl-CoA, which is a chemically reactive metabolite that may inactivate b-oxidation enzymes (136,137). This CYPinvolving pathway could explain why the hepatotoxicity of valproate is enhanced by the concomitant administration of the CYP-inducing antiepileptic drugs, phenytoin and carbamazepine, which increase the formation of 4-ene-valproate (138). Finally, valproic acid has an uncoupling effect, which favors MPT pore opening (Fig. 6), as discussed further on. An asymptomatic increase in serum aminotransferase activity, which normalizes with either dose reduction or drug discontinuation, is frequent during administration of this antiepileptic agent (139). A much less frequent side effect is a Reye’s-like syndrome (140), which occurs mainly (but not exclusively) in very young children and between the first and fourth month of treatment. Histologically, centri- and midzonal microvesicular steatosis is associated with centrizonal necrosis, and sometimes cirrhosis (140). This combination of microvesicular fat and liver cell death may be related to the dual effect of valproic acid, which both inhibits mitochondrial b-oxidation and opens the MPT pore (7), as discussed further on.

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The inhibition by valproate of both fatty acid b-oxidation and pyruvate-supported respiration may explain why valproate administration can aggravate both inborn b-oxidation defects (141,142) and mitochondrial cytopathies (143–145). Panadiplon Development of the anxiolytic drug panadiplon was interrupted due to increased serum transaminases activity during clinical trials (146). Panadiplon was converted into cyclopropane carboxylic acid, which sequesters coenzyme A and carnitine, and inhibits the mitochondrial b-oxidation of fatty acids (146). Pivampicillin Administration of pivampicillin results in the extensive formation and urinary excretion of pivaloylcarnitine, thus depleting free carnitine and inhibiting fatty acid oxidation (147). Tetracyclines Tetracycline itself and the various tetracycline derivatives produce extensive microvesicular steatosis of the liver in experimental animals (148,149). This is due to the dual effect of these antibiotics, which inhibit both the mitochondrial b-oxidation of fatty acids (148,149) and the hepatic secretion of VLDLs (Fig. 7) (149,150). The latter effect occurs at doses, which do not inhibit protein synthesis (150), and may be due to the inhibition of MTP activity by tetracycline (30). At presently administered oral doses, tetracycline may produce minor degrees of hepatic steatosis of no clinical severity in humans. However, severe microvesicular steatosis has occurred in the past during the intravenous administration of high doses of tetracycline (151). Predisposing factors included impaired renal function (which may decrease tetracycline elimination) and pregnancy (which may impair mitochondrial function, as discussed later on) (151). The syndrome usually appeared after four to ten days of tetracycline infusion. Microvesicular steatosis has also been observed after intravenous administration of several other tetracycline derivatives (3,151). 2-Arylpropionate Nonsteroidal Anti-inflammatory Drugs Several nonsteroidal anti-inflammatory drugs are 2-arylpropionate derivatives. Hepatic injury due to these drugs consists of hepatitis and/or microvesicular steatosis of the liver. The latter condition has been observed with pirprofen, naproxen, ibuprofen, and ketoprofen (152–155).

Fat Apo B

Fat

ER

MTP

Microsomal triglyceride transfer protein VLDL

VLDL

VLDL Fat

Fat

VLDL secretion Tetracycline, Amiodarone, Amineptine, Tianeptine, Pirprofen

MITO

CO2

FIGURE 7 Dual effects of tetracycline and some other steatogenic drugs on fat oxidation and hepatic lipid secretion. Several drugs inhibit the b-oxidation of fatty acids in MITO, and also inhibit MTP activity. The latter effect decreases the lipidation of Apo B into VLDL in the lumen of the ER, thus decreasing hepatic VLDL secretion. Abbreviations: MITO, mitochondria; MTP, microsomal triglyceride transfer protein; Apo B, apolipoprotein B; VLDL, very low-density lipoprotein; ER, endoplasmic reticulum.

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2-Arylpropionates have an asymmetric carbon and exist as either S(C)- or R(K)enantiomers. Only the S(C)-enantiomer inhibits prostaglandin synthesis, whereas only the R(K)-enantiomer is converted into the acyl-CoA derivative. Nevertheless, both the S(C)enantiomer and the R(K)-enantiomer of ibuprofen inhibit the b-oxidation of medium- and short-chain fatty acids (156). Inhibition of b-oxidation has also been observed with pirprofen, tiaprofenic acid, and flurbiprofen (157). Amineptine and Tianeptine These antidepressant drugs have a tricyclic moiety and a heptanoic side chain. They rarely cause immunoallergic hepatitis due to the formation of reactive metabolites by CYP (158,159). Exceptionally, they can also cause microvesicular steatosis, due to impaired b-oxidation (3). Both amineptine and tianeptine are metabolized by the b-oxidation of their heptanoic side chain, forming the 5-carbon and 3-carbon derivatives (160,161). In the presence of these drugs, mitochondria are thus exposed to C7, C5, and C3 analogs of natural fatty acids. These analogs reversibly inhibit the b-oxidation of medium- and short-chain fatty acids (162,163). Female Sex Hormones About 1 in 13,000 pregnant women develop microvesicular steatosis during the last trimester of pregnancy (164). Untreated, the disease progresses to coma, kidney failure, and hemorrhage, and leads to the death of the mother and child in 75% to 85% of cases. In contrast, rapid termination of pregnancy results in the delivery of a healthy child and rapid resolution of the mother’s disease in most cases (165). Both pregnancy and the administration of estradiol and progesterone alter mitochondrial ultrastructure and function in mice (166,167). However, these effects are mild; b-oxidation is only slightly impaired and microvesicular steatosis does not develop in these mice (166,167). Similarly, most human pregnancies do not cause acute fatty liver. Therefore, additional factors are probably required to trigger this syndrome. Partial deficiency of long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD), which is part of the trifunctional membrane-bound b-oxidation enzyme, has been reported in some women with acute fatty liver of pregnancy (168). Mothers with a single defective LCHAD allele, who are unlucky enough to marry a heterozygous carrier, and then to conceive a fetus with two defective alleles develop the disease (168), whereas those who bear an unaffected child usually have uncomplicated pregnancies. Although the fetus itself may not use fatty acids for energy production, the human placenta metabolizes fatty acids and may therefore release toxic 3-hydroxy, and dicarboxylic fatty acids in the feto-maternal circulation during LCHADnegative conceptions (169). However, the high frequency of LCHAD deficiency in some publications of acute fatty liver of pregnancy does not reflect the real prevalence of this association in an unselected series of acute fatty liver of pregnancy. We did not detect the most prevalent LCHAD mutation in any of 14 consecutive women with histologically confirmed acute fatty liver of pregnancy (170). These findings suggest that the LCHAD deficiency is a possible, albeit uncommon, cause of acute fatty liver of pregnancy in unselected cases. Furthermore, although one in 70 French persons are heterozygous for the A985G mediumchain acyl-CoA dehydrogenase (MCAD) mutation (171), which accounts for 89% of all deficient MCAD alleles, none of the 14 women with acute fatty liver of pregnancy carried the A985G MCAD mutation (170). These observations should not prevent screening for various defects in mitochondrial b-oxidation in women with acute fatty liver of pregnancy (particularly those with recurrent disease or with a still-born or unhealthy child). However, together with epidemiological data, they do suggest that such defects are rarely involved. Indeed, with few exceptions, when pregnancy is terminated early, the child is healthy and the acute fatty liver of pregnancy does not recur in subsequent pregnancies. Drugs, food, stress, infections, an autoimmune reaction triggered by the foreign child, or placental ischemia associated with preeclampsia may perhaps trigger the syndrome in different women.

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Glucocorticoids Glucocorticoids inhibit acyl-coenzyme A dehydrogenases and produce microvesicular steatosis of the liver in mice (172). Glucocorticoids can cause steatosis (151) and steatohepatitis (173) in humans. Calcium Hopantenate Calcium hopantenate has caused several cases of Reye’s-like syndrome in Japan (174). Pantothenic acid is a constituent of CoA, and calcium hopantenate may decrease CoA and inhibit mitochondrial b-oxidation (174). Inhibition of b-Oxidation and/or Respiration: Role in Steatohepatitis or Hepatitis Amiodarone, 4,4 0 -Diethyaminoethoxyhexestrol, and Perhexiline Amiodarone, 4,4 0 -diethylaminoethoxyhexestrol, and perhexiline are cationic amphiphilic compounds. They have a lipophilic moiety and an amine function, which can become protonated (and thus positively charged). This structure is responsible for the two liver lesions that occur with these drugs, namely lysosomal phospholipidosis and steatohepatitis (7). The uncharged, lipophilic form of these drugs crosses the lysosomal membrane (175). In the acidic lysosomal milieu, the unprotonated drug molecule is protonated and trapped, since it cannot cross back through the lysosomal membrane. It reaches high intralysosomal concentrations and forms non-covalent but tight complexes with phospholipids, thus hampering the action of intralysosomal phospholipases (175). Phospholipids are not degraded, and the phospholipids–drug complexes progressively accumulate as myelin-like figures in enlarged lysosomes (175). Although phospholipidosis is frequent and may be constant in patients receiving these drugs, it appears to have no clinical consequence, since it often occurs without clinical symptoms or biochemical disturbances (176). However, the cationic amphiphilic structure of these drugs also causes impaired mitochondrial function (Fig. 8) (177–181). The unprotonated, lipophilic form easily crosses the mitochondrial outer membrane and is protonated in the acidic intermembranous space (177–181). This positively charged, protonated form is electrophoretically “pushed” inside mitochondria by the high electrochemical potential existing across the inner mitochondrial membrane, and thus reaches high intramitochondrial concentrations (177–181). It remains unknown whether this transport occurs through inner membrane transporters or directly through the lipid bilayer, thanks to charge delocalization. The high intramitochondrial concentrations of these drugs inhibit b-oxidation (causing steatosis) and also block the transfer of electrons along the respiratory chain (177–181). Respiratory chain components become overly reduced and increasingly transfer their electrons to oxygen to form the superoxide anion radical and other ROS (181). This increased ROS formation causes lipid peroxidation (181), and, like ethanol (10), could increase cytokine production (10). Both lipid peroxidation and cytokines can cause steatohepatitis lesions (10). Prolonged administration of these three drugs can cause typical steatohepatitis lesions, with steatosis, necrosis, Mallory bodies, a mixed inflammatory cell infiltrate (containing neutrophils), fibrosis, and even cirrhosis (182–187). Tamoxifen As already mentioned, the antiestrogen, tamoxifen is a cationic amphiphilic drug, which is electrophoretically transported into the mitochondrial matrix, where it achieves high concentrations that directly inhibit both mitochondrial b-oxidation and respiration (75). In addition, tamoxifen intercalates between DNA bases, and inhibits mtDNA synthesis, which depletes mtDNA in mice (75). Tamoxifen can cause steatohepatitis (188,189) particularly in overweight women (74). Although tamoxifen has been shown to impair lysosomal acidification (190) and cause intralysosomal storage of polar lipids after administration of high doses to animals (191), phospholipidosis does not seem to have been reported in human livers.

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A (Amiodarone, perhexiline, DEAEH)

Respiratory chain

A

H+

AH+ + −

CYTOSOL

MITOCHONDRIA INTERMEMBRANE SPACE MATRIX

High concentrations Respiratory chain β-oxidation

Respiration e−

Steatosis

e−− e e−

O2

O2:

ROS

FIGURE 8 Effects of amphiphilic cationic drugs on mitochondrial function. After crossing the outer membrane, the uncharged tertiary amine (A) of amiodarone, perhexiline or DEAEH is protonated in the acidic intermembrane space. The positively charged molecule (AHC) is then electrophoretically pushed by the mitochondrial membrane potential into the matrix. High intramitochondrial concentrations inhibit both b-oxidation (causing steatosis) and oxidative phosphorylation (increasing the formation of ROS). The latter may oxidize fat deposits, causing lipid peroxidation, which, together with ROS-induced cytokine production, could cause steatohepatitis. Abbreviations: DEAEH, diethylaminoethoxyhexestrol; ROS, reactive oxygen species.

Buprenorphine This morphine analog is used as a substitution drug in heroin addicts. The sublingual route is used, to partly prevent extensive first pass metabolism in the liver. At high concentrations, buprenorphine inhibits both mitochondrial b-oxidation and respiration in rat hepatocyte mitochondria (192). Much lower concentrations are observed in humans, and the drug is usually well tolerated. However, cytolytic hepatitis and steatosis have been observed in a few patients (193). Predisposing factors could include intravenous buprenorphine misuse (resulting in higher concentrations) and concomitant exposure to viruses, other drugs or ethanol, all of which impair mitochondrial function (193). Benzarone and Benzbromarone Benzarone and benzbromarone, despite their structural analogy with amiodarone, are not cationic drugs but phenolic compounds. Benzarone and benzbromarone both uncouple and inhibit respiration at low concentrations (194). Although these two drugs also impair mitochondrial b-oxidation, this effect requires higher concentrations (194). In humans, benzarone and benzbromarone can cause hepatocellular liver injury (195,196). Antimalarial Drugs Chloroquine and most other antimalarial drugs are cationic compounds which accumulate in the acidic vacuole of the malaria parasite to disrupt its function due to alkalinization (197). Chloroquine also accumulates at high concentrations in the lysosomes of the host and can cause phospholipidosis (198). Chloroquine, primaquine, and quinine have been shown to impair respiration in rat liver mitochondria (199). In addition, some antimalarial drugs, including primaquine (200) and amodiaquine (201) form reactive metabolites. Metabolic activation may play an important role in amodiaquine-induced agranulocytosis and hepatitis (202).

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Inhibition of ATP Synthase or the ANT When cells lack energy, cytosolic ADP is high, and ADP enters the mitochondrial matrix through the ANT. The intramitochondrial ADP stimulates the reentry of protons through ATP synthase, and the energy, which is liberated by this reentry, is harnessed in the synthesis of ATP from ADP. The ANT then extrudes the formed ATP, in exchange for cytosolic ADP. Concomitantly, the decrease in the membrane potential due to the reentry of protons allows more electrons to flow within the respiratory chain, thus increasing mitochondrial respiration. Like drugs blocking electron flow within the respiratory chain, the inhibitors of the ANT or of ATP synthase also inhibit the mitochondrial respiration, which is triggered by ADP. However, unlike respiratory chain inhibitors, the inhibitors of the ANT or of ATP synthase do not inhibit the respiration triggered by uncouplers. The latter compounds stimulate respiration by causing the reentry of protons directly through the inner membrane, thus bypassing the need for either the ANT or ATP synthase. Inhibitors of ATP Synthase ATP synthase is a wonderful biological engine (203). Its transmembrane, F0 portion contains a nonrotating moiety, and a rotor made of c subunits (c-ring rotor). The reentry of protons through a channel located at the junction of the non-rotating moiety and the c-ring, triggers the rotation of the c-ring and the attached g stalk. The g stalk rotates within a cap made of three pairs of alternating a and b subunits (a3b3 cap), which is kept immobile by the a/b2/d stator (203). The stalk is asymmetrical. When it rotates within the a3b3 cap, it causes rhythmic deformations of the catalytic b subunits, which may alternatively tighten around ADP and Pi, close to form ATP and fully open to release ATP. Importantly, ATP synthase is a reversible machine. When the mitochondrial membrane potential is low, for example during ischemia, ATP synthase can function in the reverse mode. Its ATPase activity then consumes the meager amounts of ATP formed by glycolysis to pump protons into the intermembrane space, in an attempt to partially restore the mitochondrial membrane potential. This ATP-depleting effect can be attenuated by the binding of a matrix protein, which is termed the inhibitor of F1ATPase (IF1) (204). During ischemia, anaerobic glycolysis causes lactate formation and matrix acidification, which triggers the binding of IF1 to the F1 moiety of ATP synthase thus inhibiting its ATPase activity. This attenuates the selfish use of cell ATP by de-energized mitochondria. Organotin compounds (205) and several natural toxins, such as apoptolidin (206), aurovertin (207), citreoviridin (208), efrapeptins (209), oligomycin, and venturicidin (205), are potent inhibitors of ATP synthase. These toxins block aerobic ATP formation by mitochondria, and can damage cells, which cannot synthesize enough ATP through glycolysis. However, these toxins also block anaerobic ATP consumption by hypoxic mitochondria. Paradoxically, they can transiently protect hypoxic cells from necrosis by preventing ATP synthase from further depleting ATP in hypoxic cells (210). ATP synthase activity is also inhibited by high, supraphysiological concentrations of estrogens (211), and by several phenolic phytochemicals present in human diet, such as resveratrol, curcumin, genistein, or quercetin (212). Inhibitors of the ANT The ANT exchanges ATP for ADP across the inner membrane, and also modulates MPT (213). The ANT achieves two different conformational states (214). Atractyloside (or carboxyatractyloside) binds to, and stabilizes the cytosolic c-state of the transporter (214) and can trigger MPT (11). Bongkrekic acid binds to, and stabilizes the matrix m-state of the transporter (214), and inhibits MPT (11). Both inhibitors block the transport of adenine nucleotide and can decrease cell ATP. The impairment of the ANT also increases the membrane potential, blocks electron flow, causes the over-reduction of respiratory chain complexes, and increases mitochondrial ROS formation, thus causing mtDNA lesions (20).

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Like bongkrekate, the HIV protease inhibitor, nelfinavir binds the ANT on its matrix side, and inhibits MPT and apoptosis (215). In contrast, long-chain acyl-CoA esters bind and inhibit ANT from both sides of the inner membrane (216), and can trigger MPT (217). Uncoupling of Mitochondrial Respiration by Protonophoric Drugs Several drugs can translocate protons from the intermembrane space into the mitochondrial matrix (Fig. 9). This occurs with either cationic or acidic compounds, the latter including both phenolic and carboxylic compounds. Cationic drugs, such as amiodarone, 4,4 0 -diethylaminoethoxyhexestrol, perhexiline, tacrine, and buprenorphine, have an amine function (A) that is protonated (AHC) in the acidic intermembrane space. This positively charged molecule is electrophoretically pushed across the inner membrane by the membrane potential (72,181). It releases its proton in the alkaline matrix, thus reforming the uncharged molecule (A), which may cross back through the inner membrane lipid bilayer, to be protonated again in the intermembrane space, ready for another cycle of proton translocation (72). Carboxylic and other acidic compounds, including natural FFA and several nonsteroidal anti-inflammatory drugs (218,219), cross the inner membrane lipid bilayer in the uncharged form (R–COOH) and release HC in the alkaline matrix. Then the anionic form (R–COOK) is pulled into the intermembrane space by the mitochondrial membrane potential. This second crossing of the inner membrane may occur through anion transporters (220,221). Inside the acidic intermembrane space, the uncharged acid (R–COOH) is formed again, ready for another cycle of proton translocation. The reentry of protons into the intermembrane space increases basal respiration (Fig. 9). As explained above, the flow of electrons along the respiratory chain is coupled with the extrusion of protons from the mitochondrial matrix into the intermembranous state (Fig. 1).

NORMAL

UNCOUPLING

MATRIX

Consequences

DRUG

MATRIX

Heat generation

H+ O2 e−

O2

H2O Respiratory chain

e–

ATP

Respiration Electron flow

H+

H+ ADP

H2O

ATP synthase

ADP ATP H+

H+

ATP Cell dysfunction

IM

IM

Cell death

FIGURE 9 Opposite effects of uncouplers on mitochondrial respiration and ATP formation. Uncouplers translocate protons across the IM. The reentry of protons into the mitochondrial matrix decreases the membrane potential, thus unleashing the flow of electrons in the respiratory chain and increasing mitochondrial respiration. However, ATP synthase is bypassed, and this increased respiration produces heat instead of ATP. Energy deprivation can cause cell dysfunction or cell death. Abbreviation: IM, inner membrane.

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Once a high membrane potential is achieved, this high potential blocks the flow of electrons in the respiratory chain. Uncouplers cause the reentry of protons into the mitochondrial matrix and decrease the mitochondrial membrane potential (72,181), unleashing the flow of electrons in the respiratory chain and increasing basal oxygen consumption (Fig. 9). However, ATP synthase is bypassed, and this increased respiration occurs in vain, to produce heat instead of ATP. Severe uncoupling decreases cell ATP and can cause cell dysfunction or cell death (72). Unlike respiratory chain inhibitors, which impair the re-oxidation of NADH into NADC and may secondarily inhibit mitochondrial b-oxidation (which requires NADC), uncouplers increase respiration, the regeneration of NADC, and tend to increase mitochondrial b-oxidation (179). Steatosis does not occur, unless the drug has other effects on mitochondrial function. Induction of Uncoupling Protein 2 Another mechanism that could uncouple respiration is induction of the uncoupling protein 2 (UCP2). When UCP2 is highly expressed in yeast cells, this inner mitochondrial membrane protein has an uncoupling effect (222). UCP2 could sustain the cycling of FFA across the inner membrane, by permitting the transport of the charged anionic form (RCOOK) from the mitochondrial matrix into the intermembrane space, where RCOOK is protonated to RCOOH, which can cross the lipid bilayer to translocate one proton into the matrix (223). The UCP2 mRNA is expressed in hepatocytes and increased by FFA, ROS, lipopolysaccharide, TNF-a, interleukin-1, and peroxisome proliferator–activated receptor agonists in rodents (224,225). It is still unclear, however, whether the UCP2 protein itself or some other related protein is expressed in hepatocytes (226). Any uncoupling due to UCP2 is probably too mild to cause deleterious pathological changes, although it might accelerate hepatic fatty acid b-oxidation, and might also aggravate the toxicity of ATP-depleting substances (224). MITOCHONDRIAL PERMEABILITY TRANSITION Some drugs directly cause MPT, while other drugs first form reactive metabolites which then trigger permeability transition in the setting of direct toxicity or immune reactions. Parent Drugs Betulinic Acid and Lonidamide Betulinic acid is a pentacyclic triterpene proposed as an anticancer drug. Betulinic acid triggers MPT in isolated mitochondria, even without added calcium, and causes apoptosis in treated cells (227). Betulinic acid also inhibits topoisomerases I and II, which may also contribute to its proapoptotic effects (228). Lonidamide is another investigational antineoplastic agent. Lonidamide targets the ANT and triggers MPT (in the absence of added calcium) and apoptosis (229,230). Hydrochloroquine Mitochondrial effects can also occur as a consequence of lysosomal effects. Indeed, the lysosomotropic antimalarial drug, hydrochloroquine, releases cathepsin B from lysosomes, and causes the translocation of Bcl-2-associated protein x (Bax) from the cytosol to mitochondria, where Bax associates with its analog, Bak to trigger MPT and apoptosis (231). Peripheral Benzodiazepine Receptor Ligands The peripheral benzodiazepine receptor (PBR) is located on the outer mitochondrial membrane and interacts with the MPT pore. In different experimental conditions, PBR ligands can either inhibit or augment apoptosis by modulating MPT. 4 0 -Chlorodiazepam and another selective PBR ligands decreased the mitochondrial membrane potential and triggered apoptosis in hepatic stellate cells (232). In fibroblasts, PBR ligands, albeit not toxic by themselves, increased the MPT and cell death caused by proapoptotic substances, such as TNF-a (233). Likewise, a low concentration of the PBR ligand, alpidem, was not toxic to hepatocytes but increased TNFa-mediated toxicity (234).

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Salicylic Acid, Valproic Acid, Diclofenac, Nimesulide, and Other Acidic Drugs Carboxylic and other acidic drugs can transport protons across the inner mitochondrial membrane. The reentry of protons into the matrix decreases the mitochondrial membrane potential, unleashes the flow of electrons in the respiratory chain and increases mitochondrial respiration, which increases mitochondrial NADH consumption. The resulting NADH depletion can impair ROS detoxification. Let us recall that mitochondrial NADP transhydrogenase uses NADH and the mitochondrial membrane potential to regenerate NADPH from NADPC(235). In turn, NADPH is needed by GSH reductase to reduce GSH disulfide back to GSH (235). Finally, reduced GSH is needed by mitochondrial GSH peroxidase to inactivate the hydrogen peroxide formed by manganese superoxide dismutase (235). Therefore, an insufficient availability of NADH and/or a marked decrease in the mitochondrial membrane potential can impair the detoxification of hydrogen peroxide, and can help trigger MPT (235–237). The opening of the pore in a few mitochondria then allows the egress of NADH, NADC, NADPH, and NADPC, thus further impairing ROS inactivation (237). Pore opening also causes matrix swelling and outer membrane rupture, which releases cyt. c from the damaged mitochondria, thus increasing succinate-supported ROS formation by the affected mitochondria (238). Finally, MPT pore opening also releases calcium from mitochondria. Both the increased ROS formation and calcium release can then trigger MPT in other mitochondria, resulting in a self-amplifying, propagating wave. Thus, several anionic uncouplers, including salicylic acid and valproic acid, have been shown to facilitate calcium-triggered MPT in isolated mitochondria (239). In isolated rat hepatocytes incubated with very low concentrations of albumin, the acidic, nonsteroidal anti-inflammatory drug, nimesulide, uncouples mitochondrial respiration, depletes NAD(P)H, decreases the mitochondrial membrane potential, depletes ATP, and causes cell death (240). Similar effects are observed with the uncoupling drug, diclofenac, wh