Activation and Detoxification Enzymes: Functions and Implications

Activation and Detoxification Enzymes Functions and Implications

Chang-Hwei Chen

Activation and Detoxification Enzymes Functions and Implications

Chang-Hwei Chen, Ph.D. Professor, Institute for Health and the Environment Former Professor, Department of Biomedical Sciences University at Albany, State University of New York Albany, NY, USA [email protected]

ISBN 978-1-4614-1048-5 e-ISBN 978-1-4614-1049-2 DOI 10.1007/978-1-4614-1049-2 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2011939743 © Springer Science+Business Media, LLC 2012 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

This book is dedicated to my father and my mother

Preface

Humans are exposed to foreign compounds such as drugs, household products, and environmental chemicals by swallowing or breathing. Also, food is considered a foreign compound. Such foreign compounds can be nonessential and nonfunctional to life, and are commonly referred to as xenobiotics. Some xenobiotics are not toxic; however, many of them are potentially toxic or become toxic after conversion to metabolic intermediates. A considerable number of foreign compounds belong to nonpolar, lipophilic substances. Lipophilic compounds are not soluble in water. Metabolic conversion of lipophilic foreign compounds to facilitate their removal from the body is essentially carried out by biochemical reactions catalyzed by two classes of foreign compound-metabolizing enzymes, namely, activation enzymes and detoxification enzymes. Activation enzyme-catalyzed functionalization reaction introduces a functional group to a lipophilic compound. Functionalization modifies many foreign compounds to form reactive intermediates capable of interacting with cellular components (proteins, DNA, and lipids), leading to a variety of conditions for diseases. Functionalized compounds are further metabolized through detoxification enzymecatalyzed reactions, which result in an increase in the solubility of parent compounds and an inactivation of metabolic intermediates, thus facilitating their excretion from the body. To minimize the exposure of potentially toxic metabolic intermediates, it is essential to keep them at a minimum level. Extensive investigations have revealed that foreign compound-metabolizing enzymes exhibit genetic polymorphisms. Variations in their activities can produce different results as to the susceptibility to potential toxic effects. Moreover, the expressions of phase I activation enzymes and phase II detoxification enzymes are inducible. A number of chemical compounds are capable of acting as modulators for these two classes of enzymes. These findings have led to the proposal of modulating metabolizing enzymes as a useful approach for human health benefits. Importantly, many of these chemical compounds are present in human daily diets. There are many advances that have been made in the past decades toward the understanding of functions and implications of activation enzymes and detoxification enzymes. An organized, concise overview is needed for the readers who are vii

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Preface

initially exposed to this important subject, particularly for students and researchers in the areas of biomedical sciences, biochemistry, nutrition, pharmacology, and chemistry. This book is intended to serve this purpose as an introduction to the subject. Furthermore, major topics in the book, excluding catalytic reactions and structural properties, may interest other readers who have knowledge of basic sciences and understanding enzyme-related information. The book discusses subjects associated with foreign compound metabolizing enzymes with emphasis on biochemical aspects, including lipophilic foreign compounds, phase I enzymes and phase II enzymes, catalytic properties, reactive intermediates, biomedical and biochemical effects, genetic polymorphisms, enzyme inducibility, enzyme modulation for health benefits, dietary enzyme modulators, and structural characteristics of enzyme inducers. The author wishes to thank Professor David Carpenter, Director of the Institute for Health and the Environment, School of Public Health, University at Albany, for his many courtesies and assistance during the course of preparing this book. The author also thanks Professor Norman L. Strominger of Albany Medical College and University at Albany for his valuable comments, suggestions, and input. Albany, NY

Chang-Hwei Chen, Ph.D.

Contents

1

Overview .................................................................................................. 1.1 Defense Against Foreign Compounds ............................................. 1.2 Activation Enzymes and Detoxification Enzymes ........................... 1.3 Metabolic Intermediates................................................................... 1.4 Genetic Polymorphism and Inducibility of Enzymes ...................... 1.5 Enzyme Modulation for Health Benefits ......................................... 1.6 Catalytic Reactions of Metabolic Enzymes ..................................... Bibliography .............................................................................................

1 1 2 3 3 4 4 5

2

Lipophilic Foreign Compounds ............................................................. 2.1 Lipophiles ........................................................................................ 2.2 Transport Across Cell Membranes .................................................. 2.2.1 Major Transport Mechanisms .............................................. 2.2.2 Channels and Transporters ................................................... 2.3 Sites of Action.................................................................................. 2.4 Excretion of Foreign Compounds .................................................... 2.4.1 Renal Excretion .................................................................... 2.4.2 Hepatic Excretion................................................................. 2.4.3 Reabsorption in the Kidney.................................................. 2.5 Major Metabolic Pathways .............................................................. 2.5.1 Phase I Metabolism and Phase II Metabolism ..................... 2.5.2 Phase III Metabolism ........................................................... Bibliography .............................................................................................

7 7 8 9 10 10 11 12 12 13 13 13 15 15

3

Metabolic Conversion of Lipophilic Compounds................................. 3.1 Phase I Metabolism .......................................................................... 3.2 Phase II Metabolism ........................................................................ 3.2.1 Conjugation Reactions ......................................................... 3.2.2 Nonconjugation Reactions ...................................................

17 17 19 19 21

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3.3

Toxication Versus Detoxification ..................................................... 3.3.1 Activation ............................................................................. 3.3.2 Deactivation ......................................................................... 3.3.3 Activation Versus Deactivation ............................................ Bibliography .............................................................................................

22 22 23 23 24

4

Phase I Enzymes...................................................................................... 4.1 Activators ......................................................................................... 4.2 Oxidative Enzymes .......................................................................... 4.2.1 Cytochrome P450................................................................. 4.2.2 Flavin-Containing Monooxygenase ..................................... 4.2.3 Amine Oxidase..................................................................... 4.2.4 Lipoxygenase ....................................................................... 4.2.5 Alcohol Dehydrogenase ....................................................... 4.2.6 Aldehyde Oxidase ................................................................ 4.2.7 Xanthine Oxidase ................................................................. 4.2.8 Peroxidase ............................................................................ 4.3 Reductive Enzymes .......................................................................... 4.3.1 Nitroreductase ...................................................................... 4.3.2 Azoreductase ........................................................................ 4.4 Hydrolytic Enzymes......................................................................... 4.4.1 Carboxylesterase .................................................................. 4.4.2 Epoxide Hydrolase ............................................................... 4.5 Catalytic Actions.............................................................................. 4.5.1 Oxidative Reactions ............................................................. 4.5.2 Reductive Reactions ............................................................. 4.5.3 Hydrolytic Reactions............................................................ Bibliography .............................................................................................

25 26 26 27 28 28 29 29 30 30 30 30 31 31 31 31 32 32 32 34 35 36

5

Phase II Enzymes .................................................................................... 5.1 Excretors .......................................................................................... 5.2 Conjugation Enzymes ...................................................................... 5.2.1 Uridine-Diphosphate-Glucuronosyltransferase ................... 5.2.2 Glutathione S-Transferase .................................................... 5.2.3 Sulfotransferase.................................................................... 5.2.4 N-Acetyltransferase ............................................................. 5.2.5 Methyltransferase ................................................................. 5.2.6 Acyltransferase..................................................................... 5.3 Nonconjugation Enzymes ................................................................ 5.3.1 Quinone Reductase .............................................................. 5.3.2 Epoxide Hydrolase ............................................................... 5.4 Catalytic Actions.............................................................................. 5.4.1 Conjugation at O Atom ........................................................ 5.4.2 Conjugation at N Atom ........................................................ 5.4.3 Conjugation at C Atom ........................................................ 5.4.4 Conjugation at S Atom.........................................................

37 37 38 38 39 40 41 42 42 43 43 43 44 44 45 46 46

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5.4.5 Conjugation of Carboxylic Acid ........................................ 5.4.6 Nonconjugation Reactions ................................................. Bibliography .............................................................................................

46 47 47

6

Reactive Intermediate Formation.......................................................... 6.1 Reactive Intermediates ..................................................................... 6.2 Reactive Oxygen Species ................................................................. 6.3 Enzyme-Catalyzed Reactive Intermediate Formation ..................... 6.3.1 Mediation by Phase I Enzymes ............................................ 6.3.2 Mediation by Phase II Enzymes........................................... 6.4 Interactions with Cellular Components ........................................... 6.4.1 Protein Adducts .................................................................... 6.4.2 DNA Adducts ....................................................................... 6.4.3 Lipid Peroxidation................................................................ 6.4.4 Toxic Effects ........................................................................ 6.5 Defense Against Reactive Intermediates ......................................... 6.5.1 Conjugation Reactions ......................................................... 6.5.2 Glutathione ........................................................................... 6.5.3 Antioxidant Enzymes ........................................................... 6.6 Factors Affecting Xenobiotic Toxicity............................................. Bibliography .............................................................................................

49 50 51 51 52 52 52 54 54 55 55 56 56 56 57 57 58

7

Biomedical and Biochemical Effects ..................................................... 7.1 Exhibition of Foreign Compound Toxicity .................................... 7.1.1 Intrinsic Toxicity ................................................................ 7.1.2 Toxic Reactive Metabolites ................................................ 7.1.3 Induction of Toxicity.......................................................... 7.2 Oxidative Stress ............................................................................. 7.3 Oxidative Protein Damage ............................................................. 7.4 Oxidative DNA Damage ................................................................ 7.5 Lipid Peroxidation ......................................................................... 7.6 Intervention with Mitochondria Functions .................................... 7.7 Interaction with Ion Transporters ................................................... 7.8 Interference with Enzymatic Functions ......................................... 7.9 Immune Suppression and Stimulation Effects ............................... 7.10 Chemical Carcinogenesis ............................................................... Bibliography .............................................................................................

61 61 61 62 62 62 63 64 64 65 65 66 67 68 68

8

Genetic Variations in Metabolizing Enzymes ....................................... 8.1 Role of Enzyme Genetic Polymorphisms in Alcoholism ................ 8.2 Genetic Polymorphisms of Cytochrome P450................................. 8.2.1 CYP2A6 Polymorphisms Affecting Nicotine Metabolism ............................................................ 8.2.2 CYP1A1 Polymorphisms Affecting Polycyclic Aromatic Hydrocarbon Metabolism .................................... 8.2.3 CYP2E1 Polymorphisms Affecting Nitrosamine Metabolism ......................................................

71 72 72 73 74 75

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8.3 8.4

9

Genetic Polymorphisms of Glutathione-S-Transferase ................. Species Difference in Enzyme Activity ......................................... 8.4.1 Susceptibility to Aflatoxin Toxicity in humans, But Not in Mice ................................................................. 8.4.2 Resistance to Tamoxifen Toxicity in Humans, But Not in Rats .................................................................. 8.4.3 4-Ipomeanol Toxicity Differs Between Humans and Rodents .......................................... Bibliography .............................................................................................

76 77

Inducibility of Metabolizing Enzymes .................................................. 9.1 Modulation of Phase I Enzymes ...................................................... 9.2 Modulation of Phase II Enzymes ..................................................... 9.3 Life Style Modification .................................................................... 9.3.1 Alcohol ................................................................................. 9.3.2 Cigarette Smoke ................................................................... 9.4 Monofunctional and Bifunctional Inducers ..................................... 9.5 Balance Between Activation and Detoxification ............................. 9.6 Antioxidant Response Element........................................................ 9.7 Enzyme Modulation Against Potential Toxic Effects ...................... Bibliography .............................................................................................

83 84 84 86 86 86 86 87 88 89 90

77 78 79 80

10

Induction and Inhibition Compounds................................................... 91 10.1 Defense Against Potential Toxicities ............................................. 91 10.2 Sulforaphane and Isothiocyanates ................................................. 92 10.3 1,2-Dithiole-3-Thione and Derivatives .......................................... 93 10.4 Indole-3-Carbinol .......................................................................... 95 10.5 Flavonoids and Isoflavones ............................................................ 96 10.6 Polyphenols.................................................................................... 98 10.7 Organosulfur Compounds .............................................................. 98 10.8 Terpenes and Terpenoids ............................................................... 100 Bibliography ............................................................................................. 101

11

Diets Rich in Enzyme Modulators ......................................................... 11.1 Dietary Contributions to Enzyme Modulation ............................... 11.2 Vegetables Rich in Enzyme Modulators ........................................ 11.3 Fruits Rich in Enzyme Modulators ................................................ 11.4 Herbs Rich in Enzyme Modulators ................................................ 11.5 Beverages Rich in Enzyme Modulators ......................................... Bibliography .............................................................................................

103 104 104 106 108 109 110

12

Induction of Enzymes for Health Benefits ............................................ 12.1 Enzyme Modulation as a Defense Mechanism .............................. 12.2 Monofunctional and Bifunctional Inducers ................................... 12.3 Role of Antioxidant Response Element .........................................

113 114 115 115

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12.4

Dietary Inducers of Phase II Enzymes........................................... 12.4.1 Isothiocyanates ................................................................... 12.4.2 Sulforaphane ...................................................................... 12.4.3 Indole-3-Carbinol ............................................................... 12.4.4 Allyl Sulfides...................................................................... Bibliography .............................................................................................

117 117 118 119 119 120

13

Sources of Foreign Compounds ............................................................. 13.1 Foreign Compounds That Humans Are Exposed To ..................... 13.1.1 Food.................................................................................... 13.1.2 Pharmaceuticals.................................................................. 13.1.3 Smoking ............................................................................. 13.1.4 Household Products ........................................................... 13.1.5 Environmental Chemicals .................................................. 13.2 Heterocyclic Amines...................................................................... 13.3 Nitrosamines .................................................................................. 13.4 Polycyclic Aromatic Hydrocarbons ............................................... 13.5 Azo Dyes ....................................................................................... 13.6 a,b-Unsaturated Aldehydes ............................................................ 13.7 Mycotoxins .................................................................................... 13.8 Overdose of Drugs ......................................................................... 13.8.1 Acetaminophen .................................................................. 13.8.2 Xanthine ............................................................................. 13.8.3 Terfenadine ......................................................................... 13.9 Household Products ....................................................................... 13.9.1 Benzene .............................................................................. 13.9.2 Di(2-ethylhexyl)phthalate .................................................. 13.10 Environmental Chemicals .............................................................. 13.10.1 Diesel Exhausts ........................................................................ 13.10.2 Arsenic in Drinking and Underground Water ..................... 13.10.3 Polychlorinated Biphenyls ................................................. Bibliography .............................................................................................

123 123 123 124 124 124 125 125 126 127 128 129 129 130 131 131 131 132 132 132 133 133 133 134 134

14

Catalytic Reactions of Phase II Enzymes.............................................. 14.1 Cytochrome P450-Catalyzed Reactions ....................................... 14.1.1 Hydroxylation of Aliphatic or Aromatic Compound.......................................................................... 14.1.2 Epoxidation of Ether .......................................................... 14.1.3 Dehydrogenation of Alcohol or Aldehyde ......................... 14.1.4 Oxidation of N- or S-Compound........................................ 14.1.5 Dealkylation of Ether, Amide, or Carboxylic Acid............ 14.1.6 Oxidation of Carbon on Aromatic Ring ............................. 14.2 Flavin Monooxygenase-Catalyzed Reactions................................ 14.3 Amine Oxidase-Catalyzed Reactions ............................................

137 137 138 138 138 139 139 139 140 140

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Contents

14.4 Nitroreductase-Catalyzed Reactions .............................................. 14.5 Azoreductase-Catalyzed Reactions ............................................... 14.6 Molybdenum Hydroxylase-Catalyzed Reactions .......................... 14.7 Alcohol Dehydrogenase-Catalyzed Reactions .............................. 14.8 Peroxidase-Catalyzed Reactions .................................................... 14.9 Carboxylesterase-Catalyzed Reactions .......................................... Bibliography .............................................................................................

140 141 141 142 142 142 142

Catalytic Reactions of Phase II Enzymes.............................................. 15.1 UDP-Glucuronosyl Transferase-Catalyzed Conjugation Reactions ................................................................. 15.2 Glutathione S-Transferase-Catalyzed Conjugation Reactions ................................................................. 15.3 Sulfotransferase-Catalyzed Conjugation Reactions ...................... 15.4 Acyltransferase-Catalyzed Conjugation Reactions ....................... 15.5 N-Acetyltransferase-Catalyzed Conjugation Reactions ................ 15.6 Methyltransferase-Catalyzed Conjugation Reactions.................... 15.7 Quinone Reductase-Catalyzed Reactions ...................................... 15.8 Epoxide Hydrolase-Catalyzed Reactions ...................................... Bibliography .............................................................................................

145

Diversified Classes of Enzyme Modulators........................................... 16.1 Substrate–Enzyme Interactions ..................................................... 16.2 Modulator–Enzyme Interactions ................................................... 16.3 Michael Acceptor Functionalities .................................................. 16.4 Diversities of Enzyme Inducers ..................................................... Bibliography .............................................................................................

145 148 149 149 150 151 151 152 152 155 156 157 158 159 164

Conclusion ....................................................................................................... 167 Index ................................................................................................................. 171

Chapter 1

Overview

Wastes are produced when foods are converted into raw materials and energy through biochemical reactions occurring in living cells. Medicine used to fight diseases can accumulate in the body and cause unintended effects. Household products containing chemical compounds are used daily. Industrial chemicals and environmental pollutants are present in the air and rivers. And humans are constantly exposed to these foreign compounds. Many of these compounds undergo enzymatic conversion to metabolic intermediates. Many metabolic intermediates of chemical compounds are reactive and are ultimately responsible for their toxicities. The body’s major defense mechanism against xenobiotics is to minimize exposure by speedily removing them from the body. To achieve this goal, the body develops a number of enzyme systems involved in activation and detoxification of foreign compounds. The expression of activation enzymes and detoxification enzymes may vary among individuals due to genetic polymorphisms and environmental factors. Variations in genetic polymorphisms and environmental factors affect an individual’s susceptibility to foreign compound-mediated toxic effects. An unusually high expression of activation enzyme may give rise to an overload of metabolic intermediates. An extraordinary low efficiency of detoxification enzyme may result in an abnormal detoxification metabolism. To maintain reactive intermediates at a minimum level depends on relative efficacies of activation enzymes and detoxification enzymes and a delicate balance between the reactions catalyzed by these two classes of enzymes.

1.1

Defense Against Foreign Compounds

A large number of foreign compounds that find their way into the body are lipophilic (fat soluble) in nature. Unlike hydrophilic substances that are soluble in water, lipophilic compounds are nonpolar and are minimally soluble in water. Lipophilic

C.-H. Chen, Activation and Detoxification Enzymes: Functions and Implications, DOI 10.1007/978-1-4614-1049-2_1, © Springer Science+Business Media, LLC 2012

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Overview

compounds need to be converted into water soluble compounds before being eliminated and excreted from the body. Elimination of foreign compounds to which humans are exposed involves detoxification mechanisms which are important defenses that help humans to survive. To achieve speedy removal of xenobiotics, the body develops a number of enzyme systems that catalyze the conversion of fat-soluble lipophilic compounds to water-soluble hydrophilic metabolites. These enzymes are produced from information stored in the genes. Several metabolic steps are involved in the metabolism of foreign compounds before they are ready to be eliminated from the body. There are two distinctive steps in overall detoxification processes: phase I and phase II. Their enzymes are referred to as phase I enzymes and phase II enzymes, respectively. Based on the current convention, this book adopts the terminology of phase I and phase II, although there are some arguments against such a classification. For instances, phase II precedes phase I in a few cases, conjugation reactions in some cases occur directly, phase II step does not always result in detoxification, and some enzymes do not clearly fit into either of these two steps.

1.2

Activation Enzymes and Detoxification Enzymes

Phase I enzymes catalyze the conversion reaction called functionalization for a lipophilic foreign compound, where a functional group is introduced to its chemical structure through a reaction such as oxidation, hydrolysis or reduction. Functionalization increases the polarity of a lipophilic foreign compound, making it ready for the next metabolic step. The modified compound often leads to the formation of an active metabolic intermediate. Bioactivation is a metabolic process in which a metabolic intermediate is produced. Phase I enzymes that catalyze bioactivation are referred to as activation enzymes. A hydrophilic foreign compound that has a functional polar group may bypass the functionalization process. A functionalized lipophilic compound or a hydrophilic compound with a functional polar group is further metabolized by the phase II reactions. Phase II enzymes catalyze conjugation reactions. There are also phase II enzymes that involve nonconjugated reactions. In conjugation reaction, the functional group of foreign compound is combined with a chemical group of a small molecule. This small molecule is frequently the cofactor of enzyme. The conjugation reaction greatly increases the solubility and excretory potential of a foreign compound, thus facilitating its removal from the body. Detoxification is a metabolic process in which a foreign compound is detoxified. Phase II enzymes that catalyze detoxification of foreign compounds are referred to as detoxification enzymes. Lipophilic foreign compounds and their conversions are discussed in Chaps. 2 and 3. The descriptions of phase I enzymes and phase II enzymes can be found in Chaps. 4 and 5.

1.4

1.3

Genetic Polymorphism and Inducibility of Enzymes

3

Metabolic Intermediates

Detoxification of a foreign compound is an extraordinarily effective mechanism for the body’s defense against foreign compound-mediated toxic effects. The major site of detoxification processes is the liver, but many metabolic reactions also occur in other organs such as lung, kidney, and intestines. Although xenobiotic metabolic pathways are beneficial to living organisms, however, in many cases, the generated metabolic intermediate or metabolite is acutely or potentially toxic. Moreover, a significant number of reactive intermediates have potential to react with oxygen to form reactive oxygen species (e.g., free radicals). Reactive oxygen species are capable of interacting with cellular components (proteins, DNA, and lipids), leading to various disease conditions such as cancer, cardiovascular disease, and neurological disorders. Unlike those produced in detoxification processes, free radicals generated in aerobic cellular metabolism are created as intermediates during catalytic actions involving the transfer of electrons. Such free radicals are often critical for the normal operation of a wide variety of biological phenomena. An example is nitric oxide that acts as an important oxidative biological signal in a diversity of physiological functions. However, beyond normal physiological functions, it is essential to keep free radicals generated in aerobic cellular metabolism at a minimum. The body utilizes not only antioxidant enzymes but also endogenous antioxidant substances and small antioxidant molecules to achieve this goal. A functioning detoxification system appears to be critical in preventing a variety of disease conditions. A body critically relies on metabolic reactions catalyzed by activation enzymes and detoxification enzymes to reduce and remove reactive intermediates or free radicals produced in the metabolism of foreign compounds. Meanwhile, endogenous antioxidants also play a role in the removal of reactive oxygen species. Knowledge about metabolic mechanisms involving these two classes of enzymes is critical to the understanding of the potential toxic effect and the detoxification of foreign compounds. The discussions of reactive intermediates and reactive oxygen species, biomedical and biochemical effects, and sources of foreign compounds are described in Chaps. 6, 7, and 13, respectively.

1.4

Genetic Polymorphism and Inducibility of Enzymes

Studies of individual responsiveness to drugs or other foreign compounds have revealed considerable deviation, partly due to variations in their metabolisms. Among these variants is the difference in the level of expression of foreign compound-metabolizing enzymes, which may result in the observed variations in the potency of metabolic intermediates or metabolites, as well as the distinctive susceptibility of some individuals to their potentially toxic effects. Genetic polymorphisms are an important factor in attributing to individual variations in the efficacy of

4

1

Overview

phase I enzymes (e.g., cytochrome P450) and phase II enzymes (e.g., glutathioneS-transferase). An important toxicologically relevant feature associated with phase I enzymes and phase II enzymes is the potential of these enzymes for induction or inhibition by some chemical compounds. The inducibility of these enzymes makes it possible to modify their expression for health benefits. Some of enzyme modulators are present in our diets. The roles of genetic polymorphism, enzyme inducibility, and modulation compounds are critical to the understanding of functional properties of foreign compound-metabolizing enzymes. These subjects are discussed in Chaps. 8, 9, and 10.

1.5

Enzyme Modulation for Health Benefits

Dietary effects on the human health have been a subject of intensive study. Examples include diets rich in fiber and those high in saturated fatty acids. Fiber can be effective as an absorbant to bind and carry certain chemicals through the digestive system, thus preventing harm by absorption into the body. Unsaturated fatty acids are susceptible to lipid oxidation, a factor contributing to chemical carcinogenesis. The modulation of activation and detoxification enzymes to reduce foreign compound-mediated toxic effects has been a subject of interest in the past decades. Advances in the understanding of mechanisms that govern the detoxification of foreign compounds have revealed that diets can have important impacts on the efficacy of phase I and phase II enzymes. Many chemical compounds that are capable of acting as enzyme modulators are present in the daily human diet. Extensive research has been carried out to explore how these enzymes can be modulated in human diets for health benefits. To protect individuals against foreign compound-mediated toxic effects, important hypotheses involving activation and detoxification enzymes have been postulated. An increase in the intake of diet rich in inducers of detoxification enzymes is considered as a promising proposal to minimize foreign compound-mediated toxic effects. Diets rich in vegetables and fruits that contain enzyme modulators have received much attention, in particular, those rich in inhibitors of activation enzymes (particularly, cytochrome P450) and inducers of detoxification enzymes (mainly, uridine-diphosphate-glucuronosyl-transferases and glutathione S-transferases). These subjects are discussed in Chaps. 11 and 12. Chapter 16 discusses structural characteristics of compounds that are enzyme modulators.

1.6

Catalytic Reactions of Metabolic Enzymes

Phase I enzymes catalyze a variety of reactions to introduce functional groups to foreign compounds. Such varieties of reactions take place at specific atoms or groups. Similarly, phase II enzymes-catalyzed reactions also occur at specific atoms

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or groups in foreign compounds. Phase II enzymes catalyze various conjugation reactions to form conjugated compounds that facilitate the excretion of foreign compounds. There are also phase II enzymes that catalyze nonconjugated reactions. Either phase I or phase II metabolism consists of a variety of enzymes that carry out different catalytic reactions. Phase I and phase II reactions represent critical elements of detoxification mechanisms. Knowledge about these catalytic reactions and their functional characteristics is fundamental to understanding how these enzymes act on foreign compounds, what foreign compounds are metabolized by certain enzymes, and what functionalities of compounds are capable of modulating the expression of enzymes. To address these questions, Chaps. 4 and 5 describe specific atoms and groups that are involved in phase I reactions and phase II reactions. Chapters 14 and 15 discuss major catalytic reactions of activation enzymes and detoxification enzymes.

Bibliography Boelsterli UA (2007) Mechanistic toxicology. CRC Press, Boca Raton Buxton ILO, Benet LZ (2011) Pharmacokinetics: the dynamics of drug absorption, distribution, metabolism and elimination. In: Brunton LL et al (eds) Goodman & Gilman’s the pharmacological basis of therapeutics. McGraw-Hill, New York, NY Conney AH (2003) Enzyme induction and dietary chemicals as approaches to cancer chemoprevention. Cancer Res 63:7005–7031 Finley JW, Schwass DE (1985) Xenobiotic metabolism: nutritional effects. American Chemical Society, Washington DC Giacomini KM, Sugiyama Y (2011) Membrane transports and drug response. In: Brunton LL et al (eds) Goodman & Gilman’s the pharmacological basis of therapeutics. McGraw-Hill, New York, NY Gonzalez FJ, Coughtrie (2011) Drug metabolism. In: Brunton LL et al (eds) Goodman & Gilman’s the pharmacological basis of therapeutics. McGraw-Hill, New York, NY Hodgson E, Das PC, Cho TM, Rose RL (2008) Phase I metabolism of toxicants and metabolic interactions. In: Smart RC, Hodgson E (eds) Molecular and biochemical toxicology. Wiley, New York, NY Ioannides C (2002) Xenobiotic metabolism: an overview. In: Ioannides C (ed) Enzyme systems that metabolise drugs and other xenobiotics. Wiley, New York, NY Jakoby WB, Bend JR, Caldwell J (1982) Metabolic basis of detoxication. Academic, New York, NY Jakoby WB (1980) Enzymatic basis of detoxication. vl-2. Academic, New York, NY Josephy PD, Mannervik B, de Montellano PO (1997) Molecular toxicology. Oxford University, New York, NY LeBlanc GA (2008) Phase II-conjugation of toxicants. In: Smart RC, Hodgson E (eds) Molecular and biochemical toxicology. Wiley, New York, NY Lee JS, Obach RS (2003) Drug metabolizing enzymes. Dekker, New York, NY Mulder GJ (1990) Conjugation reactions in drug metabolism: an integrated approach. Taylor and Francis, London Parkinson A, Ogilvie BW (2008) Biotransformation of xenobiotics. In: Klaassen CD (ed) Casarett & Doull’s toxicology: the basic science of poisons. McGraw-Hill, New York, NY Sardesai VM (2003) Introduction to clinical nutrition. Dekker, New York, NY

Chapter 2

Lipophilic Foreign Compounds

On the basis of solubility, foreign compounds that humans ingest or inhale can be classified into two categories. One class of foreign compounds is soluble in water (hydrophilic), but not in lipid medium. Another class is soluble in lipid medium (lipophilic), but not in water. Lipophilic substances require enzymatic conversion into hydrophilic, polar species before being excreted in the urine. The more lipophilic a foreign compound is, the more difficult it becomes for excretion via the kidney. Hydrophilic compounds can be excreted in the urine without enzymatic conversion to increase their solubility. A foreign compound needs to move across biological membranes before it can enter the blood stream and distribute throughout the body. Owing to lipid bilayers serving as physical barriers for biomembranes, transport mechanism for lipophilic compounds across biomembranes is distinctive from that of hydrophilic substances. Hydrophilic molecules usually are unable to penetrate cell membranes because of their low lipid solubility. Membrane physical barriers also contribute to different sites of action between lipophilic and hydrophilic substances. Lipophilic molecules are lipid-soluble and generally can diffuse across cell membranes. The process by which foreign compounds cross cell membranes and enter the blood stream is referred to as absorption. The gastrointestinal tract is one of the most important sites where foreign compounds are absorbed. A major group of foreign compounds that are absorbed by the lungs are gases, vapors, and aerosols. The present chapter discusses the membrane transport, the metabolic conversion, the sites of action, and excretion of foreign compounds. Figure 2.1 briefly describes the entry, absorption, metabolism, and excretion of a foreign compound.

2.1

Lipophiles

Water molecule is polar species, which has a positive end and a negative end. Positive and negative ends of a water molecule display electrostatic attraction. Water owes its great superiority as a solvent for ionic substances partly due to its polarity. C.-H. Chen, Activation and Detoxification Enzymes: Functions and Implications, DOI 10.1007/978-1-4614-1049-2_2, © Springer Science+Business Media, LLC 2012

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Fig. 2.1 Entry, absorption, metabolism, and excretion of foreign compound

Lipophilic Foreign Compounds Foreign compound Ingestion, inhalation Entry into the body Across cell membranes Absorption Distribution Organs Metabolism

Excretion from the body

Table 2.1 Solubility of typical lipophilic foreign compounds Lipophilic foreign compounds Solubility in water Menadione Insoluble Acetaminophen Very slightly Benzo[a]pyrene Insoluble Quinone Slightly Polychlorinated biphenyls Insoluble Diazepam Slightly Aniline Insoluble Pebbendazole Insoluble

The polarity of water permits it to solvate ions strongly. Water has a high dielectric constant (78 at room temperature). When an ionic compound (either solid or liquid) dissolves in water, its structural units (ions) become separated from each other and the spaces in between become occupied by water molecules (hydration). Water contains the hydroxyl group (−OH). Compounds containing hydrogen attached to oxygen (hydroxyl group) or nitrogen (amine group) tend to increase solvation powers. For nonionic compounds, their solubility is dependent on their polarity. The rule of thumb is “Like dissolve like.” Nonpolar or weakly polar compounds dissolve in nonpolar or weakly polar solvents (organic solvents). Unlike water, nonpolar solvents have a low dielectric constant (e.g., 2 for benzene). Lipid bilayers serve as physical barriers for biological membranes, where lipid medium has a low dielectric constant (about 5). Nonpolar foreign compounds prefer to dissolve in low dielectric constant lipid medium, and are, therefore, referred to as lipophiles. Some lipophilic foreign compounds are shown in Table 2.1, which reveals poor water solubility for lipophiles.

2.2

Transport Across Cell Membranes

The accumulation of foreign compounds at the site of action is facilitated by absorption and distribution. Absorption is referred to the transfer of a foreign compound from the site of exposure into the general circulation. Lipid solubility of foreign

2.2

Transport Across Cell Membranes

9

compounds is usually the most important properties that influence their absorption. While transporters may contribute to the gastrointestinal absorption of some chemicals, a large majority of foreign compounds traverse epithelial barriers and reach blood capillaries by diffusion through the cells. Lipid bilayers serve as physical barriers that do not favor a spontaneous exchange of foreign compounds between the internal and external cell compartments. Living organisms use cell membranes as hydrophobic permeability barriers to control access to the internal cell compartment. The movement of foreign compounds into or out of the cells is carried out by various transport mechanisms depending on their solubility characteristics. Lipophilic, nonpolar compounds are able to move across cell membranes, while hydrophilic, polar compounds are largely restricted to the extracellular compartments and cannot enter into the cells simply by free diffusion. The uptake of hydrophilic compounds across cell membranes is mediated by channels or transport proteins, which specifically select substrates (solutes) from the extracellular medium.

2.2.1

Major Transport Mechanisms

Major mechanisms for the transport of a foreign compound (solute) across biological membranes include passive diffusion, facilitated diffusion, and active transport. Passive diffusion of solutes across cell membranes is composed of three steps: partition from the external aqueous medium to the membrane lipid phase, diffusion across membrane lipid bilayers, and partition into the internal cellular medium. In passive diffusion, the driving force for solute to move across membrane lipids into the cells is the concentration gradient, in which the concentration of solute in the external cell medium is higher than that in the internal cell medium. A large number of foreign compounds are transported across biomembranes through facilitated diffusion. In facilitated diffusion, the transport of a foreign compound across biomembranes into the cells is facilitated by transport protein (carrier). Facilitated diffusion involves no input energy and occurs downhill in accordance with the solute concentration gradient. Transport protein selects a specific solute from the extracellular medium. The binding enables the transport protein to carry the solute across cell membranes into the internal cell medium, such as in the case of glucose permeation across cell membranes mediated by glucose transporter protein. Active transport of a foreign compound across biomembranes into the cells is also mediated by membrane transporters. But, unlike facilitated diffusion, active transport requires energy input as well as the movement of solute against a concentration gradient. Depending on the driving force, active transport can be classified into primary and secondary active transports. Primary active transport is coupled with ATP hydrolysis catalyzed by Na+, K+- ATPase, which provides the energy for the uptake of solute against a concentration gradient. The unidirectional movement of a solute across biomembranes in mammalian cells is mediated by transporter proteins such as ATP binding cassette transporters (ABC transporters).

10

2

Lipophilic Foreign Compounds

In secondary active transport, the transport of solute across biomembranes uphill against its concentration gradient is coupled with the movement of another solute downhill in accordance with its concentration gradient. In this case, the driving force for the uptake of solute across biomembranes is the electrochemical potential stored in a concentration gradient of another solute. Therefore, secondary active transport takes place at the expense of a preexisting electrochemical gradient of another solute. For example, the uptake of lactose across E. coli membranes is coupled with the movement of H+ downhill in accordance with proton electrochemical potential. Another example is Na+-Ca++ exchange protein that uses the energy stored in the Na+ gradient established by Na+, K+-ATPase to export cytosolic Ca++.

2.2.2

Channels and Transporters

Facilitating membrane permeation of inorganic ions and organic compounds involves channels and transporters. Channels exist in two primary states: open and close. In the open state, channels act as pores for selected ions, allowing them to permeate across cell membranes, and then channels return to the close state. By contrast, transporter protein forms an intermediate complex with a specific solute (the substrate) on the external membrane, which induces the translocation of the substrate to the internal membrane. Transporter proteins are membrane proteins that control the influx of essential nutrients and ions as well as the efflux of cellular wastes and foreign compounds. Mechanistic difference between channels and transporters results in a marked difference in their turnover rates. The turnover rate constants of typical channels are much larger than those of transporter proteins. ATP binding cassette (ABC transporter) and solute carrier (SLC transporter) are two major families of membrane transporters for drug and other xenobiotics. Membrane transporters work in concert with foreign compound-metabolizing enzymes to mediate the uptake and efflux of xenobiotics and their metabolites. Generally, SLC transporters mediate either influx or efflux of drug, while ABC transporters mediate unidirectional efflux.

2.3

Sites of Action

The liver encounters foreign compounds such as food, drugs, and environment chemicals after they are absorbed in the intestinal tract. The liver is the major organ where foreign compounds are metabolized and eventually excreted, chemically active intermediates are produced, and toxicities are manifested. The liver is the main metabolic site for drugs. The metabolic site for drugs depends upon the presence of foreign compound metabolizing enzymes, and most of these enzymes are present in the liver. Hepatocytes contain phase I enzymes that have the capacity to generate metabolic intermediates as well as phase II enzymes that catalyze the

2.4

Excretion of Foreign Compounds

11

addition of polar groups to lipophilic compounds and target the formed conjugates to transport carriers for excretion. Although metabolites or metabolic intermediates may react at the site where they are generated, the liver is not necessarily the target organ of toxicity. The metabolites may diffuse away and react with other targets. Metabolic intermediates may be transported to other organs where they exert toxic effects. Meanwhile, before transported to other organs, metabolic intermediates may potentially cause toxicity in the liver. Besides the liver, the kidney is a frequent target organ of toxicity and is also a main site for drug metabolism. The kidney, which receives a large amount of blood, also contains a variety of foreign compound-metabolizing enzymes. The breast, lung, and colon are frequent target sites in spite of their limited metabolic ability. Metabolites of foreign compounds such as aromatic amines are also transported to the bladder, where they are released and converted to carcinogenic species.

2.4

Excretion of Foreign Compounds

Foreign compounds excreted from the body include waste products from the digestion of foods, drugs accumulated in the body, chemical substances in the environment, and industrial chemicals in the household. These compounds are excreted from the body either unchanged or being converted to metabolites. Owing to high solubility in water, hydrophilic species generally can be excreted from the body through urine or bile without chemical modification. Conversely, because of limited solubility in water, lipophilic foreign compounds require metabolic conversion into hydrophilic metabolites before they are excreted through urine. Metabolic intermediates formed in phase I reactions can overwhelm the body’s defense mechanism if they are not removed quickly. Solute metabolites are often products resulted from phase II reactions. Before excreted from the body, foreign compounds transport from the internal to the external cell compartment carried out by transport proteins in phase III metabolism (see below). Figure 2.2 illustrates different metabolic processes that foreign compounds may precede before being excreted from the body. In addition, foreign compounds may proceed from phase I directly to phase III before their excretion. Urinary and biliary systems are two primary routes for the excretion of foreign compounds and their metabolites from the body. Accordingly, renal and hepatic excretions are the two major pathways. Vectorial transport, i.e., asymmetrical transport of solute across cell membranes, plays a major role in urinary and hepatobiliary excretion of drugs from the blood. ABC transporters are able to achieve vectorial transport by extruding lipophilic xenobiotics to the exterior compartment of cells.

Foreign compound

Phase I

Phase II

Phase II

Phase III

Phase III

Excretion

Phase III Excretion

Fig. 2.2 Metabolic processes preceding foreign compound excretion

Excretion

12

2.4.1

2

Lipophilic Foreign Compounds

Renal Excretion

More foreign compounds are eliminated from the body by the kidney than by other organs. The kidney is, therefore, the most important organ for the excretion of foreign compounds including drugs, toxins, carcinogens, and their metabolites, The kidney is very efficient in the elimination of toxicants from the body and is critical in the body’s defense against foreign compounds. Renal excretion plays an important role in eliminating conjugation products resulting from phase II reactions. Renal excretion involves glomerular filtration, active tubular secretion, and passive tubular readsorption. Small compounds (<60 kD) are usually able to pass the glomerular filtration barrier, while larger metabolites are excreted via other pathways. After glomerular filtration, a foreign compound or its metabolite may remain in the tubular lumen and be excreted in urine. Transport carriers located on the basolateral membrane of proximal tubule cells can transport foreign compounds from the blood into the epithelial cells for excretion. Organic anion transporters and organic cation transporters are two major classes of secretory transporters in the mammalian kidney. Structurally diversified organic cations and anions, including positively and negatively charged drugs and their metabolites, are secreted in the proximal tubule of the kidney. Transporters in the kidney mediate the secretion or reabsorption of many foreign compounds, thereby influencing the plasma levels of their substrates. SLC transporters and ABC transporters are two major classes of secretory transporters in the mammalian kidney. SLC transporters are involved in moving organic cations across the basolateral membrane. They are also implicated in a variety of organic anions that are secreted in the proximal tubule. Major physiological functions of ABC transporters include the transport of toxic foreign compounds, and ABC transporters are also involved in the secretion of organic cations. They translocate a variety of compounds through cell membranes against concentration gradients at the expense of ATP hydrolysis. Besides SLC and ABC transporters, other transporters also participate in the excretion of organic ions. Transporters such as multidrug-resistance-associated proteins localized in the apical brush-border membrane are responsible for the excretion of conjugated metabolites.

2.4.2

Hepatic Excretion

In addition to being the main site for biotransformation of foreign compounds, the liver also plays an important role in removing xenobiotics from blood after they are absorbed in the gastrointestinal tract. Depending on molecular weights, foreign compounds or their conjugates can be excreted into bile in substantial quantities. SLC transporters mediate either influx or efflux of foreign compounds (e.g., drugs). Hepatic uptake of organic anions, cations, and bile salts can be carried out by SLC

2.5

Major Metabolic Pathways

13

transporters in the basolateral membrane of hepatocytes by either facilitated diffusion or secondary active transport. Moreover, ABC transporters in the canalicular membrane of hepatocytes mediate unidirectional efflux of foreign compounds. The excretion of drugs and their metabolites mediated by ABC transporters is carried out uphill against a concentration gradient, where ATP hydrolysis provides the driving force for the transport.

2.4.3

Reabsorption in the Kidney

Transporters in the kidney mediate the secretion or reabsorption of many foreign compounds and thereby influence the plasma levels of their substrates. Kidney excretion of hydrophilic compounds is more efficient than lipophilic compounds. Hydrophilic compounds and ions are readily excreted in the urine. While, lipophilic compounds that have high lipid/water partition coefficients can be readsorbed efficiently across the kidney tubules back into the bloodstream. Tubular reabsorption in the kidney is a major factor contributing to the difficulty in the excretion of lipophilic compounds. Poor solubility in water is another critical factor. Lipophilic compounds are not readily eliminated before they are converted into water soluble polar compounds. Living organisms, therefore, develop metabolic mechanisms with enzyme systems that are proficient in the conversion of lipophilic into hydrophilic compounds.

2.5

Major Metabolic Pathways

Foreign compounds including hydrophilic and lipophilic species are not produced in vivo. They are brought into the body via ingestion and inhalation and are subsequently metabolized by the body. The capacity of removing a wide range of lipophilic species is the major challenge of metabolic pathways for foreign compounds. This challenge is achieved by a combination of low specificity enzymatic systems and physical barriers of biomembranes. Low specificity of enzymatic systems makes it possible to metabolize a wide range of lipophilic and hydrophilic compounds. Physical barriers of biomembranes attribute to the role of membrane permeability and hydrophobicity in the metabolism of foreign compounds.

2.5.1

Phase I Metabolism and Phase II Metabolism

The metabolism of lipophilic foreign compounds involves a set of metabolic pathways. Among them are two major enzyme-catalyzed pathways: the functionalization

14

2

Lipophilic Foreign Compounds

a Functionalization Phase I enzyme Lipophilic compound

Metabolic intermediate

b Conjugation Phase II enzyme Metabolic intermediate + Small cofactor ligand Soluble metabolite

c Overall reaction Phase I enzyme Phase II enzyme Lipophilic compound Metabolic intermediated Soluble metabolite

Excretion

Fig. 2.3 Enzymatic conversion of lipophilic compounds

in phase I reactions involving the addition of a functional group to a xenobiotic and the conjugation in phase II reactions involving the coupling of an endogenous small cofactor molecule to a functionalized xenobiotic. These two pathways convert lipophilic substances into hydrophilic species by the introduction of a functional group and the conjugation with an endogenous molecule. A phase I reaction generally proceeds before a phase II reaction. However, in some cases, a phase II reaction may occur without a preceding phase I reaction or prior to a phase I reaction. Foreign compound-metabolizing enzymes are produced based on the information stored within the genes and are present in the liver at much higher concentrations than in other organs. Phase I enzyme catalyzes a functionalization reaction by means of oxidation, reduction or hydrolysis, where a functional group is introduced to the structure of a lipophilic foreign compound. While the addition of a functional polar group results in a moderate increase in the water solubility of the parent compound, the functionalization also activates the foreign compound, often leading to the formation of a metabolic intermediate. The functionalized compound then undergoes a conjugation reaction catalyzed by a phase II enzyme, which combines a small cofactor molecule with the functionalized compound to form a conjugate. These two pathways are responsible for the metabolism of foreign compounds, but generally are not implicated in the elimination of endogenous metabolites derived from normal cellular constituents. Many foreign compounds are converted to metabolic intermediates through biotransformation catalyzed by phase I enzymes. Many metabolic intermediates of chemical compounds are reactive and are ultimately responsible for their toxicities. The conjugation reaction catalyzed by a phase II enzyme detoxifies metabolic intermediate, rendering it less harmful and largely increasing its water solubility, thus facilitating the excretion of foreign compound from the body. Figure 2.3 illustrates the two major steps in the conversion of a lipophilic foreign compound into a water soluble species: step a (phase I metabolism) and step b (phase II metabolism). Step c represents the overall reaction.

Bibliography

2.5.2

15

Phase III Metabolism

Conjugates produced in phase II metabolism are hydrophilic, polar substances. Preceding their excretion, conjugates require transmembrane movement from the internal to the extracellular cell compartment. Because membrane lipids act as the physical barriers, the transport of conjugates out of the cells cannot be carried out by free diffusion. Instead, the transmembrane movement of conjugates across biomembranes requires transport proteins called export pumps. The process that facilitates the transport of conjugates and other metabolites from the internal to the external cell compartment is referred to as phase III metabolism. Phase III metabolism of foreign compounds is the step that occurs after metabolic conversion and before their excretion from the body. Conjugates may be further processed before being recognized by transport proteins and prior to moving out of the cells. A number of ATP-dependent transport proteins or export pumps have been identified in the liver. An example of ATP-binding transporters is the family of multidrug resistance proteins. The topic of phase III metabolism is not within the scope of this book and is, therefore, not further addressed in the following chapters.

Bibliography Borst P, Elferink RO (2002) Mammalian ABC transporters in health and disease. Annu Rev Biochem 71:537–592 Burckhardt BC, Burckhardt G (2003) Transport of organic anions across the basolateral membrane of proximal tubule cells. Rev Physiol Biochem Pharmacol 146:95–158 Buxton ILO, Benet LZ (2011) Pharmacokinetics: the dynamics of drug absorption, distribution, metabolism and elimination. In: Brunton LL et al (eds) Goodman & Gilman’s the pharmacological basis of therapeutics. McGraw-Hill, New York, NY Dean M, Rzhetsky A, Allikmets R (2001) The human ATP-binding cassette (ABC) transporter superfamily. Genome Res 11:1156–1166 Dresser MJ, Leabman MK, Giacomini KM (2001) Transporters involved in the elimination of drugs in the kidney: organic anion transporters and organic cation transporters. J Pharm Sci 90:397–421 Giacomini KM, Sugiyama Y (2011) Membrane transporters and drug response. In: Brunton LL et al (eds) Goodman and Gilman’s the pharmacological basis of therapeutics. McGraw-Hill, New York, NY Hediger MA, Romero MF, Peng JB et al (2004) The ABCs of solute carriers: physiological, pathological and therapeutic implications of human membrane transport proteins. Pflugers Arch 447:465–468 Koepsell H (1998) Organic cation transporters in intestine, kidney, liver, and brain. Annu Rev Physiol 60:243–266 König J, Cui Y, Nies AT, Keppler D (2000) Localization and genomic organization of a new hepatocellular organic anion transporting polypeptide. J Biol Chem 275:23161–23168 Lee W, Kim RB (2004) Transporters and renal drug elimination. Annu Rev Pharmacol Toxicol 44:137–166 Lehman-McKeeman LD (2007) Absorption, distribution, and excretion of toxicants. In: Klaassen CD (ed) Casarett & Doull’s toxicology. The Basic Science of Poisons. McGraw-Hill, New York, NY

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Pu RY, Wang Y, Chen CH (1995) Enthalpy changes in the formation of the proton electrochemical potential and its components. Biophys Chem 53:283–290 Reuss L (2000) Basic mechanisms of ion transport. In: Seldin D (ed) The kidney physiology and pathophysiology. Lippincott Williams & Wilkins, Baltimore Wright SH, Dantzler WH (2004) Molecular and cellular physiology of renal organic cation and anion transport. Physiol Rev 84:987–1049

Chapter 3

Metabolic Conversion of Lipophilic Compounds

As discussed in the previous chapter, the conversion of lipophilic, nonpolar species to polar, water-soluble (hydrophilic) compounds is essential to facilitate their excretion from the body through urea or bile. The conversion process occurs in two separate metabolic systems: phase I metabolism and phase II metabolism. In phase I functionalization reaction, a functional polar group such as –OH or –COOH is introduced on a foreign compound to form an intermediate through oxidation, reduction, or hydrolysis reaction. Functionalization modestly increases the water solubility of the parent compound, and paves the way for the next phase of metabolism. In phase II conjugation reaction, the functionalized compound is combined with an endogenous substrate (e.g., glucuronic acid, glutathione, or sulfonate) to produce a conjugate that facilitates its excretion from the body. The conjugation reaction greatly increases water solubility of a foreign compound, except methylation or acetylation conjugate. A foreign compound that is polar, water soluble can bypass the functionalization reaction and directly takes part in the phase II conjugation reaction.

3.1

Phase I Metabolism

Oxidation, reduction or hydrolysis reaction catalyzed by a phase I enzyme leads to the introduction of a functional group, which results in a modification of a foreign compound and a moderate increase in its water solubility. In the case of a drug, the introduction of a functional group can lead to an alteration in biological properties of the drug. The product of phase I metabolism subsequently serves as the substrate for the phase II conjugation reaction. Major reactions catalyzed by phase I enzymes in metabolic pathways include N-dealkylation, O-dealkylation, aliphatic and aromatic hydroxylation, N-oxidation, S-oxidation, epoxidation, and hydrolysis. Typical phase I reactions are briefly described below, where functional groups are introduced to foreign compounds. To show changes in specific functional groups, chemical equations described below may not be balanced. Symbol F or F¢ denotes an aromatic side group, and R or R¢ represents a straight or cyclic aliphatic side group. C.-H. Chen, Activation and Detoxification Enzymes: Functions and Implications, DOI 10.1007/978-1-4614-1049-2_3, © Springer Science+Business Media, LLC 2012

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Metabolic Conversion of Lipophilic Compounds

(a) N-oxidation:

(e.g., conversion of nicotine to nicotine N-oxide) N-oxidation results in forming hydroxylamine. (b) S-oxidation:

(e.g., S-oxidation of benzimidazole sulfides) S-oxidation results in producing oxysulfide. (c) Aromatic hydroxylation: R − Φ → R − Φ − OH (e.g., conversion of aromatic to phenyl compound) The reaction applies to a foreign compound that contains an aromatic ring. Aromatic hydroxylation leads to the generation of phenolic products. (d) Aliphatic hydroxylation: R − Φ − CH 3 → R − Φ − CH 2 − OH (e.g., conversion of pentobarbitone to hydroxypentobarbitone) Aliphatic hydroxylation is a pathway for many medical drugs containing, an aliphatic side chain. (e) O-dealkylation: R − Φ − OCH 3 → R − Φ − OH + CH 2 O (e.g., conversion of 7-ethoxycoumarin to 7-hydroxycoumarin) O-Dealkylation results in the loss of alkyl group attached to oxygen. (f) N-dealkylation:

(e.g., N-dealkylation of benzphetamine) N-Dealkylation results in the loss of alkyl group attached to nitrogen. (g) Hydrolysis: Φ − NH − CO − CH 2 CH 3 → Φ − NH 2 + CH 3 CH 2 COOH (e.g., conversion of 3,4-dichloroproprioanilide to 3,4-dicholoroaniline)

3.2 Phase II Metabolism

19

Table 3.1 Functional groups introduced by phase I reactions Phase I reaction Functional group formed N-oxidation N–OH S-oxidation S=O Aromatic hydroxylation F–OH Aliphatic hydroxylation R–OH O-dealkylation R–OH N-dealkylation –NH2 Hydrolysis (ester or amide) –NH2 or –COOH Epoxidation –C–C– \/ O

Hydrolysis is a pathway for a number of esters or amides such as esterase or amidase-catalyzed reactions. (h) Epoxidation:

− Φ − RC = CR’ → − Φ – RC – CHR’ | \/ H O (e.g., conversion of aflatoxin B1 to aflatoxin B1-epoxide) Epoxidation is a pathway in metabolizing many carcinogenic compounds such as aromatic hydrocarbons. Phase I reaction leads to the introduction of a functional group (e.g. –OH, –COOH, –SH, –O–, or NH2) to a lipophilic foreign compound, resulting in modifying the chemical structure of the parent compound. The modified compound serves as the substrate for phase II conjugating enzymes. Table 3.1 summarizes the functional groups that are introduced by phase I reactions.

3.2 3.2.1

Phase II Metabolism Conjugation Reactions

Metabolic pathways in phase II reactions include glucuronide, glutathione, sulfonate, and amino acid conjugation reactions, which are catalyzed by respective phase II transferase enzymes. A conjugate is formed in the combination of the substrate (the phase I product) with a small endogenous molecule. Conjugation reactions usually require the substrate to have oxygen, nitrogen or sulfur atom serving as acceptor site for the hydrophilic moiety (e.g., glutathione, glucuronic acid, sulfonate, or acetyl group). Phase II reactions generally inactivate potentially toxic metabolites generated in phase I reactions and greatly improve water solubility of foreign compounds, facilitating the elimination of drugs and other xenobiotics.

20

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Metabolic Conversion of Lipophilic Compounds

Major conjugation reactions catalyzed by phase II enzymes in metabolic pathways include glucuronide, sulfate, glutathione, acetyl, methyl, and amino conjugations. Representative phase II conjugation reactions are briefly described below, where the chemical equations may not be balanced. Symbol F or F¢ denotes an aromatic side group and may be replaced with a straight or cyclic aliphatic side group (R or R¢). Table 3.2 lists typical cofactors and conjugation groups in phase II reactions. (a) Glucuronide conjugation: −Φ − OH + UDP - glucuronic acid → −Φ − O − glucuronide + UDP (e.g., the conversion of hydroxydiazepam to hydroxydiazepam glucuronide) Glucuronide conjugation reaction transfers glucuronic acid from the cofactor UDP-glucuronic acid to a substrate (functionalized foreign compound) to form glucuronide metabolite. The functional group that is ready for glucuronide conjugation includes phenol and aromatic amines. (b) Glutathione conjugation: HO − Φ − OH + GSH → HO − Φ − GSH (e.g., the conversion of acetaminophen to acetaminophen glutathione) Glutathione is a tripeptide (glutamate-cycteine-glycine) containing a sulfhydryl group. Glutathione conjugation transfers glutathione to a functionalized foreign compound, where GSH is the reduced form of glutathione. (c) Sulfonate conjugation: −Φ − OH + PAPS → −Φ − O − SO 2 − OH + PAP (e.g., the conversion of 7-hydroxycoumarin to 7-hydroxycoumarin sulfonate) Sulfonate conjugation transfers sulfonate from 3¢-phosphoadenosine-5¢phosphosulfate (PAPS) to the –OH group of aromatic or aliphatic compound. Sulfonate conjugation is also an important pathway in the metabolism of amino group. (d) Amino acid conjugation: −Φ − COCl + H 2 NCH 2 COOH → −Φ − CONHCH 2 COOH + HCl (e.g., the conversion of benzoyl chloride to hippuric acid) Amino acid conjugation reaction is important in the metabolism of an organic acid, where the carboxylic group of an organic acid conjugates with an amino acid (e.g., glycine). Glycine is the most common amino acid in amino acid conjugation reaction. (e) N-acetyl conjugation: −Φ − NH 2 + acetyl - CoA → −Φ − NH − acetyl + CoA

3.2 Phase II Metabolism

21

Table 3.2 Cofactors and transferring groups in phase II conjugation reactions Phase II reaction Cofactor Conjugation group Glucuronidation UDP-glucuronic acid Glucuronic acid Sulfonation 3¢-phosphoadenosine-5¢-phosphosulfate Sulfonate Glutathione conjugation Glutathione Glutathione Acetylation Acetyl-Coenzyme A Acetyl group Methylation S-adenosyl-methionine Methyl group Amino acid conjugation Glycine Amino acid

(e.g., conversion of dapsone to N-acetyl dapsone) N-acetyl conjugation is important in the metabolism of drugs and environmental chemicals containing an aromatic amine or hydrazine group. N-acetyl conjugation transfers acetyl from the cofactor acetyl-CoA to –NH2 group of metabolite. N-acetyl conjugate often is less water soluble than the parent compound. (f) Methyl conjugation: −Φ − R − NH 2 + AdoMet → −Φ − R − NH − CH3 (e.g., conversion of –NH2 group on dopamine to –NH–CH3) Foreign compounds can undergo O-, N-, or S-methylation using S-adenosylmethionine (AdoMet) as the methyl donor. Methyl conjugate also has less water solubility than the parent compound.

3.2.2

Nonconjugation Reactions

Nonconjugation reactions involved in detoxification metabolism include quinone reductase and epoxide hydrolase. In some classifications, the reactions catalyzed by these two enzymes are considered belonging to phase II reactions. (a) Quinone reductase: R − Φ = O → R − Φ − OH (e.g., converstion of quinone to hydroquinone) Quinones can be reduced to hydroquinones by NADPH-quinone oxidoreductase. The two-electron reduction of quinone is a nontoxic reaction (not associate with semiquinone formation) if the produced hydroquinone is sufficiently stable to undergo glucuronidation or sulfonation.

22

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Metabolic Conversion of Lipophilic Compounds

(b) Epoxide hydrolase: Φ – HC − CH → Φ – HC − CH 2 2 \/ || O

HO OH

(e.g., converstion of styrene 7,8-epoxide to styrene 7,8-glycol) Many epoxides are intermediary metabolites formed during CYP450-dependent oxidation of unsaturated aromatic or aliphatic compounds. Epoxide hydrolases play an important role in detoxifying electrophilic epoxides.

3.3

Toxication Versus Detoxification

The metabolism of lipophilic foreign compounds is composed of two processes: activation and deactivation. Activation is referred to as the metabolic process in which foreign compounds are converted to form reactive intermediates or metabolites. The topic of metabolic intermediate is further discussed in Chapter 6. If the produced intermediates are highly active, some of them can interact with cellular components before phase II reactions have taken place. Such interactions could potentially result in cell damages. Toxication is the process of metabolism in which the metabolite of a foreign compound is more toxic than the parent one. Phase II reactions generally yield stable, nonreactive metabolites that are readily available for excretion from the body. Metabolic conversion of metabolic intermediates to metabolites that are no longer capable of interacting with cellular components is referred to as deactivation. Detoxification is the process of metabolism in which the metabolite toxicity of a foreign compound is detoxified.

3.3.1

Activation

The overall metabolism of lipophilic foreign compounds is a detoxification process. However, some lipophilic compounds are metabolized and converted to form activated intermediates during phase I metabolism. A chemically active intermediate is potentially a more reactive and toxic species than the parent compound. Generally, reactive intermediates are generated through functionalization reactions mediated by CYP450 and other phase I enzymes. In minor cases, conjugation reactions catalyzed by phase II enzymes are also involved. CYP450 isozymes are most implicated in phase I-mediated activation, where foreign lipophilic compounds, which are initially harmless and nonreactive, turn into reactive and potentially harmful metabolites. Chemically active intermediates are capable of interacting with cellular components (e.g., proteins, DNA, RNA, and lipids) and are ultimately responsible for the

3.3

Toxication Versus Detoxification

23

toxicity of foreign compounds. A typical example is the transformation of 4- Ipomeanol by CYP4B1 to active intermediate, a,b-unsaturated dialdehyde that exhibits toxicity, leading to 4-ipomeanol-induced lung injuries. Highly active intermediates generated by foreign compounds also include epoxides, radicals, and carbonium ions. Moreover, chemically active intermediates can further induce toxicity or carcinogenicity by interacting with molecular oxygen to yield reactive oxygen species (e.g., hydrogen peroxide and hydroxyl (OH⋅) and superoxyl anion (O2⋅) radicals). Reactive oxygen species, especially free radicals, are believed to be involved in degenerative diseases and cancers.

3.3.2

Deactivation

A body’s major defensive mechanisms against foreign compounds or their metabolites include enzymatic reactions and nonenzymatic reactions. Phase II conjugation reaction carries out enzymatic defense by combining a functionalized foreign compound with an enzyme cofactor, thus facilitating the excretion of xenobiotic from the body. Nonenzymatic reaction utilizes endogenous compounds such as glutathione. Glutathione, an antioxidant, prevents damage to cellular components caused by reactive oxygen species (e.g., peroxides and free radicals). While the vast majority of phase II enzyme-catalyzed metabolizing reactions results in the deactivation of foreign compounds, in minor cases, phase II enzymes may be implicated in the toxicity of foreign compounds. A typical example of phase II enzyme-mediated toxicity is the glutathione conjugation of epoxide, which exhibits toxic effects in the kidneys. Besides, there are other defense mechanisms against chemically active intermediates and reactive oxygen species. These include antioxidant enzymes (e.g., glutathione peroxidase and superoxide dismutase) and small antioxidant molecules (e.g., uric acid and vitamin E).

3.3.3

Activation Versus Deactivation

The amount of chemically active intermediate present in a body largely depends on the competing pathways between activation and deactivation. Therefore, the body requires a delicate balance between the rates of activation and deactivation. Factors that interrupt this delicate balance will affect the metabolism of foreign compounds and their mediated toxicity. Activation pathways overwhelm deactivation pathways when phase I enzymes are over expressed. In this case, activation pathways assume a greater role and an over production of reactive intermediates happens. Consequently, the accumulation of reactive intermediates in the body occurs, resulting in a potential increase in the interaction of foreign compounds with cellular components. Foreign compounds rely heavily on phase II enzymes for the detoxification. A deficiency in phase II conjugation pathways may lead to an increased vulnerability to the toxicity of foreign compounds. Activation pathways may become more dominant

24

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Metabolic Conversion of Lipophilic Compounds

than deactivation pathways under two circumstances: (a) one related to conjugation reactions and (b) the other associated with environmental factors. A low expression of conjugating enzyme activity leads to a low rate of conjugation reactions. An insufficiency of cofactors (e.g., glutathione, sulfonate, or glucuronic acid) can also result in a deficiency in conjugation pathways because the rate of utilization exceeds the rate of supply of a cofactor. Furthermore, the environmental factors can also attribute to activation pathways over deactivation pathways under two conditions: when enzyme systems that catalyze activation pathways are selectively induced and when enzyme systems that catalyze deactivation pathways are inhibited due to the exposure of a large quantity of chemicals (e.g., smoking, alcohol, or drugs).

Bibliography Cavalieri EL, Rogan EG (2005) The approach to understanding aromatic hydrocarbon carcinogenesis. The central role of radical cations in metabolic activation. Pharmacol Ther 55:183–199 Cribb AE, Peyrou M, Muruganandan S et al (2005) The endoplasmic reticulum in xenobiotic toxicity. Drug Metab Rev 37:405–442 Hayes JD, Flanagan JU, Jowsey IR (2005) Glutathione transferases. Annu Rev Pharmacol Toxicol 45:51–88 Ioannides C (2002) Xenobiotic metabolism: an overview. In: Ioannides C (ed) Enzyme systems that metabolise drugs and other xenobiotics. Wiley, New York, NY Meisel P (2002) Arylamine N-acetyltransferases and drug response. Pharmacogenomics 3:349–366 Negishi M, Pedersen LG, Petrotchenko E et al (2001) Structure and function of sulfotransferases. Arch Biochem Biophys 390:149–157 Park BK, Kitteringham NR, Maggs JL et al (2005) The role of metabolic activation in drug-induced hepatotoxicity. Annu Rev Pharmacol Toxicol 45:177–202 Parkinson A, Ogilvie BW (2008) Biotransformation of xenobiotics. In: Klaassen CD (ed) Casarett & Doull’s toxicology: the basic science of poisons. McGraw-Hill, New York, NY Pumford NR, Halmes NC (1997) Protein targets of xenobiotic reactive intermediates. Annu Rev Pharmacol Toxicol 37:91–117 Tukey RH, Strassburg CP (2000) Human UDP-glucuronosyltransferases: metabolism, expression, and disease. Annu Rev Pharmacol Toxicol 40:581–616 Weinshilboum RM, Otterness DM, Szumlanski CL (1999) Methylation pharmacogenetics: catechol O-methyltransferase, thiopurine methyltransferase, and histamine N-methyltransferase. Annu Rev Pharmacol Toxicol 39:19–52

Chapter 4

Phase I Enzymes

A large majority of chemical reactions in living cells valuable to an organism exhibit kinetic barriers that prevent the reactions from occurring spontaneously. Kinetic barriers can be overcome by a variety of enzymes that act as catalysts, which make the reactions energetically favorable. Consequently, the reactions can occur at the rates required for maintaining cell functions. Enzymes make up the largest and most highly specialized class of protein molecules. They are highly effective catalysts for an enormous diversity of chemical reactions. The substrate of an enzyme is a small chemical molecule on which the enzyme exerts its catalytic action. On the basis of the nature of chemical reactions that enzymes catalyze, enzymes can be divided into six major classes: (a) oxidoreductases that catalyze the oxidation and reduction reactions, (b) transferases that catalyze the transfer of functional groups, (c) hydrolases that catalyze the hydrolysis reactions, (d) lyases that catalyze the reactions involving the removal or addition of a group to double bonds, (e) isomerases that catalyze the reactions involving the intramolecular rearrangement, and (f) ligases that catalyze the reactions that join together two molecules. Foreign compound-metabolizing enzymes (phase I enzymes and phase II enzymes) belong to three of six classes described above: oxidoreductases, hydrolases, and transferases. Foreign compound-metabolizing enzymes are primarily present in the endoplasmic recticulum of the cells and/or in the cytosol. They are produced from the information stored within the genes, and are present in most tissues with the highest levels located in liver and intestines. Phase I enzymes are composed of oxidases, hydrolases, and reductases. Oxidases include CYP450s, flavin-containing monooxygenases, amine oxidases, lipoxygenases, aldehyde and xanthine oxidases, alcohol dehydrogenases, and peroxidases. Reductases include nitroreductases and azoreductases. Hydrolases include carboxylesterases and epoxide hydrolases. In the metabolism of foreign compounds, phase I enzymes that catalyze functionalization reactions and bring about active metabolic intermediates are referred to as activation enzymes or activators.

C.-H. Chen, Activation and Detoxification Enzymes: Functions and Implications, DOI 10.1007/978-1-4614-1049-2_4, © Springer Science+Business Media, LLC 2012

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4.1

4

Phase I Enzymes

Activators

A large majority of foreign compounds are lipophilic compounds that are capable of passing through biomembranes and entering into cells, and then are transported by lipoproteins in body fluids. The ultimate goal of foreign compound-metabolizing enzymes is to convert lipophilic foreign compounds into water-soluble species so as to facilitate the elimination of xenobitoics from the body. Phase I enzymes catalyze functionalization reactions which introduce functional groups to foreign compounds by means of the oxidation, hydrolysis or reduction reaction catalyzed by oxidoreductases or hydrolases. In many cases, the addition of a functional group results in the formation of an intermediate which is more chemically active and more toxic than the parent compound. Highly reactive electrophilic metabolites are able to react with cellular molecules (e.g., proteins, DNA or lipids), causing cell and organ toxicity. Hence, phase I enzymes are, therefore, referred to as activation enzymes or activators. Among the three major groups of phase I enzymes, oxidases and reductases catalyze oxidation and reduction reactions, respectively, while hydrolases catalyze hydrolysis reactions by the introduction of water. Relatively, enzymatic reduction reactions are less investigated as compared to oxidation and hydrolysis reactions. A large majority of organic chemicals undergo oxidation reactions during metabolism. For instance, a foreign compound containing a benzene ring (e.g., phenyl) commonly undergoes aromatic hydroxylation reaction, leading to the formation of phenolic products. Other foreign compounds that contain nitrogen may be oxidized to form nitrooxides or hydroxylamines. Many drugs are metabolized through aliphatic or aromatic hydroxylation. Major phase I enzymes in metabolic activation of foreign compounds are summarized in Table 4.1. There are more oxidative enzymes than reductive or hydrolytic enzymes.

4.2

Oxidative Enzymes

Table 4.1 shows that cytochrome P450, flavin monooxygenase, amine oxidases, lipoxygenases, alcohol dehydrogenase, aldehyde and xanthine oxidases, and peroxidase are classified as oxidative enzymes (oxidases). However, some of them Table 4.1 Major phase I enzymes in foreign compound metabolism Oxidative enzymes Reductive enzymes Cytochrome P450 Nitroreductase Flavin-monooxygenase Azoreductase Amine oxidase Lipoxygenase Alcohol dehydrogenase Aldehyde oxidase Xanthine oxidase Peroxidase

Hydrolytic enzymes Carboxylesterase Epoxide hydrolase

4.2

Oxidative Enzymes

27

(e.g., alcohol dehydrogenase, aldehyde oxidase, and CYP450) can catalyze both oxidative and reductive reactions depending on the substrate or conditions. The functional properties of these oxidative enzymes are briefly discussed below:

4.2.1

Cytochrome P450

Among foreign compound-metabolizing enzymes, cytochrome P450 (CYP450) is the most actively studied phase I enzyme. CYP450 refers to a unique family of heme proteins. P450 is designated because its reduced form binds carbon monoxide to produce a maximum optical absorption around 450 nm. The family of CYP450 is complex and diverse in catalytic activities. There are over 50 CYP450 isozymes that have been identified in humans. CYP450s are present predominantly in the liver, but also occur in other organs such as intestine, lung, and kidney. CYP450s are the major oxidative enzymes. These enzymes are the most important enzyme family in phase I metabolism because they are responsible for metabolizing the large majority of therapeutic drugs and other foreign compounds. CYP450 contains a heme that is bound to the polypeptide chain. The heme contains one atom of iron. CYP450s utilize O2 and H+ (from NADPH) to carry out the oxidation of a substrate (foreign compound). CYP450 functions as a monooxygenase, which catalyzes the insertion of one atom of oxygen molecule into the substrate. A molecule of oxygen first binds to the heme moiety and subsequently is cleaved. One oxygen atom forms by binding with the substrate. The remaining oxygen atom is reduced to yield water. Two electrons that stem from NADPH also participate in CYP450-catalyzed reactions, where the reaction products are oxidized substrate and water. The family of CYP450 catalyzes the metabolism of a large number of structurally diverse chemicals, including N-and O-dealkylation, aliphatic and aromatic hydroxylation, N-and S-oxidation, deamination, cleavage of esters, and epoxidation of a double bond. A diversity of foreign compounds is catalyzed by CYP450; these include drugs, food additives, and industrial and environmental chemicals. In addition, CYP450s also catalyze the reactions involving endogenous compounds, such as cholesterol, steroid hormones, and fatty acids. CYP450s play a key role in adverse effects of foreign compounds. Microsomal CYP450 isozymes involved in metabolizing foreign compounds are responsible for the breakdown of medications, which occurs mostly in the liver. Liver microsomes contain numerous CYP450 isozymes, such as CYP1A2, CYP1A2, and CYP2A6. Each of these enzymes has the potential to catalyze various types of reactions. CYP450 isozymes are very important for the pharmaceutical industry and have far reaching implications in medicine, especially in the activation of therapeutic agents. Importantly, CYP450s catalyze the conversion of foreign chemicals to reactive intermediates and the formation of many electrophilic metabolites. Moreover, environmental and genetic factors can play a role in the expression of CYP450 activities. CYP450 expression in its catalytic reactions can differ markedly

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Phase I Enzymes

as a result of exposure to dietary and environmental enzyme inducers. There are significant variations in the levels of enzyme expression present in CYP450 isozymes among individuals due to the presence of genetic polymorphisms and differences in gene regulation. Genetic variants in CYP450 have a potentially functional impact on the efficacy and adverse effects of drugs. The superfamily of CYP450s consists of over 50 functional genes with the 1, 2, and 3 families. Different ethnic groups may exhibit variations in the distribution of these genes.

4.2.2

Flavin-Containing Monooxygenase

Flavoproteins are flavin-dependent enzymes that consist of monooxygenases, oxidases, and dehydrogenases. Flavin-containing monooxygenases (FMO) are another superfamily of phase I enzymes involved in the metabolism of foreign compounds. FMOs are expressed at high levels in the liver and are bound to the endoplasmic reticulum. The family of FMO (e.g., FMO1, FMO2, FMO3, and FMO4) consists of a group of enzymes that catalyze chemical reactions through the binding of cofactor flavin. FMOs are oxidative enzymes that catalyze the oxygenation of nitrogen-, sulfur-, phosphorus-, and other nucleophilic heteroatomcontaining chemicals. FMOs have been found to associate with the detoxification of nucleophilic heteroatom-containing foreign compounds (e.g., drugs, chemicals, and food components). The reaction catalyzed by FMO involves one-step two-electron substrate oxygenation, as opposed to two sequential one-electron oxidations catalyzed by CYP450. FMO-catalyzed reactions usually convert foreign compounds into relatively polar metabolites. Kinetics studies revealed that the mechanisms of FMO1, 2, 3, and 4 isozymes are similar, but differ in the substrate specificities. Such differences can be attributed essentially to the variations in the dimensions of the cleft or channel, which limit the access to hydroperoxy flavin. Among FMO isozymes, FMO3 is most associated with FMO-mediated foreign compound metabolism. FMO3 allelic variation could contribute to the difference in FMO3-dependent metabolism of chemicals among individuals. Although many advances have been made in the understanding of CYP450-catalyzed activation of drugs or environmental chemicals, limited information is available for FMO-mediated activation. Human FMO oxygenates nucleophilic heteroatom-containing chemicals. The oxidation reaction generally converts foreign compounds into harmless, polar metabolites. However, FMO sometimes activates xenobiotics into metabolic intermediates that can cause toxic effects.

4.2.3

Amine Oxidase

Amine oxidases are widely distributed throughout the body in the liver, intestines and other organs. Amine oxidases are involved in the oxidative deamination of primary, secondary, and tertiary amines. Among amino oxidases, monoamine

4.2

Oxidative Enzymes

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oxidases are most extensively studied in terms of their involvement in foreign compound metabolism. Monoamine oxidases (MAOs) contain the covalently bound cofactor FAD and are classified as flavoproteins. MAOs are composed of two structurally related flavin-containing enzymes (MAO-A and MAO-B). MAO-A and MAO-B are mitochondrial outer membrane-bound flavoproteins that catalyze the oxidative deamination of amines. Oxygen is used to remove an amine group from a molecule in MAO that catalyzes oxidation of aliphatic and aromatic amines. The oxygen incorporated into the substrate (amine) is derived from water. Hepatic monoamine oxidases have a key defense role in the detoxification of amines. Foreign compounds which are metabolized by monoamine oxidases include tyramine in foods and beverages, 2-phenylethylamine in dietary sources, benzylamine in mouth washes, and 2-phenylpropanolamine in decongestants and cough medicines. The products of these MAO-catalyzed reactions are ammonia, hydrogen peroxide and aldehyde, which are potentially toxic. The produced aldehyde may be further metabolized by aldehyde dehydrogenase or aldehyde oxidase to form the corresponding carboxylic acid.

4.2.4

Lipoxygenase

Lipoxygenases also play a role in the metabolism of foreign compounds. These enzymes are a family of nonheme-iron-containing enzymes that catalyze the oxygenation of polyenoic fatty acid to corresponding lipid hydroperoxides. Lipoxygenase-catalyzed reactions involve a fatty acid substrate with two cis double bonds separated by a methylene group. Lipoxygenases are present in many mammalian tissues including liver, lung, kidney, and colon. They are major enzymes in the oxidation of foreign compounds. Lipoxygenases mediate oxidative metabolism of foreign compounds including industrial chemicals, pesticides, and drugs. Unlike CYP450 that inserts one oxygen atom into the substrate, lipoxygenases oxidize arachidonic acid, an essential polyunsaturated fatty acid, by inserting two oxygen atoms. Lipoxygenases also catalyze N-dealkylation and epoxidation. Examples of lipoxygenase-mediated N-demethylation of drugs include aminopyrine and chlorpromazine. Lipoxygenases are also important in the final step of epoxidation of polycyclic aromatic hydrocarbons (e.g., carcinogenic benzo[a]pyrene).

4.2.5

Alcohol Dehydrogenase

Alcohol dehydrogenases (ADH) are oxidative enzymes composed of a group of several isozymes. ADHs break down alcohols and are probably the most important dehydrogenases involved in the metabolism of alcohols. ADHs catalyze the conversion of primary and secondary alcohols to aldehydes or ketones, respectively. The conversion

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Phase I Enzymes

reaction involves the reduction of the coenzyme nicotinamide dinucleotide (NAD+ to NADH). ADH-catalyzed reaction is reversible. The produced carbonyl compound can be reduced to alcohol. The conversion product, aldehyde, is usually toxic. Further oxidation of aldehyde to acid is a vital detoxification reaction.

4.2.6

Aldehyde Oxidase

Aldehyde oxidase, a molybdozyme, is important in drug oxidation, activation and detoxification. The enzyme exhibits oxidase activity toward various aliphatic and aromatic aldehydes. High levels of aldehyde oxidase activity are present in the liver. Unlike CYP450, the ultimate source of oxygen inserted into a substrate is water, not O2. While aldehyde oxidase catalyzes the oxidation of certain xenobiotics, the enzyme is also involved in the reduction reaction for some others, such as reduction of nitroaromatics to hydroxylamines.

4.2.7

Xanthine Oxidase

Similar to aldehyde oxidase, xanthine oxidase is a molybdozyme, but differs in substrate specificities. Xanthine oxidase is also important in drug oxidation, activation and detoxification. Many features of aldehyde oxidase apply to xanthine oxidase. Xanthine oxidase also exhibits oxidase activity toward various aliphatic and aromatic aldehydes. High levels of xanthine oxidase activity are found in other tissues besides the liver.

4.2.8

Peroxidase

The family of peroxidases includes several peroxidases in addition to prostaglandin H synthase. Prostaglandin H synthase is a dual-function enzyme consisting of a peroxidase and a cyclooxygenase. It is one of the most extensively studied peroxidases involving the activation of foreign compounds, in particular in tissues with low CYP450 activity. In addition to peroxide oxidation, prostaglandin H synthase also catalyzes a number of diverse oxidation reactions involving phenols and aromatic amines.

4.3

Reductive Enzymes

There is less research information available for enzymatic reduction reactions in foreign compound metabolism as compared to enzymatic oxidation, hydrolysis and conjugation reactions. Nitroreductases and azoreductases are two known phase

4.4

Hydrolytic Enzymes

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I reduction enzymes (reductases). These enzymes catalyze reduction reactions for nitro- and azo-compounds, respectively. Nitro-compounds are found in chemicals such as industrial solvents, insecticides and food preservatives. Azo-compounds, Ar–N = N–Ar¢, are strongly colored. They are widely used as colorants in foods, cosmetics, pharmaceuticals, and textile and printing industries. The functional properties of nitroreductases and azoreductases are briefly discussed below.

4.3.1

Nitroreductase

Nitroreductase catalyzes the reduction of nitro (−NO2) groups in a wide range of substrates (foreign compounds) to produce the corresponding hydroxylamines. In metabolism of nitro-compounds, the nitro group is converted to primary amine metabolites, where nitro is initially reduced to nitroso (−NO), and then to hydroxylamine (−NHOH). Hydroxylamine can finally be converted to primary amine (−NH2).

4.3.2

Azoreductase

Azoreductases catalyze the reduction reaction of azo-compounds to primary amine metabolites. Azo is initially reduced to hydrazo and finally to primary amine. In azoreductase-catalyzed reactions, two equivalents of NAD(P)H are used to reduce one equivalent of the substrate (azo-compound). During reduction reaction, azo-compounds are converted into hydrazo-compounds, where the nitrogen–nitrogen double bond is sequentially reduced. The most important reaction of azo-compounds is cleavage, which yields two amines.

4.4

Hydrolytic Enzymes

Hydrolytic enzymes (hydrolases) include carboxylesterases and epoxide hydrolases. Carboxylesterases are found in both the endoplasmic reticulum and the cytosol of the cells. These enzymes are involved in metabolic activation of various drugs and other xenobiotics. Epoxide hydrolases are present in two forms: soluble and microsomal epoxide hydrolases, which carry out the hydrolysis of epoxides usually produced by CYP450-catalyzed reactions.

4.4.1

Carboxylesterase

Carboxylesterase isozymes, carboxylesterase 1 and carboxylesterase 2, belong to the family of hydrolases. These two carboxylesterase isoforms engage in the hydrolysis of esters and amides, especially acting on carboxylic ester bonds.

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Phase I Enzymes

Carboxylesterases catalyze the hydrolysis reaction of carboxylic ester to form alcohol and carboxylate. The hydrolysis of carboxylic amide yields N-hydroxide and carboxylate. Carboxylesterase-catalyzed reactions do not always lead to a detoxification process since some xenobiotics are converted to chemically active metabolites (e.g., the conversion of urea derivative to diazonium hydroxide).

4.4.2

Epoxide Hydrolase

Epoxide hydrolases play an important role in the detoxification of electrophilic epoxides generated from oxidative activation, such as CYP450-catalyzed epoxidation. The functional properties of epoxide hydrolases are not discussed here, since epoxide hydrolases are commonly classified as phase II enzymes.

4.5

Catalytic Actions

Phase I enzyme-catalyzed reactions may act on different atoms or groups of foreign compounds (e.g., carbon, nitrogen, oxygen, sulfur, and unsaturated hydrocarbon, ester, amide, and epoxide). Typical examples of catalytic actions of activation enzymes on some specific atoms or groups are presented below. While detailed reactions catalyzed by individual phase I enzymes are further discussed in Chap. 14. The reactions described below emphasize the conversion of specific atoms or groups of foreign compounds to different functional groups in activation reactions catalyzed by phase I enzymes. Consequently, the described equations may or may not be balanced. In these reactions, R and F represent aliphatic and aromatic derivatives, respectively, and either R or F is applicable to most of reactions described below.

4.5.1

Oxidative Reactions

4.5.1.1

Oxidation at Carbon Atom

Oxidation reactions can occur at a carbon atom on an aromatic ring, alcohol, aldehyde or other functional group of a foreign compound. Phase I enzymes that catalyze oxidation reactions include CYP450, alcohol dehydrogenase, aldehyde dehydrogenases, xanthine oxidases, and peroxidases. Typical examples of such oxidation reactions are described below: (a) Cytochrome P450 An oxygen atom is inserted in a C–H bond of an aromatic compound to form phenol.

4.5

Catalytic Actions

33

F - H + O 2 + NADPH + H + ® F - OH + H 2 O + NADP + (b) Alcohol dehydrogenase Oxidation reaction converts aliphatic, aromatic, or cyclic alcohol to aldehyde by removing two hydrogens: one attached to carbon and another attached to oxygen. R - CH 2 OH + NAD + ® R - CHO + NADH + H + Further reaction oxidizes aldehyde to form carboxylic acid. R - CHO + H 2 O + NAD + ® R - COOH + NADH + H + (c) Aldehyde oxidase An oxygen atom is inserted in C–H bond of aldehyde to yield carboxylic acid. R - CHO + H 2 O + O2 ® R - COOH + H 2 O2 (d) Peroxidase Alcohol is oxidized to aldehyde by peroxide R - CH 2 OH + H 2 O2 ® R - CHO + 2H 2 O

4.5.1.2

Oxidation at Nitrogen Atom

Oxidation reaction can occur at the nitrogen atom of amine or amino group of a foreign compound. Metabolic oxidations at a nitrogen atom are catalyzed by oxidases (e.g., CYP450 and flavin-containing monoxygenase). Enzymatic oxidation reactions at nitrogen atoms include N-hydroxylation and N-oxidation. N-hydroxylation involves the substitution of one hydrogen atom in an amino group with a hydroxy group. N-oxidation involves the addition of oxygen to an amino group. Typical examples of oxidation at a nitrogen atom are shown below: (a) Cytochrome P450 Amine is oxidized to form hydroxyl amine.

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Phase I Enzymes

Imine is oxidized to form oxime in N-hydroxylation.or

(b) Flavin monooxygenase Oxygen atom is added to nitrogen in an amine to form a hydroxyl group.

4.5.1.3

Oxidation of Unsaturated Hydrocarbon

Oxidation of an unsaturated hydrocarbon is an important step in foreign compound metabolism. Microsomal CYP450-dependent monooxygenase catalyzes oxidation reaction to convert unsaturated aliphatic or aromatic hydrocarbon to form epoxide.

The produced epoxide can undergo further enzymatic reaction catalyzed by epoxide hydrolase to yield hydrodiol. While, unhydrolyzed epoxide can either react with proteins or DNA or form conjugate with glutathione. In CYP450-catalyzed epoxidation, an oxygen atom is inserted into a C = C bond of unsaturated aliphatic or aromatic hydrocarbon to form epoxide.where R or R¢ denote aliphatic derivative, which can be replaced with F or F¢ (aromatic derivative). The produced epoxide can be converted to dihydrodiol according to the following reaction.

4.5.2

Reductive Reactions

Foreign compound containing an azo or nitro group can carry out reduction reaction by interacting with a reducing agent such as NADPH. The reductive reaction catalyzed by azoreductase or nitroreductase occurs at nitrogen atom. Other enzymes such as alcohol dehydrogenase can catalyze either reductive or oxidative reaction, depending on the substrate and conditions. The reductive reaction catalyzed by

4.5

Catalytic Actions

35

alcohol dehydrogenase occurs at carbonyl group. Azoreductase, nitroreductase, and alcohol dehydrogenase-catalyzed reductive reactions are shown below. 4.5.2.1

Reduction at Nitrogen Atom

(a) Azoreductase N = N in an azo is reduced to form an amine. F - N = N - F ¢ + 2 NADPH + 2H + ® F - NH 2 + H 2 N - F ¢ + 2 NADP + (b) Nitroreductase A nitro (−NO2) group is reduced to form an amine. R - F - NO2 + 2 NADPH + 2H + ® R - F - NH 2 + H 2 O + 2NADP + 4.5.2.2

Reduction of Carbonyl Group

Carbonyl group is reduced to form hydroxyl group catalyzed by alcohol dehydrogenase.

4.5.3

Hydrolytic Reactions

Carboxylesterase is known as serine esterase because its catalytic site contains a serine residue that participates in the hydrolysis of ester or amide. A substrate containing an ester or amide group can undergo hydrolytic reaction catalyzed by carboxylesterase or hydrolase to produce carboxylic acid and alcohol or carboxylic acid and amine, respectively. Enzymatic hydrolysis of amides generally occurs more slowly than that of esters. 4.5.3.1

Hydrolysis of Ester

Carboxylesterase catalyzes hydrolytic reaction to convert an ester to carboxylic acid and alcohol.

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4.5.3.2

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Phase I Enzymes

Hydrolysis of Amide

Carboxylesterase catalyzes hydrolysis reaction to convert an amide to carboxylic acid and amine.

Bibliography Abell CW, Kwan SW (2000) Molecular characterization of monoamine oxidases A and B. Prog Nucl Acid Res Mol Biol 65:129–156 Beedham C (1997) The role of non-P450 enzymes in drug oxidation. Pharm World Sci 19:255–263 Brash AR (1999) Lipoxygenases: Occurrence, functions, catalysis, and acquisition of substrate. J Biol Chem 274:23679–23682 Cashman JR (2002) Human flavin-containing monooxygenase (form 3): polymorphisms and variations in chemical metabolism. Pharmacogenomics 3:325–339 Cashman JR, Zhang J (2006) Human flavin-containing monooxygenases. Annu Rev Pharmacol Toxicol 46:65–100 Edmondson DE, Mattevi A, Binda C et al (2004) Structure and mechanism of monoamine oxidase. Curr Med Chem 11:1983–1993 Gershater MC, Cummins I, Edwards R (2007) Role of a carboxylesterase in herbicide B ioactivation in Arabidopsis thaliana. J Biol Chem 282:21460–21466 Guengerich FP (1991) Reactions and significance of cytochrome P-450 enzymes. J Biol Chem 266:10019–10022 Guengerich FP (2002) Cytochrome P450. In: Ioannides C (ed) Enzyme System that Metabolize Drugs and Other Xenobiotics. Wiley, New York, NY Ioannides C (2002) Xenobiotic Metabolism:An Overview. In: Ioannides C (ed) Enzymes Systems that Metabolise Drugs and Other Xenobiotics. John Wiley & Son, New York, NY Kulkarni AP (2001) Lipoxygenase-a versatile biocatalyst for biotransformation of endobiotics and xenobiotics. Cell Mol Life Sci 58:1805–1825 Meijer J, DePierre JW (1988) Cytosolic epoxide hydrolase. Chem Biol Interact 64:207–249 O’Brien PJ (2000) Peroxidases. Chem Biol Interact 129:113–139 Parkinson A, Ogilvie BW (2007) Biotransformation of xenobiotics. In: Klaassen CD (ed) Casarett & Doull’s Toxicology: The Basic Science of Poisons. McGraw-Hill, New York, NY Satoh T, Hosokawa M (2006) Structure, function and regulation of carboxylesterases. Chem Biol Interact 162:195–211 Satoh T, Hosokawa M (1998) The mammalian carboxylesterases: from molecules to functions. Annu Rev Pharmacol Toxicol 38:257–288 Strolin Benedetti M, Tipton KF (1998) Monoamine oxidases and related amine oxidases as phase I enzymes in the metabolism of xenobiotics. J Neural Transm Suppl 52:149–171 Tipton KF, Benedetti MS (2002) Amine oxidases and the metabolism of xenobiotics. In: Ioannides C (ed) Enzyme System that Metabolize Drugs and Other Xenobiotics. Wiley, New York, NY Ziegler DM (2002) An overview of the mechanism, substrate specificities, and structure of FMOs. Drug Metab Rev 34:503–511

Chapter 5

Phase II Enzymes

Functionalization reaction catalyzed by a phase I enzyme incorporates a functional group to a foreign compound, resulting in the formation of an intermediate metabolite. Many intermediates contain highly reactive chemical groups, which have the potential to react with cellular components (proteins, lipids, and DNA). The continuous presence of chemically active intermediates can lead to adverse health effects and various disease conditions. In detoxification process, intermediate metabolites undergo phase II metabolism to form highly hydrophilic and less reactive compounds, facilitating their excretion from the body through urine or bile. A foreign compound that already possesses a functional group can bypass phase I metabolism and directly take part in phase II metabolism before being eliminated from the body. Unlike phase I enzymes serving for activation metabolism, phase II enzymes deactivate and detoxify foreign compounds and are referred to as detoxification enzymes.

5.1

Excretors

Phase I enzyme-catalyzed reactions provide a foreign compound with an appropriate functional group for a succeeding phase II metabolic reaction. Phase II enzymecatalyzed reactions serve as a detoxification process by conjugating the incorporated functional group in a foreign compound with a small, endogenous molecule (called conjugating ligand). As a result of this, on the one hand, phase II conjugation reactions reduce the reactivity of intermediate metabolites, thus diminishing their potential toxicity to the cells. On the other hand, conjugation reactions greatly increase the aqueous solubility of foreign compounds, thus facilitating their elimination from the body. Phase II enzymes that catalyze reactions to detoxify and facilitate the excretion of foreign compounds are referred to as excretors. Although conjugation reactions generally result in a great increase in the solubility of foreign compounds, there are exceptional cases (methylation and acetylation) that produce conjugates with less solubility. However, the efficiency of the detoxification C.-H. Chen, Activation and Detoxification Enzymes: Functions and Implications DOI 10.1007/978-1-4614-1049-2_5, © Springer Science+Business Media, LLC 2012

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process depends on the functions of both phase II enzymes and phase I enzymes. Phase II enzymes mainly belong to the class of transferases, which are responsible for catalytic conjugation reactions in phase II metabolism. Besides transferases, a few nonconjugation enzymes that engage in detoxification processes are also classified as phase II enzymes.

5.2

Conjugation Enzymes

Phase II detoxification enzymes catalyze conjugation reactions, making metabolites less reactive toward cell components, more soluble in water, and easier to eliminate in the urine. Phase II conjugation enzymes are composed of a set of transferase enzymes, including uridine-diphosphate-glucuronosyltransferases (UGTs), glutathione S-transferases, sulfotransferases, acetyltransferases, methyltransferases, and acyltransferases. Among them, UGTs, glutathione S-transferases, and sulfotransferases are the major phase II enzymes. The functional properties of these conjugation enzymes are described as follows.

5.2.1

Uridine-Diphosphate-Glucuronosyltransferase

UGT is one of the most important transferases that catalyze conjugation reactions in phase II metabolism. The enzyme is located in the endoplasmic reticulum and contains regions of a membrane association and the transmembrane domain. UGT activity is therefore highly dependent on lipids. UGTs are a family of enzymes that catalyze the covalent binding of glucuronic acid to a wide range of lipophilic chemicals. Glucuronidation reaction involves the transfer of glucuronic acid from uridine-diphosphate (UDP)-glucuronic acid to a functional group of foreign compound (substrate). The liver is the major site of glucuronidation in the living organism. Other tissues where glucuronidation occurs include the kidney, gastrointestinal tract, and lungs. A major consequence of glucuronidation is a significant increase in the aqueous solubility of foreign compound. As a major pathway in phase II metabolism, glucuronidation represents one of the most important phase II reactions involving the conversion of lipophilic xenobiotics and their metabolites into hydrophilic conjugates, thus facilitating their excretion. Glucoronidation accounts for about 35% of drug conjugations. The site of glucuronidation is generally at electron-rich nucleophilic atom (e.g., O, N, or S). The substrates for glucuronidation consist of functional groups such as aliphatic alcohols and phenols, carboxylic acids, or aromatic and aliphatic amines. For example, glucuronic acid conjugates with phenolic, carboxylic, or sulfhydryl group of a substrate to form O-glucuronide and with an amino group of a substrate to form N-glucuronide. Glucoronidation is therefore the primary metabolic reaction for many compounds containing functional groups such as –OH, –COOH, –SH, and –NH2. Lipophilic foreign compounds catalyzed in phase I reactions are converted into electrophilic or nucleophilic metabolites. Electrophilic metabolites are often conjugated

5.2 Conjugation Enzymes

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Fig. 5.1 Schematic representation of phase O, I, II, and III metabolisms

Lipophilic foreign compound Phase O metabolism Entering into cells Phase I metabolism

Electrophilic metabolite

Nucleophilic metabolite

Phase II metabolism Conjugates ATP-dependent exporter Phase III metabolism Excreting from cells Eliminating from the body

by glutathione S-transferases, while nucleophilic metabolites are mainly conjugated by UGTs, sulfotransferases, and other transferases. Since UGTs catalyze conjugation reactions to generate products that are more polar, more soluble in water, and more readily available for excretion, glucuronidation generally detoxifies foreign compounds and is considered to be beneficial. Nevertheless, in certain cases, glucuronidation of xenobiotics is not simply just a detoxification process, and glucuronidation reaction may produce potentially toxic conjugate such as acylglucuronides. After glucuronidation, the produced glucuronides are transported to the kidneys and are available for excretion in the urine or through the apical surface of the liver hepatocytes into the bile ducts. The excretion of conjugates from cells requires ATPdependent export pumps such as multidrug resistance proteins, which is carried out by phase III mechanism. Opposite to phase O metabolism that is in charge of entering lipophilic foreign compounds into the cells, phase III metabolism is responsible for the export of conjugates from the cells. Schematic representations of phases O, I, II, and III metabolism are shown in Fig. 5.1.

5.2.2

Glutathione S-Transferase

Mammalian glutathione S-transferase (GST) families consist of cytosolic, mitochondrial, and microsomal GSTs. GST families are therefore differentiated into cytosolic enzymes and membrane-bound enzymes. Human GSTs are a multigene

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family of enzymes that are involved in the metabolism of a wide range of electrophilic foreign compounds including arene oxides, unsaturated carbonyls, organic halides, and other xenobiotics. GSTs catalyze the conjugation of glutathione (GSH) to a variety of foreign compounds, and are generally recognized as detoxification enzymes. GSTs detoxify not only electrophiles derived from xenobiotics, but also endogenous electrophiles. Endogenous electrophiles reacting with oxygen are usually the consequence of free radical that can cause damages to proteins, lipids, and DNA. Besides serving as a catalyst for conjugation reactions, GST also involves the transfer of glutathione, a reaction that occurs in the cytosol of the liver. The tripeptide glutathione is synthesized from g-glutamic acid, cysteine, and glycine. Glutathione is abundant in most cells, especially in the liver. Glutathione has the ability to bind many hydrophobic substances, such as bilirubin, steroids, and polycyclic aromatic hydrocarbons. Glutathione S-transferases catalyze the conjugation of reduced glutathione (GSH) with a xenobiotic through the formation of a thioether bond between the sulfur atom of GSH and carbon or nitrogen in an electrophilic foreign compound. The conjugate is subsequently metabolized to form cysteine or N-acetylcysteine. Substrates for GST-catalyzed reactions commonly exhibit hydrophobic and electrophilic characteristics. Glutathione conjugation is an extremely important mechanism in metabolizing electrophilic foreign compounds. Electrophiles are potentially toxic species, since they can bind to nucleophiles such as proteins and nucleic acids, leading to cellular damage and genetic mutation. Foreign compounds that undergo GST-catalyzed conjugation reactions include alkyl and aryl halides, isothiocyanates, a,b-unsaturated carbonyls, and epoxides. GSTs also play an important role in the detoxification of a broad spectrum of electrophilic metabolites such as aflatoxin B1 and benzo[a]pyrene, which may lead to cytotoxicity or mutagenicity. In many cases, GST-catalyzed conjugation reactions inactivate reactive metabolites or intermediates, and facilitate their excretion from the body. However, in some cases, GSH conjugation may become an activation step, due to the formation of active intermediates or the degradation of metabolites. In addition, the conjugation with glutathione may also be involved in the activation of some carcinogens.

5.2.3

Sulfotransferase

More than fourty cytosolic sulfotransferases have been identified from mammals, which constitute five different families. Sulfotransferases, located in the cytosol, catalyze sulfonate conjugation reactions of relatively small lipophilic xenobiotics. Sulfotransferase-catalyzed conjugation reaction is another major pathway of phase II metabolism. Sulfonation reaction involves the transfer of a sulfonate group (SO3−) from the donor, 3¢-phosphoadenosine 5¢-phosphosulfate (PAPS), to the acceptor, a nucleophilic group of foreign compounds. PAPS is the cofactor of the enzyme for the sulfonation reaction. It is synthesized in tissues to make available an activated

5.2 Conjugation Enzymes

41

form of sulfonate for sulfotransferase-catalyzed reactions. The synthesis is dependent on the availability of sulfonate and the activity of the enzymes involving in its synthesis. The concentration of PAPS is relatively low, limiting the capacity for sulfonation of foreign compounds, and making sulfonation a high-affinity, but low capacity pathway for xenobiotic conjugation. A variety of functional groups in xenobiotic molecules are substrates for sulfotransferases (e.g., hydroxyl group of aliphatic and aromatic compounds). Sulfonation conjugation reaction involves the nucleophilic attack of oxygen or nitrogen atom in a foreign compound on sulfur atom in PAPS, resulting in the cleavage of the phosphosulfate bond. Many foreign compounds (e.g., aromatic and aliphatic alcohols) that are involved in O-glucuronidation also undergo sulfonation. Sulfonation is not limited to aliphatic and aromatic compounds that contain a hydroxyl group. Other compounds involving sulfonation conjugation include aromatic amines such as aniline. Sulfonation generally produces a highly water-soluble sulfuric acid ester. The introduction of negative charge (SO3−) to a xenobiotic affects not only its aqueous solubility, but also its interaction with transport proteins such as ATP-dependent multidrug-resistance proteins. Generally, sulfonation conjugation is a detoxification reaction, which results in facilitating the elimination of foreign compounds from the body. Therefore, sulfonation is an effective means of decreasing the toxic activities of foreign compounds. However, the sulfonate is an electron withdrawing group and certain sulfonate conjugates are chemically unstable and are degraded to form potent electrophilic species. For instance, in drug metabolism, chemically active conjugates may degrade to generate reactive electrophilic cations. As a result of this, in certain cases, sulfonation conjugation increases the toxicity of foreign compounds. Thus, sulfonation affects many different physiological processes including deactivation and bioactivation of xenobiotics.

5.2.4

N-Acetyltransferase

N-Acetyltransferases (NAT) are cytosolic enzymes found in liver and other tissues. Two functional NAT genes are present in human, NAT1 and NAT2, which are closely related to proteins with an active site cysteine residue. There are over 25 allelic variants of NAT1 and NAT2. NATs catalyze the transfer of the acetyl group of acetyl-coenzyme A (acetyl-CoA) to aromatic amine or hydrazine group of a foreign compound. Acetyl-CoA acts as the cofactor in the reaction. NATs are therefore responsible for the metabolism of chemicals that contain aromatic amine or hydrazine. These chemicals are converted to aromatic amide or hydrazide, respectively. In NAT-catalyzed reaction, the acetyl group of acetyl-CoA is initially transferred to a cysteine residue in NAT active site, which is then relocated to the amino group of the substrate (foreign compound). Unlike UGT, GST, or ST-catalyzed reactions, acetylation is not associated with increased aqueous solubility of xenobiotics. The addition of the acetyl group often

42

5

Phase II Enzymes

leads to a conjugate that is less soluble in water because of the covalent binding of the acetyl group to amine or hydrazine group. Generally, acetyl conjugation is a detoxification reaction, resulting in facilitating the elimination of foreign compounds from the body. Nevertheless, in some cases, acetylation of substrate may also be an activation process. For example, aromatic amines can be both activated and deactivated by NATs. Activation occurs when aromatic amines are initially hydroxylated by CYP450 to form hydroxyaromatic amines and are then converted to form esters.

5.2.5

Methyltransferase

Methyltransferases are primarily cytosolic enzymes, but are also present in the endoplasmic reticulum. Methylation is usually a minor metabolic route in foreign compound metabolism. A conjugation reaction catalyzed by methyltransferase involves the transfer of the methyl group attached to the sulfonium ion of S-adenosylmethionine (SAM) to the functional group of the substrate to form a methyl conjugate. SAM acts as the methyl donor and the functional group of the xenobiotic acts as the acceptor. Xenobiotics can undergo O-methylation, N-methylation, and S-methylation through S-adenosyl-methionine-dependent conjugation reactions. Accordingly, the functional groups of a substrate that are involved in methyl conjugation include phenol (O-methylation), aromatic amines (N-methylation), and sulfhydryl-containing group (S-methylation). Pharmacogenetic studies have characterized several methyltransferases including catechol O-methyltransferase, thiopurine methyltransferase, and histamine N-methyltransferase. The methylation of inorganic arsenic to monomethylarsonic acid and dimethylarsinic acid has been considered to be the major pathway for inorganic arsenic biotransformation and detoxification. Similar to acetylation catalyzed by N-acetyltransferases, methylation generally decreases the water solubility of xenobiotics. Methyl conjugation usually carries out a detoxification metabolism which results in a more rapid elimination of foreign compounds. However, human methyltransferases have been reported to exhibit inherited variations, which may contribute to individual’s variation in arsenic metabolism and, perhaps, arsenicdependent carcinogenesis in humans.

5.2.6

Acyltransferase

Acyltransferases are present in mammalian mitochondria, and exist in two distinct forms involving the conjugation of acyl portion of carboxyl group with various amino acids. Amino acid conjugation occurs in both liver and kidney, and has been reported to occur with glycine, glutamine, arginine, and taurine. Acyltransferasecatalyzed conjugation reaction carries out the conjugation of carboxylic acid in xenobiotics with CoA in acyl-CoA to produce an acyl-CoA thioester, which then

5.3

Nonconjugation Enzymes

43

reacts with the amino group of an amino acid to form an amide linkage. Amino-acid conjugation of foreign compounds that contain carboxylic acid is considered to be an alternative to glucuronidation conjugation. Acyltransferase-catalyzed amino acid conjugation of carboxylic acid typically results in detoxification and elimination of xenobiotics, while in some cases it is not simply just a detoxification process.

5.3

Nonconjugation Enzymes

Most foreign compounds require activation catalyzed by phase I enzymes (e.g., CYP450) to reactive electrophilic intermediates in order to exert their toxic and neoplastic effects. In addition to conjugation enzymes that play crucial roles in detoxification of xenobiotics by conjugation of metabolite intermediates with small endogenous molecules, there are also nonconjugation detoxification enzymes. Quinone reductase and epoxide hydrolase are also classified as phase II enzymes. These two nonconjugation enzymes are briefly described below.

5.3.1

Quinone Reductase

Quinone reductase, a flavoprotein, is one of several phase II enzymes that are involved in the nonoxidative metabolism of a wide variety of xenobiotics. The enzyme acts on NADH or NADPH with a quinone. The substrates of quinone reductase are NADPH, H+, and quinone. The reaction products are NADP+ and semiquinone. Quinones are among the toxic products of oxidative metabolism of aromatic hydrocarbons. Reduction of electrophilic quinones by quinone reductase is an important detoxification pathway. This enzyme displays a broad specificity for structurally diverse hydrophobic quinines, which results in facilitating the metabolism of quinones to readily excreted conjugates.

5.3.2

Epoxide Hydrolase

Epoxide hydrolases exist in two forms. Soluble epoxide hydrolase is expressed in the cytosol, while microsomal epoxide hydrolase is present in the endoplasmic reticulum. Epoxide hydrolases catalyze hydrolytic reactions for epoxides. Epoxides are organic compounds that consist of a three-membered oxygen. Phase I oxidative reactions catalyzed by CYP450 can generate epoxides as metabolite intermediates during the metabolism of foreign compounds containing unsaturated carbon–carbon bonds. The generated epoxide intermediates are unstable and chemically reactive and have been implicated as ultimate mutagenic and carcinogenic initiators.

44

5

Phase II Enzymes

As a typical detoxification enzyme, mammalian microsomal epoxide hydrolase exhibits a high expression level in the liver and a broad spectrum of substrates. Epoxide hydrolase is capable of inactivating structurally different, highly reactive epoxides, and thus it is important in the defense against adverse effects of foreign compounds. Epoxide hydrolase catalyzes the hydration of chemically reactive epoxides to the corresponding dihydrodiol products. Dihydrodiols can then be conjugated and excreted from the body. Epoxide hydrolases are essential in the detoxification of foreign compounds that contain unsaturated carbon–carbon bonds, resulting in the inactivation of drugs and other xenobiotics.

5.4

Catalytic Actions

Phase II enzyme-catalyzed reactions can occur at different atoms or groups of foreign compounds, including phenol, epoxide, polyphenol, carboxylic acid, and amino acid. Typical examples of catalytic actions of phase II enzymes are schematically presented below, including conjugation reactions as well as nonconjugation reactions. More detailed descriptions of reactions catalyzed by individual phase II enzymes are further discussed in Chap. 14. To emphasize specific atom or functional group involved in the reaction, the presented equation may or may not be balanced. For general description, R and j denote aliphatic and aromatic derivatives, respectively. Most of the reactions may apply to either aliphatic or aromatic compounds.

5.4.1

Conjugation at O Atom

(a) Uridine-diphosphate (UDP)-glucuronosyltransferases-catalyzed reaction: Oxygen atom of hydroxyl group in phenol is conjugated with glucuronic acid (GA) to form a glucuronide derivative. F - OH + UDP - GA ® F - O - GA + UDP (b) Sulfotransferase-catalyzed reaction: Oxygen atom of hydroxyl group in alcohol or phenol is conjugated with sulfonate to form a sulfonate derivative. F - OH + PAPS ® F - O - SO3 H + PAP where PAPS and PAP denote 3-phosphoadenosine 5-phosphosulfate and 3-phosphoadenosine 5-phosphate, respectively. (c) Methyltransferase-catalyzed reaction: Oxygen atom of hydroxyl group in phenol is conjugated with methyl group of S-adenosylmethionine to form methyl conjugate.

5.4

Catalytic Actions

45

F - OH + SAM ® F - O - CH 3 + SAH Methyl group in SAM binds to O atom in phenol, where SAM and SAH denote S-adenosylmethionine and S-adenosyl-l-homocysteine, respectively.

5.4.2

Conjugation at N Atom

(a) Uridine-diphosphate (UDP)-glucuronosyltransferases-catalyzed reaction: Glucuronic acid (GA) attaches to N atom of amine to form a glucuronide conjugate.

(b) Sulfotransferase-catalyzed reaction: Sulfonate binds to N atom of aromatic amine to form sulfonate conjugate.

(c) Methyltransferase-catalyzed reaction: Methyl group of SAM attaches to N-atom of amine to form methyl conjugate. See Sect. 5.4.1, point (c) for abbreviations for SAM and SAH.

(d) Acetyltransferase-catalyzed reaction: Acetyl group of acetyl-CoA binds to N atom of amine to form acetyl conjugate. F - NH 2 + Acetyl - CoA ® F - NH - COCH 3 + CoA where Acetyl-CoA serves as the cofactor of the enzyme.

46

5.4.3

5

Phase II Enzymes

Conjugation at C Atom

(a) Glutathione S-transferase (GST)-catalyzed reaction: Reduced glutathione (GSH) binds to C atom of epoxide to form a glutathione conjugate.

5.4.4

Conjugation at S Atom

(a) Uridine-diphosphate (UDP)-glucuronosyltransferases (GT)-catalyzed reaction: Glucuronic acid (GA) binds to the S atom of thio compound to form a glucuronide conjugate. R - SH + UDP - GA ® R - S - GA + UDP (b) Methyltransferase-catalyzed reaction: The methyl group attached to the sulfonium ion in SAM binds to the S atom of thio compound to form methyl conjugate. F - SH + SAM ® F - S - CH3 + SAH

5.4.5

Conjugation of Carboxylic Acid

(a) Uridine-diphosphate (UDP)-glucuronosyltransferases-catalyzed reaction: Glucuronic acid (GA) reacts with carboxylic acid to form a glucuronide conjugate.

(b) N-Acyltransferase-catalyzed reaction: An amino acid reacts with a carboxylic acid group to form an amide, where acyl-CoA serves as the cofactor for N-acyltransferase in the conjugation reaction.

Bibliography

5.4.6

47

Nonconjugation Reactions

(a) Quinone reductases-catalyzed reaction: NADPH is involved in the reduction of quinone to hydroquinone.

(b) Epoxide hydrolase-catalyzed reaction: Epoxide is hydrolyzed to form dihydrodiol.

Bibliography Arand M, Cronin A, Adamska M, Oesch F (2005) Epoxide hydrolases: structure, function, mechanism, and assay. Methods Enzymol 400:569–588 Benson AM, Hunkeler MJ, Talalay P (1980) Increase of NAD(P)H:quinone reductase by dietary antioxidants: possible role in protection against carcinogenesis and toxicity. Proc Nat Acad Sci USA 177:5216–5220 Decker M, Arand M, Cronin A (2009) Mammalian epoxide hydrolases in xenobiotic metabolism and signalling. Arch Toxicol 83:297–318 Dekant W, Vamvakas S (1993) Glutathione-dependent bioactivation of xenobiotics. Xenobiotica 23:873–887 Evans D (1992) N-acetyltransferase. In: Kalow W (ed) Pharmacogenetics of drug metabolism. Pergamon, New York Fretland AJ, Omiecinski CJ (2000) Epoxide hydrolases: biochemistry and molecular biology. Chem Biol Interact 129:41–59 Gamage N, Barnett A, Hempel N et al (2006) Human sulfotransferases and their role in chemical metabolism. Toxicol Sci 90:5–22 Grant DM, Blum M, Meyer UA (1992) Polymorphisms of N-acetyltransferase genes. Xenobiotica 9–10:1073–1081 Hayes JD, Flanagan JU, Jowsey IR (2005) Glutathione transferases. Annu Rev Pharmacol Toxicol 45:51–88 Kato R, Yamazoe Y (1994) Metabolic activation of N-hydroxylated metabolites of carcinogenic and mutagenic arylamines and arylamides by esterification. Drug Metab Rev 26:413–429 King C, Rios G, Green M, Tephly T (2000) UDP-glucuronosyltransferases. Curr Drug Metab 1:143–161 Klaassen CD, Boles JW (1997) Sulfation and sulfotransferases 5: the importance of 3¢-phosphoadenosine 5¢-phosphosulfate (PAPS) in the regulation of sulfation. FASEB J 11:404–418 Mannervik B, Danielson UH (1988) Glutathione transferases – structure and catalytic activity. CRC Crit Rev Biochem 23:283–337

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Meech R, Mackenzie PI (1997) Structure and function of uridine diphosphate glucuronosyltransferases. Clin Exp Pharmacol Physiol 24:907–915 Miners JO, Knights KM, Houston JB et al (2006) In vitro–in vivo correlation for drugs and other compounds eliminated by glucuronidation in humans: pitfalls and promises. Biochem Pharmacol 71:1531–1539 Negishi M, Pedersen LG, Petrotchenko E et al (2001) Structure and function of sulfotransferases. Arch Biochem Biophys 390:149–157 Rao PV, Krishna CM, Zigler JS Jr (1992) Identification and characterization of the enzymatic activity of zeta-crystallin from guinea pig lens. A novel NADPH:quinone oxidoreductase. J Biol Chem 267:96–102 Talalay P (2000) Chemoprotection against cancer by induction of phase 2 enzymes. Biofactors 12:5–11 Weinshilboum RM, Otterness DM, Szumlanski CL (1999) Methylation pharmacogenetics: catechol O-methyltransferase, thiopurine methyltransferase, and histamine N-methyltransferase. Annu Rev Pharmacol Toxicol 39:19–52 Wilce MC, Parker MW (1994) Structure and function of glutathione S-transferases. Biochim Biophys Acta 1205(1):1–18 Wildfang E, Zakharyan RA, Aposhian HV (1998) Enzymatic methylation of arsenic compounds. VI. Characterization of hamster liver arsenite and methylarsonic acid methyltransferase activities in vitro. Toxicol Appl Pharmacol 152:366–375 Wood TC, Salavagionne OE, Mukherjee B et al (2006) Human arsenic methyltransferase (AS3MT) pharmacogenetics: gene resequencing and functional genomics studies. J Biol Chem 281:7364–7373 Zakharyan RA, Wildfang E, Aposhian HV (1996) Enzymatic methylation of arsenic compounds. Toxicol Appl Pharmacol 140:77–84

Chapter 6

Reactive Intermediate Formation

Some foreign compounds are acutely or potentially toxic, and others initially exhibit no intrinsic toxicity, but become harmful after metabolic conversion. Although foreign compound-metabolizing enzymes aim to produce water soluble metabolites, facilitating the excretion of foreign compounds from the body, however in many cases, metabolic conversion produces reactive intermediates. As a result of this, many toxic effects of foreign compounds do not result from the parent compounds, instead from reactive intermediates or metabolites that are formed inside cells. Reactive intermediate formation during metabolic activation is an important mechanism attributing to foreign compound-mediated toxicity. For example, the toxicity of most organic chemicals is associated with their enzymatic conversion to toxic metabolic intermediate; a process is commonly referred to as bioactivation (metabolic activation). Bioactivation can be brought about by either phase I reactions or phase II reactions. Consequently, a toxic effect of a foreign compound is determined by not only the chemical nature of a foreign compound, but also the enzyme reaction that metabolizes it. The increased reactivity of metabolic intermediate or metabolite is primarily the result of conversion into electrophiles or free radicals. Most metabolic intermediates or metabolites are electrophiles. CYP450 is the most important enzyme that catalyzes the formation of electrophiles. The conversion of foreign compounds into nucleophiles is relatively not common. Although some nucleophiles are reactive, many are further activated by conversion to electrophiles. There are nonionic and cationic electrophiles. Nonionic electrophiles include aldehydes, ketones, epoxides, quinones, sulfoxides, nitroso compounds, and acyl halides. Cationic electrophiles include carbonium ions and nitrenium ions. Lipophilic compounds are metabolized into two phases. In phase I, a functional group (e.g., hydroxyl or carboxyl) is introduced into a foreign compound by the activation of enzyme-catalyzed reaction. Subsequently, in phase II, an endogenous molecule (e.g., glucuronic, sulfuric, or amino acid) is added to the functionalized foreign compound by detoxification of the enzyme-catalyzed reaction. Figure 6.1 illustrates reactive intermediate formation in foreign compound metabolisms. Enzymes important in catalyzing bioactivation mainly involve phase I enzymes C.-H. Chen, Activation and Detoxification Enzymes: Functions and Implications, DOI 10.1007/978-1-4614-1049-2_6, © Springer Science+Business Media, LLC 2012

49

50

6 Reactive Intermediate Formation Inactive metabolite Phase II Excretion detoxification

Phase I Foreign compound activation Reactive intermediate

Interaction with cellular components ( proteins, DNA, lipids ) Toxic effects

Fig. 6.1 Reactive intermediate and foreign compound-mediated toxicity

(e.g., CYP450, flavin-containing monooxygenase, prostaglandin synthetase, and alcohol dehydrogenase), but in some cases phase II enzymes (e.g., glutathione S-transferase, uridine-diphosphate-glucuronosyltransferases, and sulfotransferase), are also involved. Figure. 6.1 reveals that phase I metabolism either yields inactive metabolites that are ready for phase II metabolism before their excretion or produces highly reactive intermediates that are capable of interacting with cellular components if not quickly detoxified by phase II metabolism.

6.1

Reactive Intermediates

Functionalization reactions catalyzed by phase I enzymes often convert foreign compounds into less harmful substances. However, a significant number of foreign compounds are converted to metabolic intermediates that are substantially more reactive than the parent compounds. While foreign compound metabolism serves as an important protective biological process, in some cases, it leads to producing adverse effects within the organism. When chemically active intermediates are created in sufficient quantities and are not promptly stabilized by endogenous substrates or other mechanisms, damage of cells, and tissues can occur. It is therefore essential to minimize the presence of reactive intermediates in intracellular levels and quickly remove them from the body. Reactive intermediates are frequently generated by chemical decomposition of foreign compounds. In order to exert their toxic and neoplastic effects, most chemical carcinogens require metabolic activation catalyzed by phase I enzymes. In a number of cases, phase II enzymes are also implicated. Nevertheless, many other foreign compounds are also oxidized by CYP isozymes to nontoxic metabolites. Thus, the same enzyme that detoxifies a foreign compound can be responsible for the activation of another. The best documented example of enzymatic processes that produce reactive intermediates is the oxidation of foreign compounds by the family of CYP450 enzymes. Metabolic intermediates produced by phase I activation reactions are often electrophilic species, free radicals, or modified chemicals. Electrophile is a compound

6.3

Enzyme-Catalyzed Reactive Intermediate Formation

51

Activation Oxygen Foreign compound Reactive intermediate Reactive oxygen species

Fig. 6.2 Formation of reactive oxygen species

that is attracted to electrons and hence is a Lewis acid. Most electrophiles are positively charged, have an atom carrying a partial positive charge, or possess an atom not having an octet of electrons. Electrophiles include cations (e.g., H+ and NO+), polarized neutral molecules (e.g., HCl, alkyl halides, acyl halides, and carbonyl compounds), and polarizable neutral molecules (e.g., Cl2 and Br2). An electrophile takes part in a chemical reaction that involves binding to a nucleophile.

6.2

Reactive Oxygen Species

Reactive intermediates can interact with oxygen molecules to produce reactive oxygen species. Reactive oxygen species are activated reactive intermediates, including highly reactive cationic electrophiles (e.g., carbonium, iminium, and nitrenium ions) as well as free radicals. Peroxidases are a major enzyme that catalyzes free radical formation. Free radicals formed in metabolic activation include superoxide anion radical (O2−•) and hydroxyl radical (HO•). Figure 6.2 shows the formation of reactive oxygen species from reactive intermediates. It is important to note that the mechanism underlying free radical formation produced from reactive intermediates of foreign compounds is different from that generated from aerobic electron transfer chains in living organisms. Reactive oxygen species are continuously produced during metabolic processes of foreign compounds, and simultaneously are detoxified by a variety of defense mechanisms. When the process of generation is more favor than that of detoxification, damages of cellular macromolecules occur. Although a few reactive oxygen species may be implicated in cell signaling and in the immune system as a way to attack pathogens, highly reactive ions and free radicals are able to interact with cellular components (proteins, membrane lipids, or nucleic acids), resulting in damage to cellular molecules and their functions.

6.3

Enzyme-Catalyzed Reactive Intermediate Formation

CYP450 is the most important phase I activation enzyme attributing to the generation of reactive intermediates (e.g., epoxides, carbonium, iminium and nitrenium ions, and free radicals). Other phase I enzymes including peroxidase and flavin monooxygenases also participate in catalytic reactions that produce reactive intermediates. In certain cases, conjugation reactions catalyzed by phase II enzymes are also involved. Examples of reactive intermediate formation mediated by phase I enzymes and phase II enzymes are briefly discussed below.

52

6.3.1

6 Reactive Intermediate Formation

Mediation by Phase I Enzymes

Typical examples of reactive intermediate formation mediated by phase I activation enzymes are shown in Table 6.1, where the listed foreign compounds primarily are drug ingredients. The two most known compounds in the list are aflatoxin and acetaminophen. Aflatoxins are mycotoxins. Various aflatoxins are present in nature, including aflatoxin B1 produced by Aspergillus, a fungus. Following prolonged exposure to a high humidity environment, host crops are particularly susceptible to infection by Aspergillus. Aflatoxins are among the most carcinogenic substances, with aflatoxin B1 being considered the worst. Figure 6.3 shows reactive intermediate of aflatoxin B1 and its toxic effects on DNA. Acetaminophen (paracetamol) is a major ingredient in widely used over-the-counter analgesic and antipyretic drugs. It is commonly used as cold and flu remedies for the relief of fever, headache, and pain. Acetaminophen is also used in the management of severe pain when it is combined with nonsteroidal anti-inflammatory drugs and opioid analgesics. Acute overdose of acetaminophen (over 1,000 mg per single dose or 4,000 mg per day for adults) can cause potentially fatal liver damage. The risk is heightened by alcohol consumption. Figure 6.4 illustrates potential toxic effects of metabolic intermediate of acetaminophen on the liver and kidney.

6.3.2

Mediation by Phase II Enzymes

Chemically active intermediates are mainly generated by phase I enzyme-catalyzed reactions. However, in certain cases, conjugation reactions catalyzed by phase II enzymes may also be involved in their formation. For example, the formation of benzo[a]pyrene 7,8-dihydrodiol-9,10-epoxide, benzo[a]pyrene metabolic intermediate, is catalyzed by epoxide hydrolase, a nonconjugation phase II enzyme. In some cases, reactive intermediate formation is also mediated by phase II conjugation enzymes (e.g., UDP-glucuronosyltransferase, sulfotransferase, glutathione S-transferase, and N-acetyltransferase). Examples of conjugation enzyme-mediated reactive intermediate formation are listed in Table 6.2.

6.4

Interactions with Cellular Components

When produced intracellularly in sufficient quantities, reactive intermediates, particularly reactive oxygen species, have the capacity to interact with cellular components (proteins, DNA, and lipids), ultimately leading to toxic effects, which may trigger alterations in the structure and function of target molecules. Reactive intermediates have therefore been implicated in various disease conditions (e.g., carcinogenesis, tissue allergic responses, and tissue necrosis). When electrophilic metabolic intermediates are extremely active and are not effectively deactivated by

6.4

Interactions with Cellular Components

53

Table 6.1 Examples of metabolic intermediates mediated by phase I enzymes Foreign compound Activation enzyme Reactive intermediate Acetaminophen CYP450 N-Acetyl-p-benzoquinoneimine 2-Acetylaminofluorene CYP450 3,7,9-Hydroxyacetylaminofluorene Acetylhydrazine FMO Acetyl radical or cation Acrylamide CYP450 Glycidamide CYP450/Peroxidase Aflatoxin B1-8,9-epoxide Aflatoxin B1 Benzo[a]pyrene CYP450/epoxide hydrolase Benzo[a]pyrene 7,8-dihydrodiol-9,10-epoxide Bromobenzene CYP450 Bromobenzene 3,4-oxide 4-Ipomeanol CYP450 a,b-Unsaturated dialdehyde Menadione CYP450 Semiquinone 2-Naphthylamine CYP450 N-Hydroxy-naphthylamine N-Nitrosodimethylamine CYP450 Carbonium ion Polycyclic aromatic CYP450/epoxide hydrolase PAH epoxides hydrocarbon (PAH) Vinyl acetate Carboxylesterase Acetaldehyde CYP450 cytochrome P450, FMO flavin monooxygenases

Fig. 6.3 Metabolic intermediate and toxicity of aflatoxin B1

Aflatoxin B1

CYP450 Peroxidase

Aflatoxin B1 8,9-epoxide Glutathione

DNA

Inactivation DNA binding Liver tumors

Sulfotransferase UDP-glucuronosyl transferase Acetaminophen sulfate Acetaminophen Acetaminophen glucuronide CYP450 PHS Quinoneimine

Semiquinoneimine radical

Binding to hepatic proteins Damage to liver

Binding to renal proteins

Damage to kidney

Fig. 6.4 Metabolic intermediates and toxicity of acetaminophen

nucleophilic species in the cells, covalently binding to proteins or DNA and oxidation of lipids occur, leading to the formation of protein adducts, DNA adducts, and lipid peroxidation. The interactions of reactive intermediates with cellular components can be either specific or nonspecific. Specific interaction involves the binding to a specific site in a receptor, enzyme, or transport protein.

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6 Reactive Intermediate Formation

Table 6.2 Examples of metabolic intermediates mediated by phase II enzymes Conjugation Foreign compound enzyme Reactive intermediate 2-Aminonaphthalene UGT N-Hydroxy-2-naphthylamine Arylamine NAT Aryl nitrenium ion Carboxylic acid UGT Acyl glucuronide Dibromoethane GST Episulfonium ion Dichloromethane GST Formaldehyde Hexachlorobutadiene GST Mercapturic acid 7-Hydromethyl,12-methyl-benz[a] SULT Carbonium ion anthracene N-Hydroxy-2-acetylaminofluorene SULT Nitrenium ion N-Hydroxy-2-aminofluorene NAT Nitrenium ion Tamoxifen SULT Reactive carbocation UGT UDP-glucuronosyltransferases, SULT sulfotransferase, GST glutathione S-transferases, NAT N-acetyltransferases

6.4.1

Protein Adducts

The primary binding sites of protein adducts are sulfur-containing amino acid residues (cysteine and methionine), nitrogen of lysine, or histidine residues. Although not all metabolic intermediate bindings to protein are toxic in nature, protein adducts may be the source of toxicity caused by foreign compounds. The binding may result in the inactivation of enzymatic functions. For example, the binding of microcystins (produced from blue-green algae) to phosphatase leads to the inactivation of the enzyme.

6.4.2

DNA Adducts

Reactive intermediate, particularly reactive oxygen species, binding to DNA has drawn more attention than protein binding. DNA binding occurs when metabolic intermediate interacts with DNA base. Electron-dense sites, like nitrogen and oxygen atoms in nucleic acid, are primary binding sites. Any of four bases in DNA may participate in the interaction. Among them, guanine residue is the predominant base for the binding. DNA adduct is not always stable. For example, hydrolysis can cleave the bonding between electrophilic metabolite and purine compound. DNA adduct formation can affect DNA base pairing. When DNA undergoes replication, erroneous base pairing can result in base mismatch. Mutation can occur if base mismatch is not repaired during DNA replication.

6.4

Interactions with Cellular Components Protein Protein adduct Reactive intermediate DNA Reactive intermediate DNA adduct

55

Cytotoxicity Mutation

Lipid Reactive oxygen species Lipid peroxidation

Malignancy

Tissue damages

Fig. 6.5 Interactions of reactive intermediates with cellular molecules

6.4.3

Lipid Peroxidation

Lipid peroxidation refers to the oxidative degradation of membrane lipids. Cell damage occurs when free radicals react with membrane lipids. Lipid peroxidation most often affects polyunsaturated fatty acids that contain multiple double bonds between methylene groups and possess reactive hydrogens. The mechanism of lipid peroxidation consists of three major steps: initiation, propagation, and termination. Initially, fatty acid free radical is produced when reactive oxygen species (e.g., OH) reacts with unsaturated lipid. The resulting unstable fatty acid free radical can react with oxygen to yield peroxyl-fatty acid radical, which can then react with another fatty acid free radical to produce a different fatty acid radical and lipid peroxide. To terminate this propagation process, living organisms develop defense systems (e.g., antioxidant enzymes and small molecules) that neutralize free radicals to produce nonradical species to protect cell membranes. If the produced free radicals are not terminated fast enough, damage to the cell membranes can occur. The end products of lipid peroxidation may be mutagenic and carcinogenic, due to the capacity of free radicals to react with DNA.

6.4.4

Toxic Effects

Figure 6.5 reveals the interactions of chemically active intermediates with proteins, DNA, and lipids, leading to the formation of protein adducts, DNA adducts, and the peroxidation of lipids, respectively. The figure also shows the potential toxic effects as a result of interactions with cellular components. The toxic effects caused by the accumulation of reactive intermediates and reactive oxygen species in the body could contribute to a variety of disease conditions, such as aging, cancer, cardiovascular disease, and neurological disorders. The liver is not only the principal site for the activation of foreign compounds and the center of production of metabolic intermediates, but also the primary site of toxic effects. Nevertheless, metabolic intermediates may also be exported to other tissues and cause harmful effects there. For example, aromatic amines are potential urinary bladder carcinogens.

56

6.5

6 Reactive Intermediate Formation

Defense Against Reactive Intermediates

The protection against harmful effects of reactive intermediates or reactive oxygen species is to minimize their presence in intracellular levels. To achieve this goal, the body develops several defense systems that include conjugation reactions, glutathione, and antioxidant enzymes. These defense systems are briefly described below.

6.5.1

Conjugation Reactions

To reduce the intracellular levels of reactive intermediates or reactive oxygen species, the body depends heavily on conjugation reactions catalyzed by phase II detoxification enzymes, including uridine-diphosphate-glucuronosyltransferases, glutathione S-transferases, and sulfotransferases. Conjugation reactions generally greatly increase aqueous solubility of foreign compounds (with exception such as acetylation), thus facilitating the excretion of foreign compounds from the body. Metabolic conjugation usually represents a detoxification process as shown in a large majority of studies. Hence, conjugation of foreign compound with glucuronic acid, glutathione, or sulfonate in phase II reactions was originally thought to represent exclusively as a detoxification process for drugs and other chemicals. However, in a number of cases, it has been found that the resulting conjugates are harmful (see Table 6.2, for examples).

6.5.2

Glutathione

In most cases, reactive metabolites or intermediates are electrophiles that contain an intramolecular center of low electron density (positive center). For inactivation, an electrophilic intermediate prefers to interact with a nucleophilic species that contains an intramolecular center of high electron density (negative center). To remove electrophilic intermediates, the body also relies on endogenous molecules. Glutathione, a nucleophilic species, is the most important endogenous molecule for neutralizing free radicals produced by foreign compound metabolism. Here, glutathione acts as an antioxidant, unlike in glutathione conjugate reaction where glutathione serves as the donor compound. By either neutralizing or combining with chemically active intermediate, glutathione reacts to produce water-soluble compounds, facilitating their excretion from the body through urine or bile. As an antioxidant, glutathione is oxidized to glutathione disulfide, leading to the depletion of glutathione.

6.6

Factors Affecting Xenobiotic Toxicity

Fig. 6.6 Relative rates of activation and detoxification

57

Activation rate Foreign compound Reactive intermediate Detoxification rate Conjugate / metabolite Excretion Transport protein

6.5.3

Antioxidant Enzymes

In detoxifying free radicals produced in aerobic cellular metabolism, the body utilizes antioxidant enzymes as the primary line of defense. Among antioxidant enzymes present in the living organisms, superoxide dismutase converts the superoxide radical to form hydrogen peroxide and oxygen. Catalase and glutathione peroxidase work simultaneously with glutathione to reduce hydrogen peroxide to water and oxygen. Moreover, the body also utilizes small antioxidant molecules in detoxifying free radicals produced in aerobic cellular metabolism. Small antioxidant molecules can neutralize free radicals by accepting or donating an electron, leading to the elimination of the state of unpaired electron in free radicals. For instance, vitamin E protects cell membranes from oxidation damage by free radicals. Vitamin C scavenges free radicals and works with vitamin E to quench free radicals. Betacarotene is the best quencher of singlet oxygen. Besides serve as the primary line of defense in destroying free radicals produced in aerobic cellular metabolism, antioxidant enzymes can also effectively break down reactive oxygen species derived from metabolic conversion of foreign compounds. For examples, glutathione peroxidase catalyzes the reaction in which glutathione breaks down hydrogen peroxide and organic peroxide, while superoxide dismutase catalyzes the reaction that breaks down superoxide anions.

6.6

Factors Affecting Xenobiotic Toxicity

The amount of metabolic intermediates that are present within the cells and the degree of toxicity that exerts on cellular components are dependent on relative rates of activation reaction and detoxification reaction. The role of relative rates of activation and detoxification in foreign compound-mediated toxicity is illustrated in Fig. 6.6. While the metabolism of foreign compounds is supposed to represent a protective biological process, an accumulation of metabolic intermediates within the cells occurs if the rate of activation is higher than that of detoxification. The balance between activation and detoxification processes can often determine whether a foreign compound exerts toxic effects or not. When metabolic intermediate or metabolite is responsible for the toxic effect of the parent compound, the toxicity of a foreign compound depends not only on the chemical nature of the parent compound, but also on the enzymes that catalyze

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its metabolic reactions. Furthermore, foreign compound-metabolic enzymes are affected not only by the genetic makeup, but also by life style modifications. Environmental factors such as nutrition and chemical exposure are able to modulate the activities of these enzymes. Consequently, the extent of foreign compoundinduced toxicity to an individual depends on at least four factors: the chemical nature of the foreign compound, the activity of its activation enzyme, the activity of its detoxification enzyme, and the environment that is exposed.

Bibliography Amacher DE (2006) Reactive intermediates and the pathogenesis of adverse drug reactions: the toxicology perspective. Curr Drug Metab 7:219–229 Anders MW (1985) Bioactivation of foreign compounds. Academic Press, New York Anders MW (2007) Chemical toxicology of reactive intermediates formed by the glutathionedependent bioactivation of halogen-containing compounds. Chem Res Toxicol 21:145–159 Baird WM, Hooven LA, Mahadevan B (2005) Carcinogenic polycyclic aromatic hydrocarbon– DNA adducts and mechanism of action. Environ Mol Mutagen 45:106–114 Boelsterli UA (2007) Mechanistic toxicology. CRC, Boca Raton Bogdanffy MS, Taylor ML (1993) Kinetics of nasal carboxylesterase-mediated metabolism of vinyl acetate. Drug Metab Dispos 21:1107–1111 Chandrasekara A, Shahidi F (2011) Inhibitory activities of soluble and bound millet seed phenolics on free radicals and reactive oxygen species. J Agric Food Chem 59:428–436 Dekant W, Vamvakas S (1993) Glutathione-dependent bioactivation of xenobiotics. Xenobiotica 23:873–887 Eaton DL, Gallagher EP (1994) Mechanisms of aflatoxin carcinogenesis. Annu Rev Pharmacol Toxicol 34:135–172 Glatt H (2000) Sulfotransferases in the bioactivation of xenobiotics. Chem Biol Interact 129:141–170 Guengerich FP (2001) Common and uncommon cytochrome P450 reactions related to metabolism and chemical toxicity. Chem Res Toxicol 14:611–650 Hinson JA, Forkert PG (1995) Phase II enzymes and bioactivation. Can J Physiol Pharmacol 73:1407–1413 James LP, Capparelli EV, Simpson PM et al (2008) Acetaminophen-associated hepatic injury: evaluation of acetaminophen protein adducts in children and adolescents with acetaminophen overdose. Clin Pharmacol Ther 84:684–690 Kalgutkar AS, Dalvie DK, O’Donnell JP et al (2002) On the diversity of oxidative bioactivation reactions on nitrogen-containing xenobiotics. Curr Drug Metab 3:379–424 Kim SY, Suzuki N, Laxmi YR et al (2004) Genotoxic mechanism of tamoxifen in developing endometrial cancer. Drug Metab Rev 36:199–218 Koob M, Dekant W (1991) Bioactivation of xenobiotics by formation of toxic glutathione conjugates. Chem Biol Interact 77:107–136 Levi PE, Hodgson E (2008) Reactive metabolites and toxicity. In: Smart RC, Hodgson E (eds) Molecular and biochemical toxicology. Wiley, New York MacKenzie EL (2008) Reactive oxygen/metabolites and toxicity. In: Smart RC, Hodgson E (eds) Molecular and biochemical toxicology. Wiley, New York McLemore TL, Litterst CL, Coudert BP et al (1990) Metabolic activation of 4-ipomeanol in human lung, primary pulmonary carcinomas, and established human pulmonary carcinoma cell lines. J Natl Cancer Inst 82:1420–1426

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Parkinson A, Ogilvie BW (2008) Biotransformation of xenobiotics. In: Klaassen CD (ed) Casarett and Doull’s toxicology: the basic science of poisons. McGraw-Hill, New York Perlow RA, Kolbanovskii A, Hingerty BE et al (2002) DNA adducts from a tumorigenic metabolite of benzo[a]pyrene block human RNA polymerase II elongation in a sequence- and stereochemistrydependent manner. J Mol Biol 321:29–47 Raucy JL, Kraner JC, Lasker JM (1993) Bioactivation of halogenated hydrocarbons by cytochrome P4502E1. Crit Rev Toxicol 23:1–20 Ritter JK (2000) Roles of glucuronidation and UDP-glucuronosyltransferases in xenobiotic bioactivation reactions. Chem Biol Interact 129:171–193 Shimada T (2006) Xenobiotic-metabolizing enzymes involved in activation and detoxification of carcinogenic polycyclic aromatic hydrocarbons. Drug Metab Pharmacokinet 21:257–276 Smith BJ, Curtis JF, Eling TE (1991) Bioactivation of xenobiotics by prostaglandin H synthase. Chem Biol Interact 79:245–264 Stadtman ER (2006) Protein oxidation and aging. Free Radic Res 40:1250–1258 Turrens JF (2003) Mitochondrial formation of reactive oxygen species. J Physiol 552:335–344

Chapter 7

Biomedical and Biochemical Effects

Potential toxic foreign compounds or metabolites are capable of interacting with an endogenous target, triggering perturbation in cellular functions, and mediating a biochemical or biomedical effect. The cells initially respond to such perturbation with repair and adaptive mechanisms. When the induced perturbation exceeds the repair and adaptive capacity, toxic effects occur. In most cases, foreign compounds are absorbed and distributed to target organs, where they exert harmful effects. Liver and kidneys are frequent target organs of toxicity. The exhibition of foreign compound toxicity including biomedical and biochemical effects may occur under the following circumstances.

7.1

Exhibition of Foreign Compound Toxicity

The exhibition of foreign compound toxicity mainly occurs under the following three circumstances: (1) the presence of intrinsic toxicity in a parent compound, such as microtoxins, (2) the generation of reactive intermediates in activation metabolism that ultimately exhibits toxicity, such as quinoneimine and epoxides, and (3) the induction of toxicity through modulation by environment factors such as smoke and alcohol. Examples presented below describe the above three circumstances that attribute to foreign compound-mediated toxicity.

7.1.1

Intrinsic Toxicity

Microtoxins are produced by fungi that exist in nature and occur frequently in food supplies due to mold infestation of susceptible agricultural products. When toxic fungi are present in foods in sufficiently high levels, fungal metabolites can have toxic effects ranging from acute (liver or kidney deterioration) to chronic disease conditions (e.g., liver cancer). Other foreign compounds that exhibit natural toxicity include nicotine, ethylene oxide, heavy-metal ions, strong acids or bases, and carbon monoxide. C.-H. Chen, Activation and Detoxification Enzymes: Functions and Implications, DOI 10.1007/978-1-4614-1049-2_7, © Springer Science+Business Media, LLC 2012

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7.1.2

7

Biomedical and Biochemical Effects

Toxic Reactive Metabolites

Acetaminophen is a primary ingredient in cold and pain relief remedies. An overdose of acetaminophen can trigger potential liver damage, resulting in major acute liver failure. In the liver, CYP450s are responsible for metabolism of acetaminophen. CYP450-catalyzed reaction metabolizes acetaminophen to produce reactive intermediates (quinoneimine and semiquinoneimine), which cause toxic effects by binding to hepatic and renal proteins.

7.1.3

Induction of Toxicity

Studies of the effect of chronic ethanol consumption have revealed that acetaminophen toxicity as manifested by liver enlargement and congestion is significantly increased in the ethanol-treated group. The enhanced toxicity is a result of alcohol’s effects on hepatocyte membranes, which renders the cells more susceptible to toxic injury. The exposure to alcohol enhances the vulnerability of acetaminophen to hepatotoxicity. Alcohol can activate CYP450 isozyme (CYP2E1) which catalyzes the activation of acetaminophen. Chronic ethanol consumption can potentiate acetaminophen hepatotoxicity through enhanced N-acetyl-p-benzoquinone imine formation, CYP2E1 induction, and selective depletion of mitochondrial glutathione.

7.2

Oxidative Stress

Major foreign compound-mediated biomedical and biochemical effects include oxidative stress, lipid peroxidation, mitochondrial function intervention, interaction with ion transporters, enzymatic function interference, immune suppression and stimulation effects, and chemical carcinogenesis. Oxidation stress is among the fundamental mechanisms that contribute to the toxicity of many foreign compounds. The production of toxic intermediate occurs in metabolic activation of a foreign compound, which requires phase I enzyme such as CYP450, flavinmonooxygenase, lipoxygenase, or xanthine oxidase. Reactive oxygen species form when chemically active intermediates react with oxygen molecule. Moreover, intrinsic toxicity such as nitric oxide (a radical) also plays a role in foreign compound-induced oxidative stress. Nitric oxide is generated enzymatically by nitric oxide synthase from l-arginine. Detoxification reactions catalyzed by phase II enzymes serve as a defense system to detoxify reactive oxygen species. However, an imbalance between activation and detoxification occurs if the rate of reactive oxygen species production is significantly larger than that of detoxification reaction. Furthermore, if the overly generated reactive

7.3

Oxidative Protein Damage

63 Protein Impairment in enzyme and other protein functions

Oxidation DNA Reactive oxygen species Lipid

DNA base pair mismatch Lipid peroxidation

Mutation

Tissue damage

Fig. 7.1 Oxidation damages by reactive oxygen species

oxygen species are not compensated by the body’s antioxidant defense systems, the production of such oxidants prevails. As a result of these, oxidative stress occurs since biological systems are unable to readily detoxify reactive oxygen species and to repair the resulting damages. Other than occurring during the processes of metabolic conversion, oxidative stress can also occur when the amount of free radicals produced in mitochondria through aerobic electron transfer chains is more than that needed for normal physiological functions, and when the presence of cellular antioxidants cannot provide enough scavengers for these unwanted, excessive free radicals. Reactive oxygen species such as hydrogen peroxide, nitric oxide, hydroxyl radical, and superoxide anion radical can directly damage cells or tissues. Oxidative cell damage has been implicated in many disease conditions. Reactive oxygen and nitrogen species are known to contribute as pathogenic factors to the development of chronic progressive diseases at various stages. Major targets for oxidative damages are cellular molecules (proteins, DNA, and lipids). Xenobiotic-induced oxidative protein damage can cause an impairment of enzyme catalytic function or other protein functions. Oxidative DNA damage affects DNA base pairing, causing a mismatch in the DNA base pair transformation and resulting in mutation. Oxidation of cell membranes leads to peroxidation of membrane lipids, a primary mechanism of tissue injury. The interactions of reactive oxygen species with cellular molecules are illustrated in Fig. 7.1.

7.3

Oxidative Protein Damage

Reactive oxygen species and other strong oxidants mediated by foreign compounds can interact with cellular proteins, resulting in oxidative protein damage. If the oxidative damage is not repaired, loss of catalytic function for enzymes and impairment of protein function can occur. For example, hemoglobin is a frequent target for oxidative damage. Xenobiotic-induced oxidation of two globin chains of hemoglobin can lead to disulfide bond formation, resulting in the interference of protein folding and the loss of protein function. Among amino acid residues, the sulfur-containing amino acids, cysteine, and methionine, are particularly susceptible to oxidation by reactive oxygen species.

64

7.4

7

Biomedical and Biochemical Effects

Oxidative DNA Damage

The oxidation of nucleic acids by reactive oxygen species is another consequence of oxidative stress in cells. Reactive oxygen species are capable of inducing DNA damage, which eventually may contribute to cell transformation and tumor initiation. The oxidation of DNA base by reactive oxygen species has severe consequences on DNA base pairing. For example, the oxidation at the guanine residue can result in the insertion of a wrong base (adenine) in DNA. If the change in base pairing persists without repair during the next replication cycle, DNA damage can occur. Reactive oxygen and nitrogen species are also implicated in tumor promotion and progression. Oxidative DNA damage can subsequently lead to mutations and eventually tumors. Moreover, metals are one group of foreign compounds that are associated with oxidative DNA damage. Nickel sulfides and other nickel compounds can give rise to the production of highly reactive hydroxyl radicals that can attack DNA bases, causing oxidative DNA damage. Nickel compound binding to DNA poses an increased risk of respiratory cancer.

7.5

Lipid Peroxidation

When reactive oxygen species are generated in the membrane compartment of the cell, membrane lipids can be oxidized, leading to membrane peroxidation and oxidative lipid damage. The mechanism of lipid peroxidation involves the oxidation of fatty acyl carbon chains and the formation of lipid peroxyl radicals. Lipid peroxidation is a propagating chain reaction resulting in damaging lipid bilayers, the backbone of biomembranes. Furthermore, the peroxidation reaction also produces a reactive aldehyde that can expand the toxicity of foreign compounds. Membrane lipid peroxidation has been implicated as one of the mechanisms that cause xenobiotic-induced tissue injury. The prevention of lipid peroxidation is an essential process in aerobic organisms since the product of lipid peroxidation can cause DNA damage. For example, carbon tetrachloride, an industrial solvent, undergoes activation metabolism catalyzed by CYP450 isozyme to form trichloromethyl radical which triggers membrane lipid peroxidation. The liver and kidneys are major organs associated with tetrachloride-induced toxicity. Peroxidation of membrane phospholipid acyl chains generates chemically active intermediate carbonyl species, mainly alpha, beta-unsaturated aldehydes, dialdehydes, and keto-aldehydes. Molecular modifications induced by reactive carbonyl species play a causal role in the aging process, and most of their biological effects are due to their capacity to react with cellular constituents, especially lipid peroxidation. For example, 4-hydroxy-2-nonenal, a common product of polyunsaturated fatty acid oxidation and decomposition, reacts extensively with DNA and proteins, depletes intracellular glutathione, alters cell signaling cascades, and affects multiple stress signaling pathways. Moreover, reactive carbonyl species are stable and can even diffuse

7.7

Interaction with Ion Transporters

65

from the cell to attack targets far away from the site of formation. They behave as mediators and propagators of oxidative stress that causes cellular and tissue damages.

7.6

Intervention with Mitochondria Functions

Mitochondria are crucial to the maintenance of many cellular functions, particularly energy utilization. The production of ATP is central to the supply of energy needed for biosynthesis and transport processes. Inhibition of mitochondrial functions subsequently causes a decrease in the production of cellular ATP, leading to an impairment of energy supply. Foreign compounds impair mitochondria functions through their interference with electron transport chains or by inhibiting ATP synthetase activity. For example, pentachlorophenol, a wood preservative, is absorbed into the body by occupational exposure, causing toxic effects through its ability to uncouple mitochondrial oxidative phosphorylation. Pentachlorophenol is endogenously formed by metabolism of hexachlorobenzene. Feeding rats hexachlorobenzene for 2 months was reported to result in uncoupling of oxidative phosphorylation of liver mitochondria. Mitochondrial dysfunction has also been implicated in the pathogenesis of valproic acid (an antiepileptic drug)-mediated hepatotoxicity. Acute treatment of freshly isolated rat hepatocytes with valproic acid was found to result in oxidative stress. Glutathione provides protection to hepatocytes against mitochondrial damage by valproic acid. Rotenone is another foreign compound that is capable of intervening with mitochondria function. It occurs naturally in the roots and stems of several plants such as vine plants, and its solution is used as an insecticide and pesticide. Rotenone is an inhibitor of oxidative phosphorylation because it blocks electron transfer in NADH-dependent oxidoreductase and thus prevents the utilization of NADH as a substrate. Inhibition of electron transport chain also inhibits ATP synthesis. In the presence of uncouples, ATP is not formed by mitochondrial ATP synthase because the protonmotive force across mitochondria membranes is dissipated. For example, 2,4-dinitrophenol can uncouple the coupling of electron transport and phosphorylation in mitochondria. It has the ability to greatly increase metabolic rate and cause the rapid loss of body fat. However, concerns about its dangerous side-effects have resulted in discontinuing the use of 2,4-dinitrophenol for weight loss purposes.

7.7

Interaction with Ion Transporters

Channels and pumps are ion transporters. These membrane proteins have the capability of transporting specific ions across biological membranes. Interference of a channel or a pump at its binding site with a foreign compound may inhibit ion transporter capacity. Na+, K+ pump is an ATP-dependent transporter which mediates the efflux

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Biomedical and Biochemical Effects

of 3 Na+ in exchange of influx of 2 K+, resulting in building up a Na+ gradient across cell membranes with higher extracellular Na+. The generated Na+ gradient can drive Na+-dependent secondary transport. For example, in cardiomyocytes, the influx of extracellular Na+ into the cell drives the efflux of Ca2+ from the cell. The concentration of intracellular Ca2+ appears to associate with contraction in cardiac muscle cells. Digitoxin mediates the inhibition of Na+, K+ pump. By binding to the outer surface of ATPase, digitoxin blocks Na+ and K+ translocation events as well as ATP hydrolysis. Specific inhibition of Na+, K+ pump by digitoxin results in indirectly causing an increase in the intracellular Ca2+ levels. Increasing intracellular Ca2+ concentration appears to directly stimulate contraction in cardiac muscle cells. Therefore, digitalis is used in treating cardiac conditions such as congestive heart failure. However, an overdose of digitoxin can cause adverse drug reactions because of over-stimulation of cardiac muscle cells, which can lead to cardiac arrhythmia. Digitoxin overdose incidence occurs especially in aging populations. Another example of foreign compound interaction with ion transporter is tetrodotoxin; a potent neurotoxin produced by marine bacteria and is present in organs of puffer fish. Poisoning with tetrodotoxin occurs after ingestion of various species of puffer fish. Tetrodotoxin specifically binds and inhibits a sodium channel in excitable nerve tissues. It blocks action potentials in nerves by binding to the pores of the voltage-dependent sodium channels in nerve cell membranes, thus essentially preventing the affected nerve cells from firing. Blocking of Na+ channels has potential medical use in treating some cardiac arrhythmias.

7.8

Interference with Enzymatic Functions

Interference with enzymatic function refers to certain conditions when the enzyme cannot carry out its normal function as the catalyst for a specific reaction. Many enzymes and proteins contain a sulfhydryl group at their catalytic site. These enzymes are vulnerable to thio-reactive compounds such as heavy metals. Heavy metals exhibit high affinity for the thiol group. For example, arsenic has been known to selectively inhibit pyruvate dehydrogenase. Pyruvate dehydrogenase is an enzyme complex which links the glycolytic pathway with the citrate cycle. The binding of nonmetallic compounds at a different site may also cause enzyme inactivation. When inhibited or damaged by a toxic foreign compound, an enzyme may no longer carry out the conversion of the substrate. For instance, organophosphates are important in agriculture industries as pesticides to protect plants. Organophosphates are lipophilic compounds that are readily absorbed and distributed in the body. Some of the organophosphates exhibit acute neurotoxicity. Binding of organophosphate to acetylcholinesterase inhibits the enzyme activity. Acetylcholinesterase is a serine esterase whose function is to hydrolyze the neurotransmitter acetylcholine. The serine hydroxyl group is normally attacked by the substrate acetylcholine.

7.9

Immune Suppression and Stimulation Effects

67

However, in the presence of organophosphate, the active serine hydroxyl group of acetylcholinesterase is attacked by phosphorus of the organophosphate, resulting in the inhibition of acetylcholinesterase. Enzymatic function can also be interfered by life style modifications such as chronic consumption of alcohol. CYP2E1 is one pathway involved in oxidative stress produced by ethanol. CYP2E1 contributes significantly to ethanol metabolism and formation of acetaldehyde, the highly reactive metabolite. Moreover, a number of potential toxic foreign compounds and their metabolites are able to weaken the body’s antioxidant defense by interacting with antioxidant enzymes. Antioxidants are substances that either directly or indirectly protect cells against adverse effects of oxidants. Antioxidant enzymes provide scavengers for reactive oxygen species and serve as a brake to prevent their over abundance. For instance, superoxide dismutase, a typical antioxidant enzyme, scavenges a superoxide free radical by converting it to peroxide, which can then be destroyed by catalase.

7.9

Immune Suppression and Stimulation Effects

Repression of an immune cell development by toxic xenobiotics or metabolites may lead to immune suppression. Immunosuppressive agents are drugs that inhibit or reduce the activation or efficacy of the immune system. Suppression of the immune system renders an organism susceptible to infections, compromising the ability of the body to fight diseases. Other side-effects of immunosuppressive agents include hypertension, peptic ulcers, liver, and kidney injury. Immune suppressants can also cause harmful effects by interacting with other medicines. Typical examples of potential immune suppressants include steroids such as cortisone and environmental chemicals such as halogenated aromatic hydrocarbons. Moreover, the environmental pollutant dioxin has a capacity of eliminating maturing T cells in the thymus. Direct addition of alkylators, such as N-nitroso-N-methylurea and dimethyl sulfate, to naive murine splenocytes has been reported to produce a dose-dependent suppression of the in vitro antibody-forming cell in response to antigens. In contrast to immunosuppressive effect, some other foreign chemicals or metabolites can cause an enhancement in immune response, leading to tissue damage and immune-mediated disease. Stimulation of the immune system may result in hyposensitivity to immunoallergic reactions. A chemical allergy is initiated by the immune system and expressed as hypersensitivity. Enhancement of the immune system can also lead to chemical hypersensitivity. Such an adverse reaction to a chemical is the result of previous sensitization to that chemical or a structurally similar compound. After an initial allergic reaction to that chemical, a small subsequent exposure can evoke a much severe response. For instance, penicillin is the most common drug that can draw an allergic response to a minority of recipients.

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7.10

7

Biomedical and Biochemical Effects

Chemical Carcinogenesis

A significant number of chemicals have been shown to be carcinogenic in animal models or cell culture experiments. Some of these carcinogens act directly on the target cells. Others take effect after these chemicals have been converted to toxic intermediates or metabolites through metabolic reactions catalyzed by phase I activation enzymes. Chemical carcinogens belong to a diverse group of compounds. A majority of known carcinogens are mutagenic. Conversely, a majority of mutagens are carcinogenic. Mutagenic chemicals are DNA damaging agents, which are considered to produce enduring alterations in the genetic substances in the cells. A variety of human carcinogens have been reported, including 2-naphthylamine, aflatoxins, arsenic and nickel compounds, asbestos, benzene, benzidine, tamoxifen, tobacco smoke, polychlorinated biphenyl, etc. Among DNA damaging agents, there are direct-acting and indirect-acting carcinogens. Direct-acting carcinogens are compounds that are intrinsically reactive and can covalently interact with DNA to form DNA adducts without requiring bioactivation as the precondition. Typical direct-acting carcinogens include N-methylN-nitrosourea and methyl methanesulfonate. In contrast, indirect-acting carcinogens require metabolic activation of parent compounds to produce reactive intermediates or reactive oxygen species before these chemicals can covalently bind to DNA to form DNA adducts. In general, reactive intermediates produced during metabolic activation are electrophilic metabolites that are capable of interacting with nucleophilic sites in DNA. Typical indirect-acting carcinogens include aflatoxin B1, dimethylnitrosamine, and benzo[a]pyrene.

Bibliography Arispe N, Diaz JC, Simakova O et al (2008) Heart failure drug digitoxin induces calcium uptake into cells by forming transmembrane calcium channels. Proc Natl Acad Sci USA 105:2610–2615 Blaikie FH, Brown SE, Samuelsson LM et al (2006) Targeting dinitrophenol to mitochondria: limitations to the development of a self-limiting mitochondrial protonophore. Biosci Rep 26: 231–243 Brzezinski MR, Boutelet-Bochan H, Person RE et al (1999) Catalytic activity and quantitation of cytochrome P-450 2E1 in prenatal human brain. J Pharmacol Exp Ther 289:1648–1653 Choi DW, Leininger-Muller B, Wellman M et al (2004) Cytochrome p-450-mediated differential oxidative modification of proteins: albumin, apolipoprotein E, and CYP2E1 as targets. J Toxicol Environ Health A 67:2061–2071 Chou AP, Li S, Fitzmaurice AG et al (2010) Mechanisms of rotenone-induced proteasome inhibition. Neurotoxicology 31:367–372 Eickhorn R, Weirich J, Hornung D, Antoni H (1990) Use dependence of sodium current inhibition by tetrodotoxin in rat cardiac muscle: influence of channel state. Pflugers Arch 416:398–405 Goetz ME, Luch A (2008) Reactive species: a cell damaging rout assisting to chemical carcinogens. Cancer Lett 266:73–83 Gonzalez FJ (2005) Role of cytochromes P450 in chemical toxicity and oxidative stress: studies with CYP2E1. Mutat Res 569:101–110

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Haggerty HG, Kim BS, Holsapple MP (1990) Characterization of the effects of direct alkylators on in vitro immune responses. Mutat Res 242:67–78 Hodgon E, Smart RC (2001) Introduction to biochemical toxicology. Wiley, New York Jaeschke H, Gores GJ, Cederbaum AI et al (2002) Mechanisms of hepatotoxicity. Toxicol Sci 65:166–176 Luís PB, Ruiter JP, Aires CC et al (2007) Valproic acid metabolites inhibit dihydrolipoyl dehydrogenase activity leading to impaired 2-oxoglutarate-driven oxidative phosphorylation. Biochim Biophys Acta 1767:1126–1133 Masini A, Ceccarelli-Stanzani D et al (1985) The role of pentachlorophenol in causing mitochondrial derangement in hexachlorobenzene induced experimental porphyria. Biochem Pharmacol 34:1171–1174 Mates JM (2000) Effects of antioxidant enzymes in the molecular control of reactive oxygen species toxicology. Toxicology 153:83–104 Pamplona R (2008) Membrane phospholipids, lipoxidative damage and molecular integrity: a causal role in aging and longevity. Biochim Biophys Acta 1777:1249–1262 Pérez MJ, Cederbaum AI (2003) Adenovirus-mediated expression of Cu/Zn- or Mn- superoxide dismutase protects against CYP2E1-dependent toxicity. Hepatology 38:1146–1158 Spracklin DK, Hankins DC, Fisher JM et al (1997) Cytochrome P450 2E1 is the principal catalyst of human oxidative halothane metabolism in vitro. J Pharmacol Exp Ther 281:400–411 Tong V, Teng XW, Chang TK, Abbott FS (2005) Valproic acid II: effects on oxidative stress, mitochondrial membrane potential, and cytotoxicity in glutathione-depleted rat hepatocytes. Toxicol Sci 86:436–443 Wells PG, Kim PM, Laposa RR et al (1997) Oxidative damage in chemical teratogenesis. Mutat Res 396:65–78 West JD, Marnett LJ (2005) Alterations in gene expression induced by the lipid peroxidation product, 4-hydroxy-2-nonenal. Chem Res Toxicol 18:1642–1653

Chapter 8

Genetic Variations in Metabolizing Enzymes

Foreign compounds to which humans are exposed undergo metabolic conversion mediated by metabolizing enzymes before they are eliminated from the body. Phase I activation enzymes catalyze oxidation, hydrolysis, and reduction reactions. The functionalized compounds then proceed with further reactions catalyzed by phase II detoxification enzymes, which generally convert functionalized foreign compounds into less reactive and water-soluble metabolites, thus facilitating their elimination from the organism. Advances in this area of research have revealed that many metabolizing enzymes exhibit genetic polymorphisms which play a crucial role in individual variations in response to foreign compound-mediated effects. Investigations of individual responsiveness to drugs or certain chemicals have shown considerable deviations, in part due to variations in the expressions of foreign compound metabolizing enzymes. In humans, interindividual differences resulting from polymorphisms in gene structure and tissue-specific expression can have a significant impact on the overall metabolism, the clearance of drugs and other chemicals and the susceptibility of individuals to foreign compound-mediated toxic effects. Many carcinogens show species differences in their toxic effects. Such species differences may have a metabolic basis since most carcinogens are metabolized to reactive electrophiles in order to elicit their toxic effects. Moreover, the expression of a foreign compound-metabolizing enzyme can also differ noticeably as a result of exposure to an enzyme inducer. Both genetic polymorphisms and enzyme inducibility are of major significance since they play an important role in the susceptibility of individuals to xenobiotic toxicity. The subject of inducibility of metabolizing enzymes is discussed in the next chapter.

C.-H. Chen, Activation and Detoxification Enzymes: Functions and Implications, DOI 10.1007/978-1-4614-1049-2_8, © Springer Science+Business Media, LLC 2012

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Fig. 8.1 Polymorphic variants in ADH and ALDH affect alcohol metabolisms

Genetic Variations in Metabolizing Enzymes Alcohol ADH2*1 ADH2*2

Low activity

High activity

Acetaldehyde ALDH2*1 ALDH2*2 High activity

Low activity Acetate

CO2 and H2O Drink more Drink less (Alcoholic) (Non-alcoholic)

8.1

Role of Enzyme Genetic Polymorphisms in Alcoholism

Alcoholism is a typical example that genetic polymorphisms affect the expression of metabolizing enzymes. The kinetics of ethanol absorption and elimination are influenced by genetic factors. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are two important enzymes involving alcohol metabolism. ADH oxidizes ethanol to acetaldehyde, and ALDH oxidizes acetaldehyde to acetate which is further metabolized to CO2 and H2O. In humans, genetic polymorphisms of ADH and ALDH have an influence on alcoholism susceptibility. ADH is present as a polygene family and has seven genes including ADH2*1, ADH2*2, and ADH2*3, which are functionally polymorphic. ALDH also exhibits genetic polymorphisms including ALDH2*1 and ALDH2*2 alleles. Genetic variations in ADH and ALDH among individuals in response to alcohol have been reported to contribute to differences in susceptibility to alcohol abuse and dependence. Particularly, the functional genetic variants of ADH that exhibit high alcohol oxidizing activity, and the genetic variants of ALDH that exhibit low acetaldehyde oxidizing activity protect against heavy drinking and alcoholism. Figure 8.1 shows polymorphic variants in ADH and ALDH affecting alcohol metabolism. Genetic polymorphisms and molecular markers can be applied to expand clinical prevention studies targeted to individuals with a high risk of developing certain disease conditions.

8.2

Genetic Polymorphisms of Cytochrome P450

Genetic variations in the levels of expression and substrate specificity of metabolizing enzymes can give rise to abnormal foreign compound metabolism. These variations may account for the differential susceptibility to potentially toxic xenobiotics. Genetic polymorphisms occur in many of the enzymes involved in metabolism of drugs and other xenobiotics. Cytochrome P450 (CYP450) and glutathione-S-transferase

8.2

Genetic Polymorphisms of Cytochrome P450

73

(GST) are presented here as typical examples of polymorphic phase I enzymes and phase II enzymes, respectively. Polymorphic variations in CYP450 genes may lead to increased toxification, whereas genetic polymorphisms in GST may result in impaired detoxification. Genetic variability in the expression of activation enzymes is an important relevant feature associated with foreign compound-mediated biochemical and biomedical effects. The differences in individual sensitivity to xenobiotic metabolites could explain many observed differential reactions to foreign compounds among individuals. Genetic polymorphisms underlying variations in enzymatic activity can have a significant effect on an individual’s susceptibility to diverse toxicity. People who possess effective phase I activation enzymes, but deficient phase II detoxification enzymes, are called pathological detoxifiers, because they exhibit severe reactions to toxic overload. There is interindividual and interethnic variability in the levels of activity of many drug metabolizing enzymes. This variation could cause differences in sensitivities to the effects and toxicity of clinically used drugs as well as environmental compounds such as nicotine and carcinogens. In many cases, polymorphisms in the gene encoding of these enzymes are the basis for this variability. Approximately 40% of human CYP450-dependent drug metabolism is carried out by polymorphic enzymes. There are significant differences in the levels of expression of CYP450 isozymes between individuals as evaluated by the analysis of human liver samples in clinical pharmacological studies. Genetic polymorphisms of CYP450 isozymes, 2A6, 1A1, and 2E1, have been investigated to examine their impacts on individual susceptibility to foreign compound toxicity. These studies are discussed below.

8.2.1

CYP2A6 Polymorphisms Affecting Nicotine Metabolism

High expression of CYP450 activity results in an undesired increase in the rate of activation of foreign compounds. This leads to over-production of reactive metabolites. Metabolisms mediated by high activity of CYP450 have been implicated in the production of toxicity and carcinogenicity of certain foreign compounds. Polymorphic variants in CYP450 genes may lead to variation in the extent of activation, thereby influencing individual sensitivity toward xenobiotic toxicity. CYP2A6 is the principle human oxidase which is involved in the metabolic activation of nicotine. CYP2A6 is present predominantly in the liver and over 22 different alleles have been characterized for this enzyme. Moreover, CYP2A6 also activates a number of structurally unrelated chemical carcinogens including aflatoxin B1, halothane, and valproic acid. CYP2A6 is responsible for the clearance of many drugs and environmental chemicals. For example, in humans, 70–80% of nicotine is oxidized by CYP450 to nicotine-iminium ions, which are further metabolized to cotinine by aldehyde oxidase. Subsequently, cotinine is converted to hydrolated products. CYP2A6 gene is highly polymorphic: it contains more than a dozen different alleles including CYP2A6*1A

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Fig. 8.2 Polymorphic variants in CYP2A6 affect nicotine metabolism

Genetic Variations in Metabolizing Enzymes Nicotine CYP2A6*1B CYP2A6*1A

Lower activation rate

Higher activation rate

Nicotine-iminium ion Aldehyde oxidase Cotinine Hydroxy-cotinine Lower risk lung cancer

Higher risk lung cancer

and CYP2A6*1B. Nicotine polymorphisms have been reported to influence the rate of nicotine elimination from the body. Interindividual and interethnic variability in the level of CYP2A6 activity is attributed to polymorphisms in the CYP2A6 gene. A relatively high allele frequency (15–20%) of the CYP2A6 gene deletion has been reported in Asian population. This population has a generally reduced CYP2A6 activity. CYP2A6*1A genotype that causes a decreased expression of CYP2A6 has been shown to a significant association of decreased risk for lung cancer. This observed decreased risk is due to a lower rate of formation of carcinogenic products by a less active CYP2A6*1A. In contrast, individuals with the CYP2A6*1B allele have higher CYP2A6 expression as well as catalytic activity. These individuals are likely to have increased risk for lung cancer. These reports demonstrate that genetic polymorphisms of CYP2A6 genotype can influence the susceptibility of individuals to lung cancer. Figure 8.2 illustrates polymorphic variants in CYP2A6 that affect nicotine metabolism. The produced nicotine-iminium ion can react with DNA, leading to higher lung cancer risk.

8.2.2

CYP1A1 Polymorphisms Affecting Polycyclic Aromatic Hydrocarbon Metabolism

Genetic polymorphism also plays an important role in the differences between individuals in response to the toxicity of polycyclic aromatic hydrocarbons. Metabolism of these aromatic compounds (e.g., benzo[a]pyrene in tobacco smoke) involves metabolic activation catalyzed by CYP450, epoxide hydrolase, and prostaglandin-H synthase to form dihydrodiol epoxide. The formed epoxide is capable of covalently binding to DNA. The detoxification of polycyclic aromatic hydrocarbons is catalyzed by GSTs. Differences in the levels of expression and catalytic activities of CYP450 and GST between individuals have been reported. Polymorphic variants in CYP450 gene, particularly CYP1A1, have an effect on the metabolism of polycyclic aromatic hydrocarbons, thereby influencing the susceptibility of individuals toward their toxicity. The frequency of the genotypes,

8.2

Genetic Polymorphisms of Cytochrome P450

Fig. 8.3 Polymorphic variants in CYP1A1 affect benzo[a]pyrene activation

75 Benzo[a]pyrene

CYP 1A1 (Ile/Ile)

Lower activation rate

CYP1A1 (Val/Val)

Higher activation rate

Benzo[a]pyrene 7,8-oxide Epoxide hydrolase Benzo[a]pyrene 7,8-dihydrodiol CYP1A1, PHS Benzo[a]pyrene 7,8-dihydrodiol-9,10-epoxide Higher risk

Lower risk

Lung cancer

Ile/Ile, Ile/Val, and Val/Val, has been assessed to help evaluate the relationship between CYP1A1 polymorphism and individual sensitivity to polycyclic aromatic compounds that may affect the incidence of lung cancer. A higher level of DNA adducts has been detected in lymphocytes of tobacco smokers with specific polymorphism of CYP1A1 gene. Specific genotype Val/Val for CYP1A1 was found to be related to the incidence of esophageal carcinoma, especially in heavy smokers. Moreover, Ile/ Val CYP1A1 polymorphism was also reported to be associated with an elevated risk of lung cancer among Japanese. Figure 8.3 illustrates polymorphic variants in CYP1A1 affecting activation of benzo[a]pyrene.

8.2.3 CYP2E1 Polymorphisms Affecting Nitrosamine Metabolism CYP2E1, another polymorphic variant in CYP450 genes, is a key enzyme in the metabolic activation of a variety of toxicants, including benzene, vinyl chloride, and halogenated solvents (e.g., trichloroethylene). CYP2E1 is one of the enzymes that metabolize ethanol to acetaldehyde. Genetic polymorphisms in CYP2E1 have been identified and linked to altered susceptibility of individuals to alcoholism. Moreover, CYP2E1 is also a key enzyme involved in the metabolism of nitrosamines (e.g., 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N¢-nitrosonornicotine) in tobacco smoke. Genetic polymorphisms of CYP2E1 may lead to interindividual differences in CYP2E1-mediated microsomal drug oxidation activities including oxidation of N-nitrosamines. The allelic frequency of three restriction fragment length polymorphisms (PstI, RsaI, and DraI) has been assessed. Figure 8.4 illustrates polymorphic variants in CYP2E1 affecting metabolism of nitrosamines. The formed reactive intermediate, carbonium ion, can react with DNA. Some studies have demonstrated an association of the CYP2E1 gene with the susceptibility of individuals to lung cancer. Genetic polymorphism of CYP2E1 DraI was found to associate with an increased risk of lung cancer in Japanese.

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Nitrosamines CYP2E1 (PstI or RsaI) Lower activation rate

CYP2E1 (DraI) Higher activation rate

α-hydroxynitrosamine Carbonium ion Lower risk Higher risk Alkylation of DNA

Fig. 8.4 Polymorphic variants in CYP2E1 affect nitrosamine activation

Chemicals inhaled by smokers Nitrosamines / polycyclic aromatic hydrocarbons Reactive intermediates GSTM1 (+) and/or GSTT1 (+) GSTM1-null and/or GSTT1 -null Detoxification Impaired detoxification

Lower risk for lung cancer

Higher risk for lung cancer

Fig. 8.5 Polymorphic variants in GST affect the risk of smokers to cancer

8.3

Genetic Polymorphisms of Glutathione-S-Transferase

Similar to activation enzymes, genetic variations in detoxification enzymes also influence the susceptibility of an organism to foreign compound-mediated toxic effects. Studies of GST in humans have revealed that there are multiplicities of GST (e.g., GSTM1 and GSTT1 genes). Genetic polymorphisms in GST may result in impaired detoxification, and therefore have significant impacts on an individual’s susceptibility to foreign compound-mediated toxicities. For example, blood cultures obtained from GSTT1 and GSTM1 null individuals were found to exhibit an increase in the sensitivity to various toxins. Individuals who carry GSTM1-null genotype were also reported to have a higher susceptibility to DNA damage induced by tobacco smoke than GSTM1-positive ones. A higher level of DNA adducts has been detected in lymphocytes of tobacco smokers who lack the GSTM1 gene. Moreover, several studies have also revealed an association of the homozygous null deletions in GSTM1 and GSTT1 with an increasing risk of lung cancer. Individuals lacking both GSTM1 and GSTT1 activities were also found to be at increased risk for gastric cancer. Figure 8.5 shows polymorphic variants in GST affecting the risk of smokers to cancer.

8.4

Species Difference in Enzyme Activity

8.4

77

Species Difference in Enzyme Activity

Variations in detoxification enzyme activity are often the basis for species differences in an organism’s susceptibility to foreign compound-mediated toxicity. A number of studies have been reported to support the concept that metabolic differences are the underlying cause of species variation in carcinogenicity. The differences in carcinogenicity may also include variations in metabolic pathways between species. It has been reported that the susceptibility to aflatoxin toxicity is different between humans and mice, and humans and rats are distinctive in their vulnerability to tamoxifen toxicity. Humans and rodents exhibit different toxic response to 4-ipomeanol. These observed differences are mainly attributed to species variations in the activities of enzymes that catalyze detoxification reactions for these foreign compounds. Species differences in the susceptibility to aflatoxin, tamoxifen, and 4-ipomeanol-induced toxicity are discussed below.

8.4.1

Susceptibility to Aflatoxin Toxicity in humans, But Not in Mice

Aflatoxin B1 is a major risk factor for hepatocellular carcinoma in humans. In humans, CYP3A4 is abundant in the liver and the intestines. The enzyme metabolizes approximately 50% of marketed drugs. Activation of aflatoxin B1 catalyzed by CYP3A4 forms a carcinogenic intermediate, aflatoxin-8,9-epoxide. Aflatoxin-8, 9-epoxide is detoxified by the conjugation reaction catalyzed by GST. GSTA3 subunit (mGSTA3) plays the crucial role in protection against aflatoxin B1 toxicity. GSTA3-catalyzed conjugation reaction that forms glutathione-conjugated aflatoxin derivative facilitates the excretion of aflatoxin B1. In contrast, unconjugated aflatoxin-8, 9-epoxide can react with DNA to form a DNA adduct. Due to relatively low expression of mGSTA3 activity for aflatoxin-8,9-epoxide, humans are highly susceptible to toxic and carcinogenic effects of aflatoxin B1. Aflatoxin B1 has therefore been reported to associate with liver cancer in humans. Conversely, mice are resistant to aflatoxin B1 carcinogenesis because of a high expression of mGSTA3 in the mouse liver. Glutathione conjugation of aflatoxin8,9-epoxide confers intrinsic resistance to aflatoxin carcinogenesis, causing no occurrence of liver cancer in mice. Such species difference in detoxifying aflatoxin B1 between humans and mice is described in Fig. 8.6. Knockout mice which have deletion of mGSTA3 gene and have the lack of mGSTA3 expression at mRNA and protein levels, have more than 100-fold of aflatoxin–DNA adduct in the liver than wild-type mice. Subsequently, knockout mice respond to toxic effects of aflatoxin B1 in a manner similar to humans. Moreover, a loss of the Nrf2 transcription factor was reported to associate with a reduction in basal expression of mGSTA3, indicating a role of Nrf2 in regulating transcription of mGSTA3.

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Genetic Variations in Metabolizing Enzymes

Aflatoxin B1 Activation

CYP3A4

Aflatoxin B1-8,9-epoxide High mGSTA3 activity

Mice

Humans

Glutathione conjugation

Excretion

Low mGSTA3 activity

DNA

DNA adduct

Fig. 8.6 Susceptibility to aflatoxin B1 toxicity occurs in humans, but not in mice

8.4.2

Resistance to Tamoxifen Toxicity in Humans, But Not in Rats

Tamoxifen is an important antiestrogenic drug used for the treatment of breast cancer. It has also been shown to decrease breast cancer incidence in healthy women who are at high risk for the disease. But, an increased risk of endometrial cancer in women has raised concerns in the application of tamoxifen. Activation of tamoxifen catalyzed by CYP3A4 involves hydroxylation, which produces oxidative metabolites including hydroxytamoxifen derivatives (e.g., a-hydroxytamoxifen). Detoxification pathways for a-hydroxytamoxifen proceed in two distinctive catalytic reactions. One pathway is a conjugation reaction catalyzed by UDP-glucuronosyl-transferase (UGT), resulting in the formation of inactive glucuronidate tamoxifen. Another detoxification pathway is a conjugation reaction catalyzed by sulfotransferase (ST), which produces tamoxifen-O-sulfonate. In detoxification, the glucuronylation pathway competes with the sulfonation pathway. Although sulfonation conjugation usually is a detoxification reaction, tamoxifen-O-sulfonate subsequently releases its sulfonate group to produce carbocation, which can interact with DNA to form tamoxifen–DNA adducts. Species differences in tamoxifen detoxification metabolism between humans and rats are presented in Fig. 8.7. Humans exhibit a much higher activity of UGT than rats, and have more effective glucuronylation of a-hydroxytamoxifen in liver microsomes than rats. The high UGT activity plays a key role for humans to defend against the toxicity of tamoxifen. A high detoxification rate in liver microsomes has been reported to correlate with a low risk of tamoxifen in causing liver carcinoma in humans. Moreover, the figure shows that a lower rate of sulfonation in human liver further protects humans from forming tamoxifen–DNA adducts. In contrast, tamoxifen has been shown to be a potent hepatocarcinogen in rats. Rats display a low activity of UGT, and meanwhile a high activity of ST. As a result of these, a-hydroxytamoxifen in rats prefer a conjugation reaction through sulfonation to glucuronidation. Sulfonation conjugation leads to the formation of tamoxifen-Osulfonate, which subsequently releases its sulfonate group to produce carbocation, a reactive species, which can react with DNA to form DNA adducts. Consequently, tamoxifen is a liver carcinogen in rats.

8.4

Species Difference in Enzyme Activity

Tamoxifen

Activation CYP3A4

79

α-hydroxytamoxifen ST

UGT glucuronide tamoxifen Human

rats

α-hydroxytamoxifen O-sulfonate

Excretion

carbocation DNA Tamoxifen-DNA adducts

DNA Mutation

Fig. 8.7 Susceptibility to tamoxifen toxicity in rats, but not in humans

Rodents CYP4B1 (lung)

Furan epoxide*

Pulmonary toxicity

4-Ipomeanol Humans

Furan epoxide* CYP3A4; CYP1A2 (Liver)

Hepatotoxicity

*Another toxic metabolic intermediate is α,β-unsaturated di-aldehyde.

Fig. 8.8 4-Ipomeanol toxicity differs between humans and rodents

Experimentally, DNA adducts were detected in hepatocytes of rats treated with tamoxifen, however, DNA adducts were not detected in tamoxifen-treated human hepatocytes. Moreover, the level of DNA adduct formation was found to be dozen times greater in rat hepatocytes treated with a-hydroxytamoxifen than those treated with tamoxifen. These findings support the proposal that the major pathway for tamoxifen activation undergoes a-hydroxytamoxifen, tamoxifen-O-sulfonate, and carbocation.

8.4.3

4-Ipomeanol Toxicity Differs Between Humans and Rodents

4-Ipomeanol is a naturally occurring furan, which is isolated from sweet potatoes infected with the fungus Fusarium solani. 4-Ipomeanol was reported to induce pulmonary toxicity in the lungs of several mammalian species (e.g., rodents and cattle). To induce cytotoxicity to rodents, 4-ipomeanol requires metabolic activation catalyzed by CYP4B1 to form a highly reactive furan epoxide in pulmonary Clara cells.

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Genetic Variations in Metabolizing Enzymes

In contrast, the risk is minimal in humans for developing pulmonary toxicity. Instead of metabolic activation by CYP4B1 in pulmonary cells, 4-ipomeanol is metabolically activated in the liver by hepatic enzymes, CYP3A4 and CYP1A2. Consequently, the produced reactive furan epoxide which causes selective hepatotoxicity in humans. Figure 8.8 illustrates distinctive responses to 4-ipomeanol toxicity between humans and rodents. The figure demonstrates that deviations in response to 4-ipomeanol toxicity between humans and rodents are attributed to not only different sites of metabolism, but also distinctive CYP450 isozymes present in the metabolic sites for activation.

Bibliography Alvarez-Diez TM, Zheng J (2004) Mechanism-based inactivation of cytochrome P450 3A4 by 4-ipomeanol. Chem Res Toxicol 17:150–157 Archer MC (1981) Reactive intermediates from nitrosamines. Adv Exp Med Biol 136:1027–1035 Baer BR, Rettie AE, Henne KR (2005) Bioactivation of 4-ipomeanol by CYP4B1: adduct characterization and evidence for an enedial intermediate. Chem Res Toxicol 18:855–864 Baum M, Amin S, Guengerich FP, Hecht SS et al (2001) Metabolic activation of benzo[c]phenanthrene by cytochrome P450 enzymes in human liver and lung. Chem Res Toxicol 14:686–693 Boocock DJ, Maggs JL, Brown K et al (2000) Major inter-species differences in the rates of O-sulphonation and O-glucuronylation of alpha-hydroxytamoxifen in vitro: a metabolic disparity protecting human liver from the formation of tamoxifen-DNA adducts. Carcinogenesis 21:1851–1858 Czerwinski M, McLemore TL, Philpot RM et al (1991) Metabolic activation of 4-ipomeanol by complementary DNA-expressed human cytochromes P-450: evidence for species-specific metabolism. Cancer Res 51:4636–4638 Falzon M, McMahon JB, Schuller HM et al (1986) Metabolic activation and cytotoxicity of 4-Ipomeanol in human non-small cell lung cancer lines. Cancer Res 46:3484–3489 Ilic Z, Crawford D, Vakharia D et al (2010) Glutathione-S-transferase A3 knockout mice are sensitive to acute cytotoxic and genotoxic effects of aflatoxin B1. Toxicol Appl Pharmacol 242:241–246 Jowsey IR, Jiang Q, Itoh K et al (2003) Expression of the aflatoxin B1-8,9-epoxide-metabolizing murine glutathione S-transferase A3 subunit is regulated by the Nrf2transcription factor through an antioxidant response element. Mol Pharmacol 64:1018–1028 Kato S, Shields PG, Caporaso NE et al (1994) Analysis of cytochrome P450 2E1 genetic polymorphisms in relation to human lung cancer. Cancer Epidemiol Biomarkers Prev 3:515–518 Kim SY, Laxmi YR, Suzuki N et al (2005) Formation of tamoxifen-DNA adducts via O-sulfonation, not O-acetylation, of alpha-hydroxytamoxifen in rat and human livers. Drug Metab Dispos 33:1673–1678 Kiss I, Sándor J, Pajkos G, Bogner B et al (2000) Colorectal cancer risk in relation to genetic polymorphism of cytochrome P450 1A1, 2E1, and glutathione-S-transferase M1 enzymes. Anticancer Res 20:519–522 Kondraganti SR, Fernandez-Salguero P et al (2003) Polycyclic aromatic hydrocarbon-inducible DNA adducts: evidence by 32P-postlabeling and use of knockout mice for Ah receptor-independent mechanisms of metabolic activation in vivo. Int J Cancer 103:5–11 Li TK (2000) Pharmacogenetics of responses to alcohol and genes that influence alcohol drinking. J Stud Alcohol 61:5–12 Neafsey P, Ginsberg G, Hattis D et al (2009) Genetic polymorphism in CYP2E1: population distribution of CYP2E1 activity. J Toxicol Environ Health B Crit Rev 12:362–388

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Nimura Y, Yokoyama S, Fujimori M et al (1997) Genotyping of the CYP1A1 and GSTM1 genes in esophageal carcinoma patients with special reference to smoking. Cancer 80:852–857 Oscarson M (2001) Genetic polymorphisms in the cytochrome P450 2A6 (CYP2A6) gene: implications for interindividual differences in nicotine metabolism. Drug Metab Dispos 29:91–95 Oscarson M, McLellan RA, Gullstén H et al (1999) Identification and characterisation of novel polymorphisms in the CYP2A locus: implications for nicotine metabolism. FEBS Lett 460:321–327 Palma S, Cornetts T, Padua L et al (2007) Influence of glutathione S-transferase polymorphisms on genotoxic effects induced by tobacco smoke. Mutat Res 633:1–12 Phillips DH, Carmichael PL, Hewer A et al (1996) Activation of tamoxifen and its metabolite alpha-hydroxytamoxifen to DNA-binding products: comparisons between human, rat and mouse hepatocytes. Carcinogenesis 17:89–94 Rothman N, Shields PG, Poirier MC et al (1995) The impact of glutathione S-transferase M1 and cytochrome P450 1A1 genotypes on white-blood-cell polycyclic aromatic hydrocarbon-DNA adduct levels in humans. Mol Carcinog 14:63–68 Sgambato A, Campisi B, Zupa A et al (2002) Glutathione S-transferase (GST) polymorphisms as risk factors for cancer in a highly homogeneous population from southern Italy. Anticancer Res 22:3647–3652 Shupe T, Sell S (2004) Low hepatic glutathione S-transferase and increased hepatic DNA adduction contribute to increased tumorigenicity of aflatoxin B1 in newborn and partially hepatectomized mice. Toxicol Lett 148:1–9 Wang J, Pitarque M, Ingelman-Sundberg M (2005) 3¢-UTR polymorphism in the human CYP2A6 gene affects mRNA stability and enzyme expression. Biochem Biophys Res Commun 340:491–497 Watanabe J, Hayashi S, Kawajiri K (1994) Different regulation and expression of the human CYP2E1gene due to the RsaI polymorphism in the 5¢-flanking region. J Biochem 116:321–326

Chapter 9

Inducibility of Metabolizing Enzymes

Besides genetic polymorphisms, another important toxicologically relevant feature associated with foreign compound metabolism is the inducibility of metabolizing enzymes. Some foreign compounds exhibit intrinsic toxicity, while some others are metabolically activated to potential toxic metabolic intermediates by activation enzymes. The generated toxic metabolites further undergo detoxification enzymecatalyzed reactions before they are ready for excretion. Activation and detoxification enzymes have the potential to be inducted or inhibited by some chemical compounds. Induction or inhibition of these metabolizing enzymes, therefore, has a significant impact on the extent of toxicity of xenobiotics. Enzyme inducibility is of major significance because it plays a role in the susceptibility of individuals to foreign compound-mediated toxicities. Strategies for protecting cells from initiation toxic events include a decrease in the expression of activation enzymes responsible for generating reactive metabolic intermediates, and an increase in the activities of detoxification enzymes that detoxify reactive oxygen species and electrophiles known to intervene in regular cellular processes. In either case, it is important to note that the consequence of enzyme induction or inhibition is also dependent on the balance between the rates of activation and detoxification. In addition, the expression of metabolizing enzymes can also differ significantly as a result of exposure to dietary inducers or environmental chemicals. Induction of activation enzymes leads to a higher expression of enzyme activity, resulting in an enhancement in the activation rate and the production of potential toxic intermediates. If the generated toxicants are not immediately removed from the body, the induction of activation enzymes would promote toxic effects of foreign compounds. In contrast, the induction of detoxification enzymes leads to a higher expression of enzyme activity and an increase in the rate of detoxifying reactions, resulting in a decrease in xenobiotic toxic effect and an acceleration of foreign compound excretion. Moreover, the effects of inhibiting activation or detoxification enzymes depend on not only the relative activities of these enzymes, but also the nature of foreign compounds. For a foreign compound whose toxicity is caused by metabolic activation,

C.-H. Chen, Activation and Detoxification Enzymes: Functions and Implications, DOI 10.1007/978-1-4614-1049-2_9, © Springer Science+Business Media, LLC 2012

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Inducibility of Metabolizing Enzymes

an inhibition of activation enzymes decreases xenobiotic toxic effects. Conversely for a foreign compound with inherent toxicity, an inhibition of detoxification enzymes would increase xenobiotic toxicity. A further discussion of the effects of enzyme modulation is presented below.

9.1

Modulation of Phase I Enzymes

When a foreign compound is metabolized to a potent metabolic intermediate, a significant induction of activation enzyme activity may increase the activation rate and promote toxic intermediate production, resulting in an enhancement in xenobiotic toxic effect. Among phase I enzymes, CYP450 is most studied with respect to enzyme modulation. A typical example of an inducer is phenobarbital, which has been used for biochemical investigations of drug metabolism. Phenobarbital was reported to induce drug metabolism in animal models and humans. It enhances the activation of cocaine, leading to the potentiation of cocaine-induced hepatotoxicity. The induction effect of phenobarbital mainly involves the activation of CYP450. Conversely, a modest inhibition of CYP450 leading to a lower enzyme activity may decrease the activation rate by producing less toxic intermediate, leading to a reduction in xenobiotic toxic effect. Nevertheless, an over inhibition of activation rate could lead to an accumulation of foreign compounds and cause impaired metabolic clearance. A typical example is the inhibition of CYP450 by grapefruit juice components (naringin and furanocoumarins). The consumption of grapefruit juice with drugs taken orally has been reported to inhibit intestinal CYP3A4 activity, which results in a decrease in the metabolism of many drugs. Figure 9.1 illustrates how induction and inhibition of a phase I activation enzyme may potentially affect xenobiotic toxicity. Whether modulation of activation enzyme increases or decreases the toxicity of a foreign compound is an issue of complexity. The figure indicates that modest inhibition of activation enzyme may result in a lower xenobiotic toxic effect, assuming that the resulted enzyme activity is enough to perform its metabolic function. In contrast, a significant induction or inhibition of activation enzyme may enhance toxic effect of foreign compounds, due to an increase in the rate of toxic intermediate production or an impairment in metabolic clearance, respectively. In either case, the effects of activation enzyme modulation depend on not only the resulting activation enzyme activity, but also the rate of respective detoxification enzyme activity.

9.2

Modulation of Phase II Enzymes

Among phase II enzymes, UDP-glucuronosyl transferase (UGT), glutathioneS-transferases (GST), and NAD(P)H quinine reductase (NQO) are most studied with respect to enzyme modulation. For instances, oltipraz, a 1,2-dithiole-3-thione

9.2

Modulation of Phase II Enzymes

85

Decreasing/increasing toxic effect Inducer

Modestly higher expression

Inducer

Higher expression

Higher metabolic rate

Activation enzyme More reactive intermediate Increasing toxic effect Decreasing toxic effect Inhibition

Modestly lower expression

Inhibition

Lower expression

Less reactive intermediate

Activation enzyme Overload of xenobiotics Impaired metabolic clearance

Fig. 9.1 Phase I enzyme modulation affecting xenobiotic toxic effects

Less xenobiotic toxic effects Inducer Detoxification enzyme Higher enzyme expression Detoxification enzymes Lower enzyme expression Inhibitor

Higher detoxifying efficiency Lower detoxifying efficiency Higher xenobiotic toxic effects

Fig. 9.2 Phase II enzyme modulation influencing xenobiotic toxic effects

derivative, was reported to cause several fold induction of constitutive hepatic and gastric activities of GST and NQO in mice. An increase in the expression of phase II genes by oltipraz attributes to a reduction in aflatoxin B1-induced hepatocarcinogenesis. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific carcinogen, exerts carcinogenic potential through alpha-hydroxylation in metabolic activation. Phenobarbital was found to induce glucuronide conjugation of 4-hydroxyl-NNK catalyzed by UGT. Induction of phase II detoxification enzymes generally enhances the rate of detoxification reactions, leading to a faster increase in forming conjugated metabolites, and resulting in decreasing xenobiotic toxic effect and facilitating the excretion of xenobiotics. Conversely, inhibition of phase II enzymes decreases the rate of detoxification reactions, resulting in an increase in toxic effects of foreign compounds. Figure 9.2 illustrates how the induction or inhibition of a detoxification enzyme affects xenobiotic toxic effect. The modulation of detoxification enzyme is less complicated than that of activation enzyme. However, when phase II detoxification enzyme is overly induced, a potential problem involving the drug interaction and the action of other metabolisms may arise: this issue requires further investigation.

86

9.3

9

Inducibility of Metabolizing Enzymes

Life Style Modification

Two most known life style modifications that are capable of modulating foreign compound-metabolizing enzymes are alcohol and cigarette smoke. The effects of alcohol and cigarette smoke on metabolizing enzymes are described below.

9.3.1

Alcohol

An enhanced vulnerability of the alcoholic to the hepatotoxicity of many foreign compounds has been recognized. The metabolisms of foreign compounds are inducible by chronic alcohol consumption. For example, heavy drinkers develop severe hepatotoxicity from carbon tetrachloride exposure, but not for nondrinkers. CYP2E1 is the primary component of alcohol oxidizing system in the hepatic microsomes. It plays a key role in alcoholic liver disease and associated oxidative stress. CYP2E1catalyzed reaction is involved in the production of reactive oxygen species. Induction of CYP2E1 by alcohol increases metabolic activation of carcinogens.

9.3.2

Cigarette Smoke

Polycyclic aromatic hydrocarbons (e.g., benz[a]pyrene) and nitrosamines produced in cigarette smoke undergo metabolic activation by phase I enzymes, particularly CYP450. Induction of CYP450 isozymes (CYP1A2 and CYP2E1) by cigarette smoking has been reported. Polycyclic aromatic hydrocarbons are believed to be responsible for the induction of CYP1A1, CYP1A2, and perhaps CYP2E1. Cigarette smoking may increase the risk of cancer by inducing the metabolic activation of carcinogens. Activation enzyme induction is a major mechanism involving the interactions between cigarette smoking and drugs.

9.4

Monofunctional and Bifunctional Inducers

Two classes of enzyme inducers have been characterized: monofunctional and bifunctional inducers. Monofunctional inducers raise the activities of phase II enzymes [e.g., GST, NAD(P)H:quinone reductase, and UDP-glucuronosyltransferases] in various tissues without significantly elevating the activity of phase I enzymes (e.g., CYP450, and flavor-containing monooxygenase). In contrast, bifunctional inducers raise the activities of both phase II enzymes and phase I enzymes. Table 9.1 lists some typical monofunctional and bifunctional inducers. The table reveals that neither monofunctional nor bifunctional inducers display common characteristics in terms of chemical structures or functional groups.

9.5

Balance Between Activation and Detoxification

87

Table 9.1 Examples of monofunctional and bifunctional inducers Monofunctional inducers Bifunctional inducers Phenols Polycyclic aromatic hydrocarbons Sulforaphane Flavonoids Coumarins Azo dyes Thiocarbamates Cinnamates 1,2-Dithiol-3-thiones

Quinone reductase carries out two-electron reductions to protect cells against the toxicity of quinones. This enzyme is induced in many tissues in coordination with other major phase II enzymes that protect against toxic intermediates. Measurements of quinine reductase activity in murine hepatoma cells and two mutants provide an efficient method for studying the potency and mechanism of phase II enzyme induction. A direct assay of the activity of quinone reductase in murine hepatoma cells has been applied to characterize monofunctional and bifunctional inducers. The two mutants are defective in either aryl hydrocarbon receptor function or aryl hydrocarbon hydroxylase expression. In quinone reductase activity assay, a monofunctional inducer elevates phase II enzyme in various tissues without significantly raising phase I enzyme (e.g., CYP450 or aryl hydrocarbon hydroxylase). The induction is independent of aryl hydrocarbon receptor function. In contrast, a bifunctional inducer induces both phase II and phase I enzymes. Bifunctional induction is dependent on aryl hydrocarbon receptor function or aryl hydrocarbon hydroxylase expression. Furthermore, a simple, efficient microtiter plate assay has also been developed for the direct measurement of quinone reductase basal activity and inducibility in human peripheral blood lymphocytes grown in suspension culture. In these cells, quinone reductase is exclusively induced by monofunctional inducers.

9.5

Balance Between Activation and Detoxification

The efficiency of foreign compound metabolisms is dependent on the rates of activation and detoxification, which are interrelated to the activities of phase I enzymes and phase II enzymes. Figure 9.3 summarizes the relative rates of activation and detoxification in relation to toxicity of foreign compounds. In general, minimal xenobiotic toxicity is found in either a fine balance rate between activation and detoxification or a higher rate of detoxification than activation. Nevertheless, when the rate of detoxification is induced to a level much higher than that of activation, the implications of such metabolism remain to be investigated. Induction of CYP450 expression is likely to either increase or decrease the toxic effects of foreign compounds, depending on the relative activity of phase II enzymes. When the induction of CYP450 results in the rate of activation comparable with that of detoxification, then the induction is likely to produce beneficial effects.

88

9

Inducibility of Metabolizing Enzymes Increasing toxic effect

> Detoxification rate

Reactive metabolites overload

Activation rate = Detoxification rate

Reactive metabolites at minimum

< Detoxification rate

Reactive metabolites at minimum

Reducing toxic effect

Reducing toxic effect

Fig. 9.3 Relative rate of enzyme reactions affecting xenobiotic toxicity

Nevertheless, if the induction of CYP450 results in the rate of activation being significantly higher than that of detoxification, then the induction may produce harmful effects, such as in the case of smoking-related induction of CYP450. Therefore, a fine balance between activation enzymes and detoxification enzymes determines whether a toxic metabolite is securely detoxified or may cause cell dysfunction or damage. Accordingly, the rate of reactive metabolite production comparable with the rate of detoxification reaction is essential to achieve this fine balance. Consequently, detoxification metabolisms that function correctly appear to be an important means of preventing foreign compound-mediated toxic effects.

9.6

Antioxidant Response Element

The nuclear transcription factor E2-related factor 2 (Nrf2) has been shown to play crucial roles in preventing foreign compound-mediated toxicity and carcinogeninduced carcinogenesis. The protective roles of Nrf2 are attributed in part to its involvement in the induction of phase II enzymes. Transcriptional control of the expression of phase II enzymes is mediated in part through the antioxidant response element (ARE) found in the regulatory regions of phase II genes. Experiments have demonstrated that when Nrf2 is deleted in knock-out mice, the basal levels of phase II enzymes are very low and are not inducible. Consequently, these mice are much more susceptible to benzo[a]pyrene carcinogenesis than their wild-type counterparts. A major role of phase II enzymes in controlling the risk of exposure to carcinogens has recently received strong support from animal studies. Phase II detoxification enzymes neutralize reactive electrophiles and act as indirect antioxidants. Induction of phase II enzymes appears to be an effective method for achieving protection against a variety of carcinogens in animals and humans. Inducers of phase II detoxification enzymes were found to disrupt the cytoplasm complex between the protein Keap1 and the transcription factor Nrf2, thereby releasing Nrf2. Nrf2 then migrates to the nucleus where it activates the ARE of phase II genes and accelerates their transcription. The induction of phase II enzymes requires the functional integrity of the repressor Keap1 and the transcription factor Nrf2.

9.7 Enzyme Modulation Against Potential Toxic Effects

89

Under oxidative stress conditions, the transcription factor Nrf2 binds to ARE and induces phase II enzymes. Loss of Nrf2-mediated transcription was found to intensify vulnerability to the neurotoxin, 6-hydroxydopamine. Activation of the Nrf2ARE pathway by the chemical inducer, tert-butylhydroquinone, can protect against 6-hydroxydopamine-mediated toxicity. Induction of this pathway by transplantation of astrocytes overexpressing Nrf2 was also reported to protect against such toxicity in mice. The transcription factor Nrf2 binding to the ARE appears to be essential for the induction of phase II detoxification enzymes such as GST and NAD(P)H:quinone oxidoreductase. Constitutive hepatic and gastric activities of GST and quinone oxidoreductase were found to be reduced to less than half in Nrf2-deficient mice compared with wild-type counterparts. Exposure of rodents to 1,2-dithiole-3-thione, a phase II enzyme inducer, triggers nuclear accumulation of the transcription factor Nrf2 and enhances Nrf2 binding to ARE, leading to transcriptional activation of a score of genes involved in carcinogen detoxification.

9.7

Enzyme Modulation Against Potential Toxic Effects

Foreign compound-mediated toxic effects occur at least in the following three circumstances: (1) xenobiotics are inherent toxicants which are detoxified by detoxification enzymes, (2) reactive metabolic intermediates produced in metabolism catalyzed by activation enzymes are not effectively detoxified by detoxification enzymes, and (3) foreign compound toxic effects are potentiated by other xenobiotics which may have little or no toxicity, but are capable of interfering effectively with detoxification enzymes. In (1) and (3) circumstances, the induction of detoxification enzymes is beneficial to protect against potential foreign compound-mediated toxic effects. While in (2) circumstance, either the inhibition of activation enzymes or the induction of detoxification enzymes may be helpful if the levels of enzyme activities are suitably retained. It has been proposed that modulation of foreign compound-metabolizing enzymes may be a useful approach in minimizing the risk of xenobiotics-mediated toxic effects. One hypothesis proposes to achieve such a protection by inhibiting activation enzymes as well as inducing detoxification enzymes. Another hypothesis proposes that the induction of detoxification enzymes alone is enough to achieve the protection against carcinogenesis and other forms of toxicities. Generally, the result of activation enzyme inhibition is more complicated than that of detoxification enzyme induction. Extensive studies have been carried out to test the hypothesis that the induction of detoxification enzymes alone is enough to achieve the protection against xenobiotic toxicity. Results of these investigations suggested that the induction of phase II detoxification enzymes appears to be an effective means for achieving protection against a variety of carcinogens. In line with these studies, it has been proposed that a promising approach to minimize foreign compound-mediated toxic effects and

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related oxidative stress is to increase the intake of dietary inducers of phase II enzymes. The subject of inducing and inhibiting compounds of importance in modulating foreign compound-metabolizing enzymes is discussed in Chap. 10. While, the topic of dietary inducers of phase II enzymes is discussed in Chap. 11. Besides the above hypothesis regarding phase II enzyme modulation, it has also been postulated that the ingestion of an excessive amount of antioxidants is presumed to shift the oxidant–antioxidant balance toward the antioxidant side. An excessive antioxidant may effectively break down free radicals. However, such a shift of balance toward the antioxidant side could result in affecting some key physiological processes that are dependent on free radicals. This is a matter of concern, which requires further investigation.

Bibliography Buetler TM, Gallagher EP, Wang C et al (1995) Induction of phase I and phase II drug-metabolizing enzyme mRNA, protein, and activity by BHA, ethoxyquin, and oltipraz. Toxicol Appl Pharmacol 135:45–57 Cuendet M, Oteham CP, Moon RC et al (2006) Quinone reductase induction as a biomarker for cancer chemoprevention. J Nat Prod 69:460–463 Jakel RJ, Townsend JA, Kraft AD et al (2007) Nrf2-mediated protection against 6-hydroxydopamine. Brain Res 1144:192–201 Kensler TW, Curphey TJ, Maxiutenko Y et al (2000) Chemoprotection by organosulfur inducers of phase 2 enzymes: dithiolethiones and dithiins. Drug Metabol Drug Interact 17:3–22 Liu Y, Kern JT, Walker JR et al (2007) A genomic screen for activators of the antioxidant response element. Proc Natl Acad Sci USA 104:5205–5210 Murphy SE, Nunes MG, Hatala MA (1997) Effects of phenobarbital and 3-methylcholanthrene induction on the formation of three glucuronide metabolites of 4-(methylnitrosamino)-1(3-pyridyl)-1-butanone, NNK. Chem Biol Interact 103:153–166 Nguyen T, Sherratt PJ, Pickett CB (2002) Regulatory mechanisms controlling gene expression mediated by the antioxidant response element. Annu Rev Pharmacol Toxicol 43:233–260 Okey AB, Roberts EA, Harper PA et al (1986) Induction of drug-metabolizing enzymes: mechanisms and consequences. Clin Biochem 19:132–141 Prochaska HJ, Talalay P (1998) Regulatory mechanisms of monofunctional and bifunctional anticarcinogenic enzyme inducers in murine liver. Cancer Res 48:4776–4782 Prochaska HJ, Santamaria AB, Talalay P (1992) Rapid detection of inducers of enzymes that protect against carcinogens. Proc Natl Acad Sci USA 89:2394–2398 Ramos-Gomez M, Kwak MK, Dolan PM et al (2001) Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice. Proc Natl Acad Sci USA 98:3410–3415 Shen G, Kong AN (2009) Nrf2 plays an important role in coordinated regulation of phase II drug metabolism enzymes and phase III drug transporters. Biopharm Drug Dispos 30:345–355 Talalay P (1989) Mechanisms of induction of enzymes that protect against chemical carcinogenesis. Adv Enzyme Regul 28:237–250 Zevin S, Benowitz NL (1999) Drug interactions with tobacco smoking. An update. Clin Pharmacokinet 36:425–438

Chapter 10

Induction and Inhibition Compounds

Enzyme systems in detoxification processes including phase I and phase II enzymes protect the cells from toxic effects mediated by a variety of foreign compounds. The hypotheses underlying the defense against xenobiotics-mediated toxicity include (a) the reduction of activation by inhibiting phase I activation enzymes, (b) the enhancement of detoxification by inducing phase II detoxification enzymes, and (c) the glutathione antioxidant protection against reactive oxygen species. Phase I enzyme inhibition is mainly concentrated on CYP450. Phase II enzyme induction is largely focused on UDP-glucuronosyl transferases, glutathione S-transferases, and quinone reductases. Glutathione is considered as not only a nucleophilic scavenger of reactive oxygen species, but also a conjugation cofactor in glutathione S-transferase catalyzed reactions.

10.1

Defense Against Potential Toxicities

CYP450 is the major activation enzyme responsible for a majority of activation reactions that involve various foreign compounds including toxins, drugs, and pesticides. The modulation of CYP450 activity as a defense against xenobiotic-mediated toxic effects is complicated and the result of modulation is associated with the expression of detoxification enzymes. The effects of CYP450 modulation depend on how the detoxification enzyme activity is comparable with the CYP450 activity. If the rate of activation is higher than that of detoxification, an overload of potentially toxic intermediates may occur. In contrast, an over inhibition of CYP450 may cause an accumulation of xenobiotics and impaired metabolic clearance. Glutathione in combination with its coupled glutathione S-transferase enzyme system is one of the most important antioxidant defense mechanisms in the body. The reactive sulfhydryl group of glutathione is responsible for glutathione antioxidant activities. A high level of intracellular glutathione maintained in many tissues plays a prominent role in antioxidant protection against reactive oxygen species. A reduction of the amount of glutathione to a lower level can impair the defense of C.-H. Chen, Activation and Detoxification Enzymes: Functions and Implications, DOI 10.1007/978-1-4614-1049-2_10, © Springer Science+Business Media, LLC 2012

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the cells against toxic effects of such species. The depletion of glutathione occurs due to a lack of essential nutrients or amino acids needed to synthesize glutathione. Environmental effects such as smoking can also deplete the level of glutathione. Considerable evidence has been accumulated to support the proposal that the induction of phase II detoxification enzymes is a useful approach for defense against the risk of exposure to xenobiotic toxicity. In mice model studies, it has been postulated that the induction of phase II enzymes is a highly effective approach for reducing susceptibility to carcinogens. The specific transcription factor (Nrf2) that binds to the antioxidant response element (ARE) is essential for the induction of phase II enzymes. As indicated in Chap. 9, when this transcription factor is deleted in knockout mice, the basal levels of phase II enzymes are very low and not inducible. Consequently, these mice are no longer protected by phase II inducers and become much more susceptible to stomach carcinogenesis by benzo[a]pyrene than their wild-type counterparts. This observation further supports the proposal that the induction of phase II enzymes is a potential defense mechanism of chemoprevention. Chemical compounds that modulate the expression of phase II enzymes may play an important role in the intervention of carcinogenic processes, particularly at the initial stage where activated reactive metabolites are involved. Known compounds of importance in the modulation of foreign compound-metabolic enzymes include (a) sulforaphane and other isothiocyanates, (b) 1,2-dithiole-3-thione and derivatives, (c) indole-3-carbinol, (d) flavonoids and isoflavones, (e) polyphenols, (f) organosulfur compounds, and (g) terpenes and terpenoids. Among these chemical compounds, oltipraz (1,2-dithiole-3-thione derivative) and sulforaphane (4-methyl-sulfinylbutane isothiocyanate) are the most studied inducers of detoxification enzymes. There are other compounds, such as geniposide, that do not belong to the above categories. Geniposide is an iridoid glycoside extracted from the fruits of Gardenia jasminoides used as an herbal medicine for treating hepatic and inflammatory diseases. Geniposide was reported to inhibit liver CYP450-dependent monooxygenases, increase hepatic glutathione, and induce glutathione S-transferase activity in the liver.

10.2

Sulforaphane and Isothiocyanates

Isothiocyanates contain –N=C=S group formed by substituting sulfur of isocyanates for oxygen. They are hydrolysis products of the enzymatic conversion of metabolites called glucosinolates, sulfur-containing compounds. Isothiocyanates are primarily metabolized through the mercapturic acid pathway, which gives rise to N-acetylcysteine conjugates. Cruciferous vegetables contain a variety of glucosinolates which form different isothiocyanates under hydrolysis reactions. Glucoraphanin is the precursor of sulforaphane. Sulforaphane is produced from glucoraphanin by myrosinase, a class of enzymes that catalyzes the hydrolysis of glucosinolates. Other isothiocyanates of interest include allyl, phenethyl, and benzyl isothiocyanates. Animals and cultured human cell studies have reported that many isothiocyanates, particularly sulforaphane, are potent inducers of phase II detoxification

10.3

1,2-Dithiole-3-Thione and Derivatives

93

enzymes including glutathione S-transferases, UDP-glucuronosyl transferases, and quinone reductase. Among isothiocyanates, sulforaphane is the most investigated and is thought to provide the most protective effect of glucoraphanin. Isothiocyanates have been shown to enhance phase II enzyme activity by increasing the transcription of genes that contain an ARE. The effects of isothiocyanates appear to be mediated by the induction of phase II enzymes which detoxify electrophilic metabolites generated by phase I enzymes and thereby destroy their ability to damage DNA. Sulforaphane has been reported to significantly induce phase II enzyme activity in human prostate cells and block mammary tumor formation in rats. Oral administration of sulforaphane also potently induces phase II enzymes in the bladder tissues in animal models. Studies have also suggested the importance of phase II enzymes in modifying the effects of allergic inflammation. Diesel exhaust particles initiate and intensify airway allergic responses through enhancing IgE production. These exhaust particles are associated with allergic respiratory disorders including asthma. Induction of glutathione S-transferases and quinone reductases by sulforaphane was reported to block diesel exhaust particles-induced enhancement of immunoglobulin IgE production. Moreover, anticarcinogenic effects of isothiocyanates also appear to be mediated by the suppression of carcinogen activation by CYP450 isozymes through inhibition and regulation of their catalytic activities. CYP450 2B1 is one of the major isozymes involved in the activation of nicotine-derived nitrosamine ketone (NNK). NNK is the most potent carcinogen present in tobacco. Inhibition of NNK-induced lung tumorigenesis by phenethyl isothiocyanate has been reported to block the metabolic activation of NNK, which results in an increase in urinary excretion of detoxified metabolites. Phenethyl isothiocyanate is also an effective inhibitor of lung tumor induction by the tobacco-specific nitrosamine, 4-(methylnitrosamine)-1-(3pyridyl)-1-butanone. A number of isothiocyanates, including sulforaphane, have been reported to modulate phase II enzymes and phase I enzymes. These isothiocyanates are listed in Table 10.1. In those studies, animal models and cultured cells were employed. The table shows that sulforaphane and other isothiocyanates act as not only the inducers for phase II enzymes, but also the inhibitors for phase I enzymes, mainly CYP450.

10.3

1,2-Dithiole-3-Thione and Derivatives

1,2-Dithiole-3-thiones (D3Ts) are naturally occurring five-member cyclic organosulfur compounds with antioxidant and chemoprotective properties. Chemoprotection is a quality of some chemicals or foods that may protect healthy tissues from the toxic effects of anticancer drugs or carcinogens. The increased expression of detoxification enzymes is of central importance to chemoprevention. The transcription

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Table 10.1 Enzyme modulation by isothiocyanates Induction of phase II enzymes Compound Enzyme induction Sulforaphane GST, QOR, epoxide hydrolase Phenylethyl isothiocyanate GST, QOR Ally isothiocyanate GST Benzyl isothiocyanate GST Inhibition of phase I enzymes Compound Enzyme inhibition Sulforaphane CYP450 Phenylpropyl isothiocyanate CYP450 Phenylhexyl isothiocyanate CYP450 Benzyl isothiocyanate CYP450 Phenylethyl isothiocyanate CYP450 Phenyl isothiocyanate CYP450

Induction and Inhibition Compounds

Model systema Animal tissue, cultured cells Animal tissue, cultured cells Animal tissue Animal tissue Model systema Animal tissue, cultured cells Animal tissue Animal tissue Animal tissue Animal tissue, cultured cells Animal tissue

GST glutathione S-transferase, QOR quinone oxidoreductase, CYP450 cytochrome P450 a Animal tissue from rat, mouse, or hamster; cultured cells from human or rat

factor (Nrf2) is essential for the induction of detoxification enzymes. D3Ts have been shown to induce Nrf2-dependent phase II enzymes. Exposure of rodents to D3Ts triggers nuclear accumulation of Nrf2, leading to transcriptional activation of genes involved in carcinogen detoxification. In contrast, D3Ts fails to induce these genes in Nrf2-deficient mice. Some derivatives of D3T have also been shown to induce phase II enzymes and to protect against chemical carcinogenesis in animals. The most extensively studied D3T derivative is oltipraz (4-methyl-5-pyrazinyl-3H-1,2-dithiole-3thione), a synthetic compound. The chemoprotective action of oltipraz may offer protection against a wide range of carcinogens. Rodent model studies have demonstrated chemoprevention of aflatoxin-induced hepatocarcinogenesis by oltipraz. Administration of oltipraz was reported to protect mice against the neoplasia induced by benzo[a]pyrene. Several other D3T derivatives (e.g., 5,6-dihydrocyclopentaD3T, 4-chloro-5-methyl-D3T, and 4-phenyl-D3T) were also found to exhibit protection against acute toxicity of many xenobiotics and offer effective inhibition of carcinogenesis. The chemoprotective effect of oltipraz is attributed, in part, to the induction of detoxification enzymes (e.g., glutathione S-transferases, UDP-glucuronosyl transferases, and quinone oxidoreductases) in the liver and other target tissues. The inhibition of activation enzymes, mainly by CYP450 isozymes, may also attribute to the effect. Table 10.2 lists D3T, oltipraz and other D3T derivatives that were reported to modulate phase II enzymes or phase I enzymes. Animal models and cultured cells were also employed in those studies. The table reveals that D3T, oltipraz, and other D3T derivatives induce phase II enzymes. While, D3T and oltipraz also inhibit the major phase I enzyme, CYP450.

10.4

Indole-3-Carbinol

95

Table 10.2 Enzyme modulation by 1,2-dithiole-3-thione and derivatives Induction of phase II enzymes Compound Enzyme induction Model systema 1,2-Dithiole-3-thione GST Animal tissue Oltipraz QOR, GST, UGT Animal tissue, cultured cell 5,6-Dihydrocyclopenta-1,2-dithiole- QOR, GST Animal tissue 3-thione 4-Chloro-5-methyl-1,2-dithiole-3QOR, GST Animal tissue thione 4-Phenyl-1,2-dithiole-3-thione QOR, GST Animal tissue Inhibition of phase I enzymes Compound Enzyme inhibition Model systema b 1,2-Dithiole-3-thione CYP450 Animal tissue Oltipraz CYP450 Cultured cells or plasma GST glutathione S-transferase, QOR quinone oxidoreductase, CYP450 cytochrome P450, Oltipraz 4-methyl-5-pyrazinyl-3H-1,2-dithiole-3-thione, UGT uridine-diphosphate glucuronosyl transferase a Animal tissue from rat, mouse, or hamster; cultured cells from human or rat b Slight inhibition or no effect

10.4

Indole-3-Carbinol

Indole-3-carbinol is the hydrolysis product of glucobrassicin found at relatively high levels in cruciferous vegetables. When plant cells are damaged by chopping or chewing, glucobrassicin interacts with myrosinase, resulting in the formation of indole-3-carbinol. Myrosinase, an enzyme that catalyzes the hydrolysis of glucosinolates, is physically separated from glucosinolates in intact plant cells. Oral consumption of indole-3-carbinol leads to the formation of acid condensation products (e.g., dimeric 3,3¢-diindolylmethane), which are responsible for the biological effects attributed to indole-3-carbinol. Indole-3-carbinol, a potential chemopreventive agent, is a compound of growing interest. Indole-3-carbinol was found to induce hepatic levels of CYP1A1, but inhibit flavin-containing monooxygenase in rat livers and intestines. Studies of CCl4-induced hepatotoxicity revealed that indole-3-carbinol induced the level of CYP450 activity; however, CCl4-produced decrease in hepatic levels of glutathione was restored to control levels by indole-3-carbinol. Indole-3-carbinol was also found to inhibit aflatoxin-induced hepatocarcinogenesis in rats. Reports of indole-3-carbinol on the modulation of activation enzymes (e.g., CYP450 and FMO) and detoxification enzymes (e.g., GST and QOR) are presented in Table 10.3. The table reveals that indole-3-carbinol affects both phase I enzymes and phase II enzymes.

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Table 10.3 Enzyme modulation by indole-3-carbinol Induction of phase II enzymes Compound Enzyme induction Indole-3-carbinol GST, QOR Inhibition of phase I enzymes Enzyme Compound Induction Inhibition Indole-3-carbinol CYP450 FMO

Model systema Animal tissue

Model systema Animal tissue Animal tissue

GST glutathione S-transferase, QOR quinone oxidoreductase, CYP450 cytochrome P450, FMO flavin-containing monooxygenase a Animal tissue from rat, mouse, or hamster; cultured cells from human or rat

10.5

Flavonoids and Isoflavones

Flavonoids are the most common group of polyphenolic compounds that are synthesized by plants. Based on chemical structure, flavonoids are grouped into flavonols, flavones, flavanones, isoflavones, catechins, anthocyanins, and chalcones. Many biological effects of flavonoids appear to be associated with their ability to modulate cell-signaling pathways. However, a number of studies have revealed the effects of flavonoids on foreign compound-metabolizing enzymes. The protective effects of flavonoids against xenobiotic toxicity are attributed in part to the modulation of these enzymes. For example, leucocyanidin is known to exhibit antioxidant and antimutagenic activities and exert a protective effect against cardiovascular disease. Its role as a chemopreventive agent against toxic or carcinogenic metabolites is associated with the induction of detoxification enzymes and the inhibition of activation enzymes. It has been proposed that modulation of detoxification enzymes (e.g., uridinediphosphate-glucuronosyltransferase, glutathione S-transferase, and quinone oxidoreductase) to accelerate detoxification of carcinogens is an important mechanism of the anticarcinogenic effects of flavonoides. For example, isoliquiritigenin is an inducer of quinone reductase and 4¢-bromoflavone significantly induces quinone reductase in addition to glutathione S-transferase. Table 10.4 lists a number of flavonoids that are reported to modulate phase II enzymes and phase I enzymes. These flavonoids induce detoxification enzymes. However, their effects on the activation enzyme CYP450 are not homogenous. The effects can be induction, inhibition or no significant effect. Isoflavones comprise a class of organic compounds related to flavonoids. Soybeans and soy products are rich sources of isoflavones in the human diet. Genistein and daidzein are two of several known isoflavones found in plants and herbs. Isoflavones were reported to regulate the expression of genes critical to drug metabolism. When rats consume a diet high in isoflavones, the activities of glutathione S-transferase in kidney and quinone reductase in the colon are higher.

10.5

Flavonoids and Isoflavones

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Table 10.4 Enzyme modulation by flavonoids Induction of phase II enzymes Compound Enzyme induction Isoliquiritigenin QOR 4¢-Bromoflavone QOR, GST Beta-naphthoflavone GST, UGT, NQO Leucocyanidin GST Anthocyanins GTS Catechin UGT Quercetin QOR, QR Myricetin QOR Inhibition/induction of phase I enzymes Enzyme Compound Inhibition Induction 4¢-Bromoflavone Beta-naphthoflavone Leucocyanidin Catechin Epigallocatechin Quercetin

CYP450

Model systema Cultured cell Cultured cell Animal tissue Animal tissue Animal tissue Animal tissue Cultured cell Cultured cell

Unaffected

Model systema

CYP450

Cultured cells Animal tissue Animal tissue Animal tissue Animal tissue Animal tissue

CYP450 CYP450 CYP450 CYP450 CYP450

GST glutathione S-transferase, QOR quinone oxidoreductase, CYP450 cytochrome P450, QR quinone reductase, NQO NADH-quinone oxidoreductase, UGT uridine-diphosphate glucuronosyl transferase a Animal tissue from rat, mouse, or hamster; cultured cells from human or rat

Table 10.5 Enzyme modulation by isoflavones Induction of phase II enzymes Compound Enzyme induction Genistein QR Daidzein QR Soy isoflavones GST, QR and UGT Inhibition/induction of phase I enzymes Enzyme Compound Inhibition Induction Genistein CYP450 Soy isoflavones CYP450

Model systema Cultured cell Cultured cell Animal gene or tissue

Model systema Animal genes

GST glutathione S-transferase, QR quinone reductase, CYP450 cytochrome P450, UGT uridinediphosphate glucuronosyl transferase a Animal tissue from rat, mouse, or hamster; cultured cells from human or rat

Genistein was found to induce the activity of quinone reductase and inhibit the expression of aromatase. The inhibition of aromatase leads to a decrease in estrogen biosynthesis, thus producing an antiestrogenic effect. Table 10.5 lists some isoflavones that are capable of modulating detoxification enzymes or phase I enzymes. In addition to inducing phase II enzymes, genistein and soy isoflavones can also affect the activity of CYP450.

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10.6

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Induction and Inhibition Compounds

Polyphenols

Polyphenols are characterized by the presence of more than one phenol group in their chemical structure. Polyphenolic compounds are a group of chemical compounds present in beverages such as wine and tea. There has been a growing interest in investigation of the role of polyphenolic compounds in the prevention of diseases such as cancer and cardiovascular diseases. Several naturally occurring plant polyphenols have also been reported to inhibit the mutagenicity of chemical carcinogens such as polycyclic aromatic hydrocarbons. For example, resveratrol, a well-known polyphenol, has been reported to exert its chemopreventive activity against carcinogenesis and to provide protection against oxidative cardiovascular disorders. Most animal studies also indicate that tea exhibits chemopreventive effects against lung tumorigenesis. It has been reported that polyphenols are capable of inducing detoxification enzymes and/or inhibition of activation enzymes. Induction of detoxification enzymes is a potential mechanism through which polyphenols carry out anticarcinogenic activities. For example, curcumin was found to modestly induce detoxification enzyme activity in the prostate in animal models. Tea polyphenols were found to increase the activity of glutathione S-transferase. Table 10.6 lists a number of polyphenols that have been reported to affect phase II and phase I enzymes. The table reveals that resveratrol is capable of inducing detoxification enzymes, while most other polyphenols induce detoxification enzymes in addition to inhibiting CYP450.

10.7

Organosulfur Compounds

In addition to 1,2-dithiole-3-thione, organosulfur compounds that have the capacity to affect activation or detoxification enzymes include diallyl sulfide, diallyl disulfide, diallyl trisulfide, and alliin. Diallyl sulfide, diallyl disulfide, and diallyl trisulfide are principal constituents of garlic oil. Among the most studied organosulfur compounds is diallyl sulfide which was found to increase the activities of QOR and GST in the tissues of stomach, colon, liver, lung, and urinary bladder. Diallyl sulfide was also reported to inhibit CYP2E1 activity, but induce the activity of CYP1A1 or CYP1A2. Moreover, diallyl sulfide was shown to inhibit chemically induced carcinogenesis and cytotoxicity in animal model systems, for instances, the inhibition of 1,2-dimethylhydrazine-induced colon and liver cancers in rodents, and the inhibition of arylamine N-acetyltransferase activity and gene expression in human colon cancer cell lines. It has also been reported that alliin, a sulfoxide, is capable of inducing UGT and GTS activities, and inhibiting CYP2E1 activity, but slightly inducing CYP1A2 activity. Naturally occurring organosulfur compounds have been recognized as potential chemopreventive chemicals. For example, diallyl sulfide and diallyl disulfide were found to inhibit aflatoxin B1-initiated carcinogenesis in rat liver. The prospective mechanisms that are responsible for such protective effects are believed to be the inhibition of carcinogen activation through modulating phase I enzymes (e.g., CYP450 and monooxygenases) and/or the induction of carcinogen detoxification through inducing phase II enzymes. Table 10.7 lists a number of organosulfur compounds

10.7

Organosulfur Compounds

Table 10.6 Enzyme modulation by polyphenols Induction of phase II enzymes Compound Enzyme induction Resveratrol GTS, QOR, UGT Curcumin GTS, QOR Turmeric GTS Carnosol GTS Carnosic acid GTS Protocatechuic acid GST, UGT, NQO Tannic acid GST, NQOb Ellagic acid GST, QR, UGT Gallic acid GST Polyphenols (tea) GST, QR Inhibition of phase I enzymes Compound Enzyme inhibition Turmeric CYP450 Curcumin CYP450 Carnosol CYP450 Carnosic acid CYP450 Protocatechuic acid CYP450 Tannic acid CYP450 Ellagic acid CYP450

99

Model systema Animal tissue Animal tissue, cultured cells Animal tissue Cultured cells Cultured cells Animal tissue Animal tissue Animal tissue Cultured cells Animal tissue Model systema Animal tissue Animal tissue Cultured cells Cultured cells Animal tissue Animal tissue Animal tissue

GST glutathione S-transferase, QOR quinone oxidoreductase, CYP450 cytochrome P450, NQO NADH-quinone oxidoreductase, QR quinone reductase, UGT uridine-diphosphateglucuronosyl transferase a Animal tissue from rat, mouse or hamster; cultured cells from human or rat b Inhibition

Table 10.7 Enzyme modulation by organosulfur compounds Induction of phase II enzymes Compound Enzyme induction Model systema Diallyl sulfide GTS, QOR Animal tissue Diallyl disulfide GTS, QOR, UGT Animal tissue, cultured cells Diallyl trisulfide GTS, QOR Animal tissue, cultured cells Alliin GTS, UGT – Modulation of phase I enzymes Enzyme Compound Model systema Induction Inhibition Diallyl sulfide CYP2E1 CYP1A1, CYP1A2 Animal tissue Diallyl disulfide CYP2E1 Animal tissue Diallyl trisulfide – – Alliin CYP2E1 CYP1A2 Animal tissue GST glutathione S-transferase, QOR quinone oxidoreductase, CYP450 cytochrome P450, UGT uridine-diphosphate glucuronosyl transferase a Animal tissue from rat, mouse, or hamster; cultured cells from human or rat

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Induction and Inhibition Compounds

Table 10.8 Enzyme modulation by terpenes and terpenoids Induction of detoxification enzymes Compound Enzyme induction

Model systema

b-Carotene Lycopene Zerumbone Canthaxanthin Astaxanthin Modulation of activation enzymes Compound

QOR QOR GST UGT, QOR UGT, QOR

Animal tissue Animal tissue, cultured cells Cultured cells Animal tissue Animal tissue

Enzyme induction

Model systema

Canthaxanthin Astaxanthin

CYP450 CYP450

Animal tissue Animal tissue

GST glutathione S-transferase, QOR quinone oxidoreductase, CYP450 cytochrome P450, UGT uridine-diphosphate glucuronosyl transferase a Animal tissue from rat, mouse, or hamster; cultured cells from human or rat

that are reported to affect foreign compound-metabolizing enzymes. The table reveals that organosulfur compounds induce detoxification enzymes, but either inhibit or induce activation enzyme CYP450, depending on its isozymes.

10.8

Terpenes and Terpenoids

Terpenes are naturally occurring hydrocarbons composed of isoprene units. Among known terpenes are limonene and carotene. Lemon and citrus fruits contain a considerable amount of limonene. Foods such as carrots and cantaloupe are rich in carotene. Carotene consists of two primary isomers, a-carotene and b-carotene, which differ in the position of double bonds in the cyclic group at the end. Carotene is known for protecting plant cells against destruction from ultraviolet light. Carotenoids belong to a larger class of chemicals called terpenoids, which are compounds related to terpenes. The best known carotenoid is lycopene. Lycopene is present in ripe fruits, especially tomatoes. Other carotenoids include canthaxanthin, astaxanthin, and zerumbone. Canthaxanthin and astaxanthin are b-carotene-related compounds. Zerumbone is a sesquiterpene phytochemical found in subtropical edible ginger. Table 10.8 lists a number of terpenes, terpenoids, and carotenoids that are reported to modulate phase I enzymes and phase II enzymes. The table reveals that these compounds exhibit the induction of phase II enzymes. Among them, canthaxanthin and astaxanthin also induce phase I enzyme CYP450. Expression of phase II enzymes requires ARE and the transcription factor Nrf2. Lycopene was found to activate the expression of reporter gene fused with ARE sequences in transiently transfected cancer cells. Other carotenoids such as b-carotene and astaxanthin exhibit a similar, but much smaller effect. Moreover, the exposure of epithelial cell lines to zerumbone results in an induction of glutathione S-transferase. Zerumbone may be a chemopreventive agent. This carotenoid was also found to induce nuclear localization of the transcript factor Nrf2 that binds to ARE.

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Bibliography Ahn D, Putt D, Kresty L et al (1996) The effects of dietary ellagic acid on rat hepatic and esophageal mucosal cytochromes P450 and phase II enzymes. Carcinogenesis 17:821–828 Ben-Dor A, Steiner M, Gheber L et al (2005) Carotenoids activate the antioxidant response element transcription system. Mol Cancer Ther 4:177–186 Brady JF, Ishizaki H, Fukuto JM et al (1991) Inhibition of cytochrome P-450 2E1 by diallyl sulfide and its metabolites. Chem Res Toxicol 4:642–647 Brooks JD, Paton VG, Vidanes G (2001) Potent induction of phase 2 enzymes in human prostate cells by sulforaphane. Cancer Epidemiol Biomarkers Prev 10:949–954 Clark J, You M (2006) Chemoprevention of lung cancer by tea. Mol Nutr Food Res 50:144–151 Crowell PL, Gould MN (1994) Chemoprevention and therapy of cancer by d-limonene. Crit Rev Oncog 5:1–22 Davenport DM, Wargovich MJ (2005) Modulation of cytochrome P450 enzymes by organosulfur compounds from garlic. Food Chem Toxicol 43:1753–1762 Dinkova-Kostova AT, Talalay P (1999) Relation of structure of curcumin analogs to their potencies as inducers of phase 2 detoxification enzymes. Carcinogenesis 20:911–914 Dinkova-Kostova AT, Fahey JW, Talalay P (2004) Chemical structures of inducers of nicotinamide quinone oxidoreductase 1 (NQO1). Methods Enzymol 382:423–448 Fahey JW, Talalay P (1999) Antioxidant functions of sulforaphane: a potent inducer of phase II detoxication enzymes. Food Chem Toxicol 37:973–979 Fong AT, Swanson HI, Dashwood RH et al (1990) Mechanisms of anti-carcinogenesis by indole3-carbinol. Studies of enzyme induction, electrophile-scavenging, and inhibition of aflatoxin B1 activation. Biochem Pharmacol 39:19–26 Fukao T, Hosono T, Misawa S et al (2004) The effects of allyl sulfides on the induction of phase II detoxification enzymes and liver injury by carbon tetrachloride. Food Chem Toxicol 42:743–749 Gradelet S, Astorg P, Leclerc J et al (1996) Effects of canthaxanthin, astaxanthin, lycopene and lutein on liver xenobiotic-metabolizing enzymes in the rat. Xenobiotica 26:49–63 Guyonnet D, Belloir C, Suschetet M et al (2001) Antimutagenic activity of organosulfur compounds from allium is associated with phase II enzyme induction. Mutat Res 495:135–145 Hamilton SM, Teel RW (1996) Effects of isothiocyanates on cytochrome P-450 1A1 and 1A2 activity and on the mutagenicity of heterocyclic amines. Anticancer Res 16:3597–3602 Hecht SS (2000) Inhibition of carcinogenesis by isothiocyanates. Drug Metab Rev 32:395–411 Kang JJ, Wang HW, Liu TY et al (1997) Modulation of cytochrome P-450-dependent monooxygenases, glutathione and glutathione S-transferase in rat liver by geniposide from Gardenia jasminoides. Food Chem Toxicol 35:957–965 Krajka-Ku niak V, Szaefer H, Baer-Dubowska W (2004) Modulation of 3-methylcholanthreneinduced rat hepatic and renal cytochrome P450 and phase II enzymes by plant phenols: protocatechuic and tannic acids. Toxicol Lett 152:117–126 Kwak MK, Egner PA, Dolan PM et al (2001) Role of phase 2 enzyme induction in chemoprotection by dithiolethiones. Mutat Res 480–481:305–315 Lançon A, Hanet N, Jannin B et al (2007) Resveratrol in human hepatoma Hep G2 cells: metabolism and inducibility of detoxifying enzymes. Drug Metab Dispos 35:699–703 Li Y, Cao Z, Zhu H (2006) Upregulation of endogenous antioxidants and phase 2 enzymes by the red wine polyphenol, resveratrol in cultured aortic smooth muscle cells leads to cytoprotection against oxidative and electrophilic stress. Pharmacol Res 53:6–15 Li Y, Mezei O, Shay NF (2007) Human and murine hepatic sterol-12-alpha-hydroxylase and other xenobiotic metabolism mRNA are upregulated by soy isoflavones. J Nutr 137:1705–1712 Mahéo K, Morel F, Langouët S et al (1997) Inhibition of cytochromes P-450 and induction of glutathione S-transferases by sulforaphane in primary human and rat hepatocytes. Cancer Res 57:3649–3652 Moon YJ, Wang X, Morris ME (2006) Dietary flavonoids: effects on xenobiotic and carcinogen metabolism. Toxicol In Vitro 20:187–210

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Munday R, Munday CM (2001) Relative activities of organosulfur compounds derived from onions and garlic in increasing tissue activities of quinone reductase and glutathione transferase in rat tissues. Nutr Cancer 40:205–210 Munday R, Munday CM (2004) Induction of phase II enzymes by 3H-1,2-dithiole-3-thione: dose– response study in rats. Carcinogenesis 25:1721–1725 Munday R, Zhang Y, Paonessa JD et al (2010) Synthesis, biological evaluation, and structureactivity relationships of dithiolethiones as inducers of cytoprotective phase 2 enzymes. J Med Chem 53:4761–4767 Nakajima M, Yoshida R, Shimada N et al (2001) Inhibition and inactivation of human cytochrome P450 isoforms by phenethyl isothiocyanate. Drug Metab Dispos 29:1110–1113 Nakamura Y, Yoshida C, Murakami A et al (2004) A tropical ginger sesquiterpene, activates phase II drug metabolizing enzymes. FEBS Lett 572:245–250 Ow YY, Stupans I (2003) Gallic acid and gallic acid derivatives: effects on drug metabolizing enzymes. Curr Drug Metab 4:241–248 Pugazhenthi S, Akhov L, Selvaraj G et al (2007) Regulation of heme oxygenase-1 expression by demethoxy curcuminoids through Nrf2 by a PI3-kinase/Akt-mediated pathway in mouse betacells. Am J Physiol Endocrinol Metab 293:E645–E655 Ramos-Gomez M, Kwak MK et al (2001) Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice. Proc Natl Acad Sci USA 98:3410–3415 Reicks MM, Crankshaw DL (1996) Modulation of rat hepatic cytochrome P-450 activity by garlic organosulfur compounds. Nutr Cancer 25:241–248 Roebuck BD, Curphey TJ, Li Y et al (2003) Evaluation of the cancer chemopreventive potency of dithiolethione analogs of oltipraz. Carcinogenesis 24:1919–1928 Rogan EG (2006) The natural chemopreventive compound indole-3-carbinol: state of the science. In Vivo 20:221–228 Seo K, Jung S, Park M et al (2001) Effects of leucocyanidines on activities of metabolizing enzymes and antioxidant enzymes. Biol Pharm Bull 24:592–593 Shih PH, Yeh CT, Yen GC (2007) Anthocyanins induce the activation of phase II enzymes through the antioxidant response element pathway against oxidative stress-induced apoptosis. J Agric Food Chem 55:9427–9435 Song LL, Kosmeder JW 2nd, Lee SK et al (1999) Cancer chemopreventive activity mediated by 4¢-bromoflavone, a potent inducer of phase II detoxification enzymes. Cancer Res 59:578–585 Steele VE, Kelloff GJ, Balentine D et al (2000) Comparative chemopreventive mechanisms of green tea, black tea and selected polyphenol extracts measured by in vitro bioassays. Carcinogenesis 21:63–67 Tsai CW, Chen HW, Yang JJ et al (2007) Diallyl disulfide and diallyl trisulfide up-regulate the expression of the pi class of glutathione S-transferase via an AP-1-dependent pathway. J Agric Food Chem 55:1019–1026 von Weymarn LB, Chun JA, Knudsen GA et al (2007) Effects of eleven isothiocyanates on P450 2A6- and 2A13-catalyzed coumarin 7-hydroxylation. Chem Res Toxicol 20:1252–1259 Wallig MA, Kingston S, Staack R et al (1998) Induction of rat pancreatic glutathione S-transferase and quinone reductase activities by a mixture of glucosinolate breakdown derivatives found in Brussels sprouts. Food Chem Toxicol 36:365–373 Wang W, Liu LQ, Higuchi CM et al (1998) Induction of NADPH:quinone reductase by dietary phytoestrogens in colonic Colo205 cells. Biochem Pharmacol 56:189–195 Xu M, Dashwood RH (1999) Chemoprevention studies of heterocyclic amine-induced colon carcinogenesis. Cancer Lett 143:179–183 Yang CS, Chhabra SK, Hong JY et al (2001) Mechanisms of inhibition of chemical toxicity and carcinogenesis by diallyl sulfide (DAS) and related compounds from garlic. J Nutr 131:1041S–1045S Yannai S, Day AJ, Williamson G et al (1998) Characterization of flavonoids as monofunctional or bifunctional inducers of quinone reductase in murine hepatoma cell lines. Food Chem Toxicol 36:623–630 Zhang W, Go ML (2007) Quinone reductase induction activity of methoxylated analogues of resveratrol. Eur J Med Chem 42:841–850

Chapter 11

Diets Rich in Enzyme Modulators

Phase I activation enzymes metabolize foreign compounds to either inactive metabolites or active intermediates. Reactive metabolic intermediates contain highly reactive chemical groups which can exert their toxicity through interacting with cell components (proteins, DNA, and lipids). While phase II detoxification enzymes are capable of detoxifying chemically active intermediates, resulting in facilitating their excretion from the body, problems arise when these two metabolizing enzyme systems are in imbalance. Furthermore, some foreign compounds, with little or no intrinsic toxicity, may act to enhance toxic effects of other compounds by interacting effectively with activation enzymes or detoxification enzymes. The detoxification metabolic systems are highly complex and are responsive to individual’s life style. Metabolic conversion of xenobiotics to toxic substances can be dramatically influenced by the nutritional status of the organism. For example, when the consumption of a specific food modulates the activity of a drugmetabolizing enzyme, food–drug interactions occur, resulting in an alteration of the pharmacokinetics of the drug metabolism. Moreover, waste products produced in the processes of converting food into raw materials and energy must be eliminated from the body. Dietary changes can affect the metabolisms of waste products and have a profound influence on human health. There has been an intensive interest in searching for chemical compounds in human diets which have potential to benefit human health through manipulations of detoxification or activation enzymes. Extensive investigations of dietary chemicals have been carried out by a number of laboratories and a significant number of dietary chemicals that are able to induce or inhibit foreign compound-metabolizing enzymes have been identified. Many of such dietary chemicals also affect cellular levels of antioxidants. This chapter discusses diets that are rich in inducers of phase II detoxification enzymes or inhibitors of phase I activation enzymes. The available evidence suggests that diets rich in vegetables and fruits have a lower risk of developing disease conditions such as aging, cardiovascular problems, and cancers.

C.-H. Chen, Activation and Detoxification Enzymes: Functions and Implications, DOI 10.1007/978-1-4614-1049-2_11, © Springer Science+Business Media, LLC 2012

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Diets Rich in Enzyme Modulators

Dietary Contributions to Enzyme Modulation

The previous chapter describes a variety of classes of chemical compounds that are inducers of phase II enzymes and/or inhibitors of phase I enzymes, which are capable of modulating the metabolism of foreign compounds. Many of such chemical compounds are present in human diets. For example, garlic and onion are rich in polyphenolic and flavonoid compounds, broccoli has a high content of isothiocyanates, and soy bean is rich in isoflavones. By increasing intake of vegetables, it is possible to modify the diet of an individual to improve the efficiency of detoxification metabolic systems or to regulate the imbalance between activation enzymes and detoxification enzymes. Diets rich in chemical compounds that are capable of modulating foreign compound metabolizing enzymes can have significant impacts on human health. Moreover, many dietary factors possess antioxidant and anti-inflammatory properties. Diets that promote oxidant scavenging by inducing detoxification enzymes have been reported to have positive influence on human health conditions such as aging associated degenerative changes. Increasing evidence has also implicated dietary factors in the progression of diseases such as diabetes, obesity, and certain cancers. Foods contain a large variety of chemical components which may be procarcinogens (e.g., nitrosamines, heterocyclic amines, and aflatoxin) or anticarcinogens (e.g., indoles, isothiocyanates, and organosulfurs). Researchers have identified numerous phytochemicals to evaluate their chemopreventive capacity. Food components were reported to modify carcinogenesis in a number of manners, including the modification of carcinogen activation by inhibiting activation enzymes, as well as the alteration of carcinogen detoxification by inducing detoxification enzymes.

11.2

Vegetables Rich in Enzyme Modulators

Besides providing excellent sources of fiber, vitamins, and minerals, vegetables also contain non-nutritive components that can provide substantial health benefits beyond basic nutrition. Many non-nutritive chemical components, such as those described in the previous chapter, are capable of modulating activation or detoxification enzymes (e.g., sulforaphane in broccoli, phenethyl isothiocyanate in watercress, organosulfurs in garlic, and isoliquiritigenin in tonka bean). Alliaceous plants (garlic or onion) are rich in organosulfur compounds such as 1,2-dithiole-3-thione. Besides 1,2-dithiole-3-thione, cruciferous vegetables are also rich in indole-3carbinol and flavonoids. Consumption of cruciferous vegetables results in the uptake of substantial quantities of glucosinolate, which produces the bioactive compound, isothiocyanate, under enzymatic hydrolysis. Cruciferous vegetables are believed to play an important role in cancer prevention. Epidemiological and dietary studies have revealed an association of high dietary intake of vegetables with reduced risk of developing a number of disease conditions such as cardiovascular problems. There are dozens of vegetables which have been reported to have an influence on phase I enzymes

11.2 Vegetables Rich in Enzyme Modulators Table 11.1 Modulation of phase II detoxification enzymes by vegetables Vegetable Phase II enzyme Organ/tissue Part 1 Broccoli GST; UGT Testicle, liver NQO; GTS Skin

Brussels sprout

Cabbage Cauliflower Cruciferous vegetablesb Garden cress Green leaf vegetablesc Part 2 Garlic Onion

Soy; soybean Horseradish Mustard seed Juice Sprouts Tropical gingerd Tonka bean Watercress

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Model systema

NQO GST GST; QR GST GST; QR NQO GST; NQR NQO GTS; QR NQR; UGT QR; GST

Liver – Liver Liver, intestine Liver Liver Breast Liver Pancreas Liver Colon

Rat Mouse, human cells Murine cell lines Human cells Rat Rat Rat Murine cell lines Human cells Murine cell lines Rat Human cell culture Human cell culture

GST; QR GST NQO QR GST; QR UGT; GST GST; QR; UGT GST; QR; UGT GST GST NQR; UGT GST GST QR NQR QR

– Liver Liver Liver – Liver Kidney, colon, liver; intestine Liver, colon, kidney; intestine Liver Liver, lung, fore stomach Liver – Liver Liver Liver Liver

Rat tissues Rat Murine cell lines Murine cells Rat Rat Rat Rat Rat Mice Human cell lines Human cells Rat Rat cell culture Human cell culture Murine cells

GST glutathione S-transferase; QOR quinone oxidoreductase; CYP450 cytochrome P450; QR quinone reductase; NQO NADH-quinone oxidoreductase; UGT uridine-diphosphate glucuronosyl transferase a Animal tissue from rat, mouse, or hamster; cultured cells from human or rat b Including broccoli, Brussels sprouts, cabbage, cauliflower, etc. c Asian green leaf vegetables d Also called pain ginger or horse ginger

and/or phase II enzymes, including broccoli, Brussels sprouts, cabbage, cauliflower, garden cress, green leaf vegetables, garlic, horseradish, mustard, onion, soy, tropical ginger, tonka bean, and water cress. The effects of vegetables listed above on foreign compound-metabolizing enzymes have been reported and are summarized in Tables 11.1 and 11.2. These vegetables exert induction effects on phase II detoxification enzymes (see Table 11.1),

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Table 11.2 Modulation of phase I activation enzymes by vegetables Phase I enzyme Vegetable Induction Inhibition Organ/tissue Broccoli CYP450 Liver Brussels sprout –a – Liver CYP450 Liver; intestine – – Liver Mustard seed CYP450 Liver Onion CYP450b CYP450b Liver

Model system Mice Rat Rat Rat Mice Rat

a

No significant change Different CYP450 isozymes

b

including glutathione S-transferase (GST), UDP-glucuronosyl transferase (UGT), NADPH-quinone reductase (NQR), quinone reductase (QR), and NADPH-quinone oxidoreductase (NQO). Investigations of the inhibition or induction of phase I enzymes are focused on CYP450 (Table 11.2). These tables also contain information on sources of enzymes and model systems used in those studies. Table 11.2 reveals that the effects of vegetables on CYP450 are more complicated than those on phase II enzymes (Table 11.1), partly because the results may vary depending on specific isozymes of CYP450. For example, broccoli exhibits inhibiting effects on CYP450, while Brussels sprouts display either induction or has no significant effect. Mustard seed shows induction effects, but onion exhibits either induction or inhibition depending on CYP450 isozymes. Investigations are needed for vegetables other than those listed in Tables 11.1 and 11.2. Vegetables that are lack in the studies for their effects on foreign compoundmetabolizing enzymes include anise, arugula, artichoke, asparagus, bean, beet, bok choy, carrot, celery, chard, cilantro, collard greens, corn, cucumber, daikon, eggplant, endive, escarole, gailon, kale, leek, lettuce, mushroom, napa, okra, parsley, parsnips, pea, pepper, pomegranate, potato, pumpkin, purslane, radicchio, radish, rapini, scallion, spinach, squash, turnip, and zucchini.

11.3

Fruits Rich in Enzyme Modulators

In addition to providing excellent sources of fiber, vitamins, and minerals, fruits also contain nonnutritive components which offer substantial health benefits beyond basic nutrition. Fruits including grapefruit, oroblanco, musa x paradisiacal, blueberry, citrus fruit, and grape have been reported to contain chemical components that are inhibitors or inducers of activation enzymes or detoxification enzymes, for example, anthocyanin in blueberry, auraptene in citrus fruit, and leucocyanidine in grape seed. Many fruits also contain flavonoids, such as hippophae fruit, which were found to inhibit benzo(a)pyrene-induced forestomach carcinogenesis in mice. The inhibition effects may involve up-regulation of detoxification enzymes and antioxidant enzymes. Moreover, leucocyanidines extracted from grape seeds are also known to exhibit antioxidant and antimutagenic activities.

11.3

Fruits Rich in Enzyme Modulators

Table 11.3 Modulation of phase II detoxification enzymes by fruits Phase II enzymes Fruit Induction Inhibition Organ/tissue b Grapefruit QR Liver Oroblancob GTS; QR Liver Musa x paradisiacalc QR Liver Blueberry QR; GST – Citrus fruit GST; QR Liver, colon Grape GST; QR Liver

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Model systema Rat Rat Mouse cultured cells Rat Rat Rat

GST glutathione S-transferase; CYP450 cytochrome P450; QR quinone reductase a Animal tissue from rat, mouse, or hamster; cultured cells from human or rat b Juice c Banana

Table 11.4 Modulation of phase I activation enzymes by fruits Phase I enzymesa Fruit Induction Inhibition Organ/tissue Grapefruit CYP450 Intestine CYP450 Liver Oroblanco CYP450 Liver Pomegranate CYP450 Prostate Grape CYP450 Liver a

Model system − Rat Rat Cell lines Rat

CYP450 isozymes

Epidemiological studies have suggested that diets rich in fruits are associated with reduced risk for a number of common cancers such as prostate cancer. Beneficial health effects have been reported for pomegranate juice, which exhibits chemoprevention and antioxidant activity. Pomegranate juice consumption was found to decrease total hepatic CYP450 isozyme content as well as the expression of CYP1A2 and CYP3A. Tables 11.3 and 11.4 list the effects of a number of fruits on phase II enzymes and phase I enzymes, respectively. These enzymes are primarily present in the liver. Animals or cell culture model systems were employed in these studies. Table 11.3 reveals that phase II enzymes (e.g., glutathione S-transferase and quinone reductase) are induced by grapefruit, oroblanco, musa x paradisiacal, citrus fruit, and grape. In contrast, these enzymes are inhibited by blueberry. Table 11.4 shows that grapefruit, oroblanco, pomegranate, and grape are capable of inducing or inhibiting the major phase I enzyme CYP450. Future research is needed to appraise fruits other than those listed in Tables 11.3 and 11.4. Fruits that are lack in the studies for their effects on foreign compound-metabolizing enzymes include almond, apple, apricot, avocado, blackberry, cantaloupe, cherimoya, cherry, cinnamon, clementine, cranberry, date, honeydew, kiwi, lemon, lime, mandarin, mango, melon, olive, orange, papaya, passion fruit, peach, peanut, pear, pineapple, plum, prune, raspberry, strawberry, tomato, tangerine, and watermelon.

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Diets Rich in Enzyme Modulators

Herbs Rich in Enzyme Modulators

Some herbs also contain chemical compounds that are capable of modulating foreign compound-metabolizing enzymes (e.g., carnosol in rosemary and carvacrol in thyme). It has been reported that rosemary extract inhibits CYP450 and induces glutathione S-transferase. Rosemary extract is therefore considered as a potent inhibitor of DNA adduct formation induced by benzo(a)pyrene or aflatoxin B1. Thyme was also found to significantly increase the levels of glutathione S-transferase and quinone reductase activities. Moreover, dandelion tea was reported to increase the activity of UDP-glucuronosyl transferase, but not that of glutathione S-transferase. Table 11.5 lists the effects of rosemary, sage tea, thyme, and dandelion tea on phase II detoxification enzymes. The table shows that these herbs induce glutathione S-transferase, quinone reductase or UDP-glucuronosyl transferase. These studies were performed on rodent or human cultured cells of the liver or lung tissue. Table 11.6 reports that rosemary extract and dandelion tea inhibit the activity of CYP450 isozymes. However, sage tea drinking was found to significantly enhance glutathione S-transferase activity in addition to CYP2E1 activity. This finding suggests possible sage tea–drug interactions, which may affect the efficacy and safety of medical therapy with drugs that are metabolized by CYP2E1. Future research is needed to evaluate herbs other than those listed in Tables 11.5 and 11.6. Herbs that are lack in the investigations for their effects on foreign compound-metabolizing enzymes include basil, bay leave, chive, dill, marjoram, mint, and tarragon. Table 11.5 Modulation of phase II detoxification enzymes by herbs Phase II enzymes Herbs Induction Inhibition Organ/tissue Rosemary GST; QR Lung Rosemary extract GST Liver Sage teab GST Liver Thyme GST; QR Liver Dandelion teab UGT Liver

Model systema Human tissue culture Human cells Rat; mice Mouse Rat

GST glutathione S-transferase; CYP450 cytochrome P450; QR quinone reductase; UGT uridinediphosphate glucuronosyl transferase a Animal tissue from rat, mouse, or hamster; cultured cells from human or rat b Considered as herb Table 11.6 Modulation of phase I activation enzymes by herbs Phase I enzymes Herbs Induction Inhibitiona Organ/tissue Rosemary CYP450 Lung Rosemary extract CYP450 Liver Dandelion tea CYP450 – Green or black tea CYP450 − a b

CYP450 isoforms Animal tissue from rat, mouse, or hamster; cultured cells from human or rat

Model systemb Human cell culture Human liver cells – Mice or rats

11.5

11.5

Beverages Rich in Enzyme Modulators

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Beverages Rich in Enzyme Modulators

The effects of green or black tea on foreign compound-metabolizing enzymes are believed to associate with the induction of detoxification enzymes or the inhibition of activation enzymes. In animal models, green tea polyphenols were found to significantly enhance the activities of glutathione S-transferase and quinone reductase. Green and black tea catechins were shown to induce UDP-glucuronosyl transferase activity. These findings are consistent with the observation that increasing Nrf2-mediated antioxidant responsive element leads to transcriptional upregulation of detoxification enzymes in liver and lungs. Moreover, inhibition of 7,12-dimethyl-benz[a]anthracene-induced hamster buccal pouch carcinogenesis by polyphenols of green and black tea was found to correlate with a significant decrease in the activity of CYP450. Mice fed diets containing 3–6% coffee for 5 days were reported to have increased level of mRNA for NAD(P)H:quinone oxidoreductase and glutathione S-transferase in the liver and small intestine. Chemoprotective effects of coffee components and whole coffee are believed to associate with the activity of glutathione S-transferase. Coffee constituents (cafestol and kahweol) have been found to induce this enzyme activity in laboratory animals. Resveratrol (3,4¢,5-trihydroxystilbene), a polyphenolic compound found in red wine, has been demonstrated to be capable of protecting against oxidative cardiovascular conditions. Such protective effect is attributed to the induction of antioxidants and phase II detoxification enzymes. Among resveratrol-induced phase II enzymes, glutathione S-transferase and quinone reductase are most notable. Resveratrol was also found to significantly inhibit the expressions of CYP1A1 and CYP1B1. Similar to tea, red wine also contains flavonoids. The inhibition of CYP450 isozymes (e.g., CYP1A1, 1A2, 2E1, and 3A4) and the induction of phase II detoxification enzymes (e.g., UDP-glucuronyl transferase, glutathione S-transferase, and quinone reductase) by flavonoids represent one major mechanism of red wine’s anticarcinogenic effects. Results of research on the effects of beverages, including tea, coffee, and red wine, on phase II detoxification enzymes and phase I activation enzymes are summarized in Tables 11.7 and 11.8, respectively.

Table 11.7 Modulation of phase II detoxification enzymes by beverages Phase II enzymes Beverages Induction Inhibition Organ/tissue Green tea GST; QR Bowel; liver Green/black tea UGT, GST, QR – Coffee GST, NQO Intestine GST Liver, bowel Red wine GST, NQO Breast

Model systema Mice Mice or rats Mice Mice Cell lines

GST glutathione S-transferase; CYP450 cytochrome P450; QR quinone reductase; NQO NADHquinone oxidoreductase; UGT uridine-diphosphate glucuronosyl transferase a Animal tissue from rat, mouse, or hamster; cultured cells from human or rat

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Table 11.8 Modulation of phase I activation enzymes by beverages Phase I enzymesa Organ/tissue Beverages Induction Inhibition Green or black tea CYP450 − Coffee CYP450 Liver Red wine CYP450 Breast a b

Model systemb Mice or rats Rat Cell lines

CYP450 isozymes Animal tissue from rat, mouse, or hamster; cultured cells from human or rat

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Chapter 12

Induction of Enzymes for Health Benefits

Phase II detoxification enzymes play an important role in antioxidant defense by eliminating electrophilic intermediates and reactive oxygen species. This is done by up regulating the mechanisms that keep reactive intermediates at a minimum level and improving the mechanisms that scavenge oxidants. The ability of individuals to remove chemically active metabolites from the body plays a crucial role in reducing the risk of a variety of disease conditions. Oxidative stress occurs when reactive electrophilic species are neither readily detoxified by phase II enzymes nor compensated for by the body’s antioxidant defense systems. It has been reported that oxidative stress causes inflammation and damages to cells and tissues, which could attribute to many disease conditions (e.g., cancer, atherosclerosis, heart disease, chronic fatigue syndrome, and Alzheimer). Diseases associated with aging also have underlying oxidative stress and inflammatory components. Detoxification systems exhibit a great deal of individual variability and are affected by genetics, lifestyles, and environmental factors. There is convincing evidence that consumption of certain dietary ingredients may favorably modulate foreign compound metabolism. The available data suggest that people whose diets are rich in vegetables and fruits have a lower risk of developing disease conditions. Reactive electrophilic species are susceptible to metabolic conjugation and other types of detoxification by phase II enzymes. Diets rich in phase II enzyme inducers have been found to promote scavenge of oxidants, resulting in a positive influence on health and related aging processes. Unlike antioxidant molecules (e.g., glutathione and vitamins C and E) that serve directly in antioxidant defense by scavenging free radicals or other oxidant molecules, inducers of phase II detoxifying enzyme act as indirect antioxidants by boosting the body’s own antioxidant systems and exerting antioxidant activities. Phase II enzymes act catalytically to neutralize reactive electrophiles. As catalysts, these enzymes are not consumed in detoxification processes. Phase II genes are regulated by transcription factor Nrf2. The Keap1/Nrf2/ARE pathway controls a network of genes that defend against the damaging effects of electrophiles, oxidative stress, and inflammation.

C.-H. Chen, Activation and Detoxification Enzymes: Functions and Implications, DOI 10.1007/978-1-4614-1049-2_12, © Springer Science+Business Media, LLC 2012

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Enzyme Modulation as a Defense Mechanism

Phase I enzymes catalyze functionalization reactions that often convert foreign compounds into active metabolic intermediates which are potentially more toxic than parent compounds. The ultimate goal of phase II enzymes is to eliminate metabolic intermediates and transform lipophilic foreign compounds into hydrophilic ones, thus facilitating their excretion from the body via urine or bile. The susceptibility of organisms to the mutagenic effects by activated metabolic intermediates depends on the balance in the efficacy between phase I enzymes and phase II enzymes. Extensive research has been focused on the modulation of metabolizing enzymes to overcome the imbalance between these two classes of enzymes. Strategies for protecting against cells include inhibition of the expression of phase I enzymes responsible for generating electrophilic intermediates, as well as induction of the expression of phase II enzymes responsible for detoxifying electrophiles and free radicals known to intervene normal cellular processes. Some researchers have focused on the inhibition of phase I enzymes to keep the production of activated reactive intermediates low enough so that even those with less effective phase II enzymes still can deal with them. However, inhibition of CYP450 enzymes to a level of activity lower than that of metabolic detoxification may hinder the metabolic activation of certain procarcinogens or toxins, leading to ineffectiveness in removing these toxic substances from the body. Other researchers work on the approach to selectively enhance the expression of phase II enzymes (e.g., UDP-glucuronosyl transferases, glutathione S-transferases, and quinone reductase), making the body better able to deal with activated reactive intermediates it encounters and defend against their toxic effects. The efficacy of phase II enzymes is associated with reduced susceptibility of animals and their cells to toxic and carcinogenic effects. Induction of phase II enzymes also elevates glutathione levels against oxidants. A combination of phase II enzymes with intracellular glutathione plays a major role in providing the cells protection against foreign compound-mediated toxic effects. Researchers have identified a significant number of dietary and synthetic compounds that are inducers of phase II enzymes. Many of these inducers that inactivate ultimate carcinogens also can cause an increase in cellular levels of antioxidants (e.g., glutathione) that protect cells from oxidative stress. This antioxidant response may be an important component of the effects of phase II enzyme inducers. Consequently, it is widely recognized that phase II enzymes and glutathione synthesis play major protective roles against electrophiles and reactive oxygen species. Investigations in rodent models for chemoprevention of aflatoxin B1-induced hepatocarcinogenesis by oltipraz, a strong phase II enzyme inducer, demonstrated that increased expression of phase II genes is of crucial importance, although inhibition of phase 1 activation of aflatoxin B(1) can also contribute to protection. Animal studies further support the proposal that induction of phase II enzymes is a sufficient condition for obtaining chemoprevention. Induction of phase II enzymes

12.3 Role of Antioxidant Response Element

115

has also been proposed as a major potential strategy for reducing the risk of chronic degenerative diseases. A potential problem associated with phase II enzyme inducers may occur if these inducing compounds alter the action of some therapeutic drugs, leading to drug interactions. Selective induction of phase II enzymes seems to offer a safer prospect for achieving protection against toxic and carcinogenic effects. Accordingly, researchers actively search for monofunctional inducers of phase II enzymes. However, among reported monofunctional phase II enzyme inducers, many of them may also inhibit or induce CYP450 isozymes.

12.2

Monofunctional and Bifunctional Inducers

Monofunctional inducers elevate detoxification enzyme activity in various tissues without significantly raising activation enzyme activity, while bifunctional inducers elevate both detoxification and activation enzyme activities. A direct assay of quinone reductase activity in wild type and mutant murine hepatoma cells has been utilized to distinguish these two classes of enzyme inducers. The mutants are defective in either aryl hydrocarbon (Ah) receptor function or aryl hydrocarbon hydroxylase (Ahh). A monofunctional inducer elevates the activity of quinone reductase without significantly raising that of CYP450 or Ahh, and the induction is independent of Ah receptor function. In contrast, a bifunctional inducer elevates both quinone reductase and CYP450 enzyme activities, and the induction is dependent on Ah receptor function or Ahh expression. Some reported monofunctional and bifunctional enzyme inducers in dietary components are listed in Table 12.1. Monofunctional inducers include polyphenols, sulforaphane, coumarins, thiocarbamates, cinnamates, and 1,2-dithiole-3-thiones that raise phase II enzymes (e.g., glutathione S-transferases, NAD(P)H:quinone reductase, UDP-glucuronosyl-transferases) in various tissues without significantly elevating the phase I enzyme CYP450. Bifunctional inducers include indole-3carbinol, polycyclic aromatic hydrocarbons, flavonoids, and azo dyes that induce both classes of foreign compound-metabolizing enzymes. Among those reported monofunctional inducers, some of them may affect CYP450 isozymes, which require further investigations.

12.3

Role of Antioxidant Response Element

Transcriptional control of the expression of phase II enzymes is mediated, at least in part, through the antioxidant response element (ARE) found in the regulatory regions of their genes. Monofunctional inducers that raise the activity of phase II

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Table 12.1 Dietary-related monofunctional and bifunctional inducersa,b Monofunctional inducer Bifunctional inducer Sulforaphane Indole-3-carbinol 1,2-Dithiole-3-thiones Beta-naphthoflavone Phenol Polycyclic aromatic hydrocarbons Coumarin Phenethyl isothiocyanatec Thiocarbamates Resveratrol Isoliquiritigenin Curcuminoid Cinnamates Alkyl sulfides Azo dyes Canthaxanthin Ethoxyquin Butylated hydroxytoluene Prenylflavonoids Flavonoids Quercetin Daidzein Genistein Catechol a Enzyme used for induction studies is quinone reductase or quinone oxidoreducatse b Model systems for studies are murine hepatoma or other cell lines c Many isothiocyanates are bifunctional inducers

enzymes without significantly raising that of activation enzymes are believed to trigger cellular signals that activate gene transcription through ARE. Thus, monofunctional inducers appear to be mediated by ARE. CYP450 are not induced by monofunctional inducers and CYP450 genes have been reported to contain no functional ARE. Nevertheless, further investigations may be needed to examine a possibility that some phase I enzymes may also be regulated by a mechanism involving ARE. The transcription factor Nrf2, which binds to the ARE, appears to be essential for the induction of phase II detoxifying enzymes. The induction of phase II enzymes requires the functional integrity of both the repressor Keap1 and the transcription factor Nrf2. Inducers of these enzymes disrupt the cytoplasm complex between the protein Keap1 and the transcription factor Nrf2, thereby releasing Nrf2 to migrate to the nucleus where it activates the ARE of phase II genes and accelerates their transcription. The role of Nrf2 binding to the ARE on the induction of phase II enzymes has received strong support from animal studies. When Nrf2 is deleted in knock-out mice, the basal levels of phase II enzymes are very low and are not inducible. Consequently, these mice are much more susceptible than their wild-type counterparts to benzo[a]pyrene carcinogenesis, and are not protected by phase II inducers. Moreover, the exposure of 1,2-dithiole-3-thione, a phase II enzyme inducer, triggers nuclear accumulation of Nrf2 and enhances its binding to ARE, resulting in transcriptional activation of genes involved in carcinogen detoxification and attenuation of oxidative stress. In contrast, Nrf2-deficient mice fail to induce many of these genes in response to 1,2-dithiole-3-thiones.

12.4

Dietary Inducers of Phase II Enzymes

12.4

117

Dietary Inducers of Phase II Enzymes

A number of edible plants contain substantial quantities of compounds that induce foreign compound-metabolizing enzymes, and thereby accelerate the metabolic disposal of toxic substances. Such dietary inducers are widely distributed among edible plants. Increasing evidence implicates dietary factors in the progression of diseases, including certain cancers, diabetes, and obesity. The available evidence supports the hypothesis that consumption of large quantities of vegetables is associated with a reduction in the risk of developing a variety of malignancies (e.g., colorectal cancer) and cardiovascular problems (e.g., hypertension and atherosclerosis). Dietary compositions have been considered to be a major determinant of various disease conditions in humans and experimental animals. In addition to the sources of fiber, vitamins, and minerals, vegetables also contain non-nutritive components that may provide substantial health benefits. Most studies show that phytochemicals in cruciferous vegetables up-regulate detoxification enzyme systems (e.g., quinone reductase and glutathione S-transferases), which offers protection against carcinogens and other toxic electrophiles. Epidemiological data also provide evidence that the consumption of cruciferous vegetables more effectively protects against cancer than does the intake of other vegetables. Broccoli and cauliflower in cruciferous vegetables and onion and garlic in Allium plants have been implicated in chemoprotective effects. It is of interest to examine a number of compounds present in substantial quantities in edible plants that regulate mammalian enzymes of foreign compound metabolisms. Dietary phase II enzyme inducers may provide novel strategies for reducing various disease conditions. Among the most actively investigated dietary enzyme inducers are the glucosinolate hydrolysis products, isothiocyanates, sulforaphane, and indole-3-carbinol in cruciferous vegetables as well as allyl sulfides in plants of the Allium family. These potent inducers of phase II enzymes are discussed below.

12.4.1

Isothiocyanates

Research has been actively pursued on dietary inducers of phase II enzymes in vegetables, especially Brassicas. Brassicas are members of the Brassicaceae family, which are also called crucifers. Brassica vegetables (cruciferous vegetables) include broccoli, spinach, cabbage, cauliflower, Brussels sprouts, kale, collard greens, mustard, and bok choi. Isothiocyanates and their naturally occurring glucosinolates are widely consumed in diets rich in cruciferous vegetables. Glucosinolates are usually broken down through the hydrolysis reaction catalyzed by myrosinase (an enzyme derived from damaged plant cells), which releases bioactive isothiocyanates. Isothiocyanates are able to affect the activities of foreign compound-metabolizing enzymes that catalyze reactions to eliminate toxic chemicals that can damage DNA and other cell components. Many isothiocyanates were reported to be potent inducers of phase II enzymes including glutathione

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S-transferases and quionone reductase in cultured cells and animal studies. Some isothiocyanates were also found to inhibit phase I enzymes (e.g., CYP2E1) that activate carcinogens. A number of isothiocyanates were reported to block chemical carcinogenesis in a variety of animal model studies by inducing phase II enzymes that inactivate toxic foreign compounds and their metabolites. Epidemiological investigations reported an inverse association between the consumption of Brassica vegetables and the risk of cancer. Organic isothiocyanates were found to block the production of tumors induced in rodents by a diversity of carcinogens such as polycyclic aromatic hydrocarbons, azo dyes, ethionine, N-2-fluorenylacetamide, and nitrosamines. Their anticarcinogenic actions appear to be mediated by (a) the suppression of carcinogen activation by CYP450, probably through down-regulation of enzyme levels in combination with a direct inhibition of catalytic activity, thereby reducing the amount of chemically active intermediates ultimately formed and (b) the induction of phase II enzymes (e.g., glutathione transferases and quinone reductase) that detoxify chemically active intermediates generated by phase I enzymes, thereby destroying their ability to damage DNA.

12.4.2

Sulforaphane

Sulforaphane (1-isothiocyanato-4-(methyl-sulfinyl)butane), a naturally occurring isothiocyanate derived from cruciferous vegetables, is the most known potent inducers of phase II enzymes. Sulforaphane is abundant in cruciferous vegetables and the amount of sulforaphane in broccoli is variable. Studies on the source of inducer activity in broccoli lead to the isolation of sulforaphane. Broccoli, a most publicized member of the Brassica family, is a rich source of glucoraphanin, the precursor of sulforaphane, which is metabolized into sulforaphane. Broccoli sprouts are a concentrated source of glucoraphanin, providing many times more by weight than mature broccoli plants. Researchers have extensively investigated the health benefits provided by sulforaphane. In various animal studies, a regular intake of sulforaphane in broccoli sprouts has been shown to increase tissue antioxidant defense mechanisms, and lower inflammatory responses, resulting in benefits to a range of disease conditions, including hypertension, cardiovascular disease, stroke and neuronal and retinal damage. Application of sulforaphane was found to up-regulate phase II enzymes in mice and human skin, providing protection against UV-induced inflammation in mice and reduction in susceptibility to erythema in humans. The degree of protection is correlated with the potencies of inducers in raising the levels of glutathione and the activities of phase II enzymes (e.g., quinone oxidoreductase). As a highly potent inducer of phase II detoxification enzymes, sulforaphane is capable of providing protection against electrophiles including carcinogens, oxidative stress, and inflammation. The mechanism of sulforaphane action is believed to involve the induction of phase II enzymes through activation of the Keap1/Nrf2

12.4

Dietary Inducers of Phase II Enzymes

119

antioxidant response pathway. Induction of phase II enzymes accelerates metabolic elimination of toxicants and carcinogenic compounds from the body. Sulforaphane is considered as a monofunctional inducer which induces phase II enzymes selectively without significantly affecting phase I activation enzymes. Nevertheless, sulforaphane was reported to appreciably down-regulate CYP3A4 expression in human hepatocytes. CYP3A4 is responsible for the hepatic and intestinal metabolism of toxicants and drugs.

12.4.3

Indole-3-Carbinol

Broccoli and other cruciferous vegetables, including Brussels sprouts, cabbage, and cauliflower, are rich sources of glucobrassicin which is the glucosinolate precursor of indole-3-carbinol, a naturally occurring component of cruciferous vegetables. Glucobrassicin makes up about 10% of the total glucosinolates and the amount of indole-3-carbinol formed from glucobrassicin depends partly on the processing and preparation of foods. Cooking deactivates myrosinase and thus limits the release of indole-3-carbinol. Indole-3-carbinol is believed to be a critical component in the chemopreventive effects of Brassica vegetables, which is consistent with the hypothesis that higher intake of cruciferous vegetables is associated with a lower cancer risk. Indole-3-carbinol is a potent modulator of phase I enzymes and phase II enzymes in the liver and intestinal epithelial cells. Studies of indole-3-carbinol have revealed its ability to induce the activity of glutathione S-transferase. The mechanisms by which indole-3-carbinol protects against cancer include altering detoxification by inducing phase II enzymes (e.g., glutathione S-transferases and quinone reductase) and decreasing carcinogen activation by inhibiting phase I enzymes (e.g., flavincontaining monooxygenase). However, the inhibition of phase I enzymes by indole3-carbinol is less conclusive than the induction of phase II enzymes, since other studies have reported that indole-3-carbinol induces hepatic levels of CYP1A1.

12.4.4

Allyl Sulfides

Allyl sulfides including diallyl sulfide, diallyl disulfide, and diallyl trisulfide are sulfur-containing substances derived from plants of the Allium family such as garlic and onions. Epidemiological evidence indicates that a high dietary intake of plants of the Allium family decreases the risk of cancer in humans. One of the hypotheses explaining the mechanisms of the chemopreventive action of allyl sulfides is the induction of phase II enzyme systems (e.g., glutathione S-transferase and quinone reductase). Diallyl disulfide has been known to increase the activities of these phase II enzymes in a variety of rat tissues.

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Significant variations in response to allyl sulfides were reported among different organs. Forestomach, duodenum, and jejunum are most sensitive to the induction of detoxification enzymes by diallyl sulfides. In these organs, notable increases in quinone reductase activity were observed at a dose level close to which may be achieved through human consumption of garlic and onions, thus providing the evidence that the induction of phase II enzymes may contribute to the protection offered by garlic and onions against cancer of the gastrointestinal tract in humans.

Bibliography Brooks JD, Paton VG, Vidanes G (2001) Potent induction of phase 2 enzymes in human prostate cells by sulforaphane. Cancer Epidemiol Biomarkers Prev 10:949–954 Conney AH (2003) Enzyme induction and dietary chemicals as approaches to cancer chemoprevention: the Seventh DeWitt S. Goodman Lecture. Cancer Res 63:7005–7031 Cuendet M, Oteham CP, Moon RC et al (2006) Quinone reductase induction as a biomarker for cancer chemoprevention. J Nat Prod 69:460–463 Dinkova-Kostova AT, Talalay P (2008) Direct and indirect antioxidant properties of inducers of cytoprotective proteins. Mol Nutr Food Res 52(Suppl 1):S128–S138 Finley JW (2003) The antioxidant responsive element (ARE) may explain the protective effects of cruciferous vegetables on cancer. Nutr Rev 61:250–254 Hatono S, Jimenez A, Wargovich MJ (1996) Chemopreventive effect of S-allylcysteine and its relationship to the detoxification enzyme glutathione S-transferase. Carcinogenesis 17:1041–1044 Hwang ES, Jeffery EH (2005) Induction of quinone reductase by sulforaphane and sulforaphane N-acetylcysteine conjugate in murine hepatoma cells. J Med Food 8:198–203 Manson MM, Ball HW, Barrett MC et al (1997) Mechanism of action of dietary chemoprotective agents in rat liver: induction of phase I and II drug metabolizing enzymes and aflatoxin B1 metabolism. Carcinogenesis 18:1729–1738 Moon YJ, Wang X, Morris ME (2006) Dietary flavonoids: effects on xenobiotic and carcinogen metabolism. Toxicol In Vitro 20:187–210 Munday R, Munday CM (1999) Low doses of diallyl disulfide, a compound derived from garlic, increase tissue activities of quinone reductase and glutathione transferase in the gastrointestinal tract of the rat. Nutr Cancer 34:42–48 Murray M (2006) Altered CYP expression and function in response to dietary factors: potential roles in disease pathogenesis. Curr Drug Metab 7:67–81 Noyan-Ashraf MH, Wu L, Wang R, Juurlink BH (2006) Dietary approaches to positively influence fetal determinants of adult health. FASEB J 20:371–373 Pantuck EJ, Pantuck CB, Anderson KE et al (1984) Effect of brussels sprouts and cabbage on drug conjugation. Clin Pharmacol Ther 35:161–169 Ramos-Gomez M, Kwak MK, Dolan PM et al (2001) Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice. Proc Natl Acad Sci U S A 98:3410–3415 Sarkar FH, Li Y (2004) Indole-3-carbinol and prostate cancer. J Nutr 134:3493S–3498S Talalay P (1989) Mechanisms of induction of enzymes that protect against chemical carcinogenesis. Adv Enzyme Regul 28:237–250 Talalay P, Dinkova-Kostova AT, Holtzclaw WD (2003) Importance of phase 2 gene regulation in protection against electrophile and reactive oxygen toxicity and carcinogenesis. Adv Enzyme Regul 43:121–134

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Chapter 13

Sources of Foreign Compounds

Organisms are constantly exposed to foreign compounds, which are either naturally occurring or man-manufactured, and are not normally produced in living cells. Some of these compounds are acutely or potentially toxic, or become toxic and exhibit long-term effects after metabolic conversion. Since their presence in the body could cause a variety of disease conditions, it is essential that the metabolisms catalyzed by phase I enzymes and phase II enzymes maintain these toxic substances at minimum levels. General descriptions of the sources of foreign compounds are presented here, including food, pharmaceuticals, smoking, household and industrial products, and environmental chemicals from the air and water pollution. Potential harmful chemicals that are specifically present in these sources are also discussed below.

13.1 13.1.1

Foreign Compounds That Humans Are Exposed To Food

Vegetables and fruits that humans ingest are largely natural plants. Natural plants generate a variety of biologically active chemicals to protect themselves. After indigestion, metabolites are generated via complex biochemical reactions that occur continuously within the cells. Moreover, chemical derivatives are produced when meat or fish is cooked at high temperature. Residues of antibiotics and hormones used to raise chickens, cattle, pigs, and sheep remain as contaminants in meat. Molds may produce secondary metabolites with the potential to produce adverse health effects. Mycotoxins are secondary fungal metabolites, which represent a diverse group of chemicals that can occur in a variety of plants used as food such as grains and fruits. Contaminants in food also include pesticides from crop sprays, fungi from storage, phthalate esters from packaging, and styrene from containers. Environmentally, fish may be contaminated with industrial wastes (e.g., mercury, PCBs, and dioxin). C.-H. Chen, Activation and Detoxification Enzymes: Functions and Implications, DOI 10.1007/978-1-4614-1049-2_13, © Springer Science+Business Media, LLC 2012

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Sources of Foreign Compounds

Pharmaceuticals

The majority of medications that humans take are synthetic chemical compounds. Individuals may experience some side effects on taking specific over-the-counter or prescription drugs. Such side effects derived from drug metabolisms make the accumulation of drugs in the body harmful. This can occur with drugs such as pain killers (pain reliever and fever reduction) and furosemide (a diuretic). Acetaminophene is a major ingredient in pain killers. An overdose of acetaminophene can cause damage to the liver. Furosemide, if given in excessive amounts, can lead to profound diuresis with depletion of water and electrolytes. The time course and the duration of action are affected by the activities of enzymes associated with the metabolisms of these drugs.

13.1.3

Smoking

Cigarette smoke contains a great variety of chemicals many of which are toxic. The association of cigarette smoke with higher levels of chronic inflammation and other disease conditions is well documented. Typical toxic chemicals produced in cigarette smoke include nicotine and benzo(a)pyrene. Nicotine in cigarette smoke is rapidly absorbed into the bloodstream and reaches the brain within 10 s. Nicotine affects the brain chemistry, resulting in a number of chemical reactions that involve hormones and neurotransmitters. Benzo[a]pyrene is found in cigarette smoke resulting from the combustion of organic materials. Benzo[a]pyrene has the capacity of binding to cell components in major organs.

13.1.4

Household Products

Since many organic solvents are volatile, humans are exposed to them mainly through inhalation. Organic solvents are present in common household and industrial products such as gasoline, paint remover, varnishes, and wood sealants. These volatile solvents are also present in dry cleaning shops, electronics industries, and scientific laboratories. A typical example of organic solvents is benzene which is known to cause human health problems. Benzene has been reported to depress the immune system and cause cancer in both animals and humans. Concern about the use of phthalates as plasticizers for polyvinyl chlorides (PVC) has persisted for years. Many countries have banned the use of the lower molecular weight phthalates in cosmetic applications and restricted in its use in toys and child-care articles.

13.2

Heterocyclic Amines

13.1.5

125

Environmental Chemicals

Industrial combustions in refinery, incineration, and coal plants produce industrial pollution including polycyclic aromatic hydrocarbons, dioxins, and PCBs. Environmental toxicants to which humans are exposed are of major concern. Transportation vehicles generate exhaust gases that also consist of toxic substances. Environmental toxicants have been reported to play an important role in the pathogenesis of lung disease and other disease conditions such as cancer.

13.2

Heterocyclic Amines

Potentially harmful compounds that are either intrinsically toxic or become toxic after metabolic conversion are present in a variety of sources. Specific examples of harmful chemical compounds are discussed below, including heterocyclic amines and polycyclic aromatic hydrocarbons from cooking and cigarette smoking; nitrosamines, azo dyes, unsaturated aldehydes, and mycotoxins from food and cooking; acetaminophene, xanthine, and terfenadine from overdose of drugs; and exhaust particles, arsenic, and polychlorinated biphenyls from environmental contaminants. Heterocyclic amines are organic compounds that contain at least one atom of carbon and at least one atom of nitrogen within aromatic or nonaromatic rings (e.g., pyridine or pyrimidine). Heterocyclic amines are formed when amino acids react with creatine in muscle when meats (beef, pork, or fish) are cooked at high temperature by frying, broiling, or barbecuing. More than a dozen heterocyclic amines have been identified. 2-Amino-1-methyl-6-phenylimidazo [4,5-beta] pyridine is the most abundant heterocyclic amine found in human diets. Many heterocyclic amines have been reported to be carcinogenic. Chronic administration of these chemicals was found to induce tumors in rats at several sites including the colon. Heterocyclic amines are enzymatically activated by phase I enzymes such as CYP1A2. Green tea and black tea that inhibit phase I enzymes were reported to impede heterocyclic amine-induced mutagenesis. Typical examples of carcinogenic heterocyclic amines are listed in Fig. 13.1. Heterocyclic amines Pyridine N

Pyrimidine

2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

N CH3 N

N

NH2 N

Fig. 13.1 Heterocyclic amines

N

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13

13.3

Sources of Foreign Compounds

Nitrosamines

Nitrosamines containing an organic functional group N–N=O are formed by the addition of N=O group to secondary or tertiary amines. They are N-nitroso derivatives of amines formed by reaction between nitrite and amines. Nitrosamines are found in trace amounts in foods such as mushrooms, fermented and smoked fish, and pickled foods. Cured meats such as bacon can also contain nitrosamines because sodium nitrite is added as a preservative. High cooking temperatures used to fry bacon also contribute to nitrosamine formation. Nitrosamines have also been found in human gastric juice, possibly formed by reaction between amines and nitrites from the diet. Many nitrosamines have been reported to be carcinogenic in a wide variety of experimental animals. For example, N-nitrosodibutylamine and its hydroxylated metabolite (N-nitrosobutyl(4-hydroxybutyl)amine) are urinary bladder-specific carcinogens. Humans seem also to be susceptible to carcinogenic properties of nitrosamines, and high temperature cooking may be responsible for high cases of colon cancer. Humans are exposed to cigarette smoke and auto exhausts more commonly than other environmental sources. Cigarette smoking contains a large number of potentially harmful chemical compounds. Among them are nitrosamines. Cigarette generated nitrosamines are composed of various amines such as nicotine, nornicotine, anabasine, and anatabine. There are also tobacco-specific nitrosamines that are found only in tobacco products and are generated during fermentation, curing, and burning of the tobacco leaf. Among the best known tobacco-specific nitrosamines are 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N¢-nitrosonornicotine. Typical examples of carcinogenic nitrosamines are shown in Fig. 13.2. Tobaccospecific nitrosamines are strongly carcinogenic in laboratory animals. Considerable evidence supports the role of tobacco-specific nitrosamines as an important contributing factor for cancers of the lung, pancreas, and esophagus in humans. Chemical carcinogens generally require metabolic activation in order to bind to DNA and cause mutation and Nitrosamines 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone O

O

N-nitrosobutyl-(4-hydroxybutyl)amine

N

OH N

CH3

N N

N O

N-nitrosonornicotine O

N N

Fig. 13.2 Nitrosamines

H

N

13.4

Polycyclic Aromatic Hydrocarbons

127

develop tumors. Human susceptibility to nitrosamine toxicity varies from individual to individual, depending on the expression of metabolic enzymes. Nitrosonornicotine was found to significantly increase hepatic and pulmonary phase I enzymes and significantly decrease liver and lung glutathione levels and glutathione S-transferase activity in rats.

13.4

Polycyclic Aromatic Hydrocarbons

Polycyclic aromatic hydrocarbons are products of incomplete combustion of organic substances at high temperatures. Humans could be exposed to polycyclic aromatic hydrocarbons by eating grilled, charred meats and contaminated foods. Major polycyclic aromatic hydrocarbons detected in charcoal meats include benzo[a]pyrene and dibenz[a,h]anthracene. A considerable number of studies over the years have documented the link between benzo[a]pyrene and cancers. Regular consumption of overcooked charcoal barbecued beef has been reported to associate with increased levels of colon cancer. Following absorption, dibenz[a,h]anthracene is distributed to various tissues with the highest accumulation in the liver and kidneys. Polycyclic aromatic hydrocarbons are metabolically activated by phase I enzymes (e.g., CYP1A1) into electrophilic species that have the capacity of interacting with DNA. There is evidence that links polycyclic aromatic hydrocarbons to the induction of phase I enzymes. In addition to their present in grilled or charred meat, polycyclic aromatic hydrocarbons also exist in cigarette smoke and auto exhausts due to the combustion of organic materials. Polycyclic aromatic hydrocarbons exhibit their genotoxic properties after metabolic conversion to chemically active intermediates. Two major carcinogens in cigarette smoke are benzo[a]pyrene and dibenz[a,h]anthracene. Benzo[a] pyrene is a major lung carcinogen. This toxicity is produced by bioactivation of benzo[a]pyrene to a toxic intermediate, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide. Dibenz[a,h]anthracene is metabolized mainly by CYP450 to form carcinogenic metabolites. Animal studies reported that dibenz[a,h]anthracene depresses immune responses in mice. Dibenz[a,h]anthracene was also shown to induce hepatic aryl hydrocarbon hydroxylase activity in mice. Typical examples of carcinogenic polycyclic aromatic hydrocarbons are listed in Fig. 13.3. Polycyclic aromatic hydrocarbons Benzo[a]pyrene

Fig. 13.3 Polycyclic aromatic hydrocarbons

Dibenz[a,h]anthracene

128

13.5

13

Sources of Foreign Compounds

Azo Dyes

Synthetic azo dyes are much more stable than most natural food dyes, making them applicable for use in foods. Azo dyes are stable upon heating and are also stable in the pH ranges of food products and their colors do not fade when exposed to light or oxygen. However, the azo linkage is the most labile portion of the azo dye molecule. Azo dyes undergo enzymatic breakdown catalyzed by azo-reductase presence in various microorganisms. Azo-reductase activity is high in the liver and the kidney. After cleavage of the azo-linkage, amine metabolites are absorbed in the intestine and excreted in the urine. Many azo pigments are nontoxic. Although the acute toxicity is rather low in consuming azo dye-colored foods, some azo dyes have been banned for food use because of side effects due to degradation products. Azo degradation products have been reported to be mutagenic or carcinogenic. Enzymatic sulfonation appears to decrease the toxicity of azo degradation products by facilitating their urinary excretion. A typical example of mutagenic pigment is orthonitroaniline orange. Its chemical structure is shown in Fig. 13.4.

Azo, aldehyde and microtoxin Dinitroaniline orange pigment

4-hydroxynonenal

O2N OH N

O

N NO2 HO

Patulin O O O

OH

Fig. 13.4 Azo, aldehyde, and mycotoxin

13.7

13.6

Mycotoxins

129

a,b-Unsaturated Aldehydes

4-Hydroxynonenal is an a,b-unsaturated hydroxyalkenal generated in the oxidation of lipids or corresponding fatty acids, and is present in higher quantities as the lipid peroxidation chain reaction increases. 4-Hydroxylnonenal can also be produced in foods during processing or storage. Cooking oils that are used repeatedly in caterings and households can generate significant amounts of oxygenated a,b-unsaturated aldehydes. Through diets, the body absorbs 4-hydroxynonenal and oxygenated a,b-unsaturated aldehydes. These compounds have received a great deal of attention because they are considered as potentially contributing agents to a number of disease conditions (e.g., inflammation, respiratory distress syndrome, diabetes, and cancer). Pretreatment with phase II enzyme inducer, resveratrol or 1,2-dithiole-3thione, was found to provide marked protection against 4-hydroxynonenal-mediated cytotoxicity in cardiomyocytes. The chemical structure of 4-hydroxynonenal is also shown in Fig. 13.4.

13.7

Mycotoxins

Mycotoxin is a toxin produced by fungi of mushrooms, molds, or yeasts. When fungi propagate into colonies, the levels of mycotoxin become high. Mycotoxin appears in food chains as a result of fungal infection of crops, and can also remain in food chains of meat and dairy products. Wild mushrooms contain an assortment of mycotoxins that can cause noteworthy health problems. Patulin, a mycotoxin produced by a variety of molds, is commonly found in rotting apples. The chemical structure of patulin is also presented in Fig. 13.4. Some studies reported that patulin is genotoxic and may be a carcinogen. Studies of rat liver tissues reported that patulin decreases glutathione S-transferase activity and markedly increases lipid peroxidation. These effects may be a result of patulin-mediated reduction in the level of glutathione. The decrease in glutathione level and glutathione S-transferase activity may be related to the presumed mutagenic or carcinogenic potential of patulin. Poultry such as turkey is quite susceptible to toxic effects of aflatoxin B1, another mycotoxin, due to a combined result of efficient activation by CYP1A and deficient detoxification by glutathione S-transferases. Aflatoxin B1 can cause health problems by acting as an allergen or irritant or by weakening the immune system. Moreover, enzyme inducers diallyl sulfide and diallyl disulfide were found to inhibit aflatoxin B1-initiated carcinogenesis in rat liver. Diallyl sulfide prevents aflatoxin B1 mutagenicity by modulating CYP450 and glutathione S-transferase, whereas diallyl disulfide acts mainly by inducing glutathione S-transferase.

130

13.8

13

Sources of Foreign Compounds

Overdose of Drugs

Active ingredients of drugs perform the needed chemical or biochemical actions, leading to therapeutic responses in the body. When the active constituent of a drug is metabolized to a chemically active metabolite, drug-mediated toxic effects occur if detoxification enzymes are inefficient in eliminating such chemically active metabolites from the body. Accumulation or overdoses of drugs are a health concern. Examples of potential overdose of drugs including acetaminophen, xanthine, and terfenadine are described below. The chemical structures of these drug ingredients are shown in Fig. 13.5.

Drugs Acetaminophen

Xanthine O H

HO N

O

N N

N

CH3 N

O

N

H H Terfenadine C(CH3)3

N OH OH

Fig. 13.5 Acetaminophen, xanthine, and terfenadine

13.8

Overdose of Drugs

13.8.1

131

Acetaminophen

Acetaminophen is one of the most common pharmaceutical agents that are involved in overdose toxicity. Acetaminophen is metabolically activated via CYP450 system. Glucuronide and sulfonate conjugates are involved in hepatic metabolism of acetaminophen elimination. In the liver, acetaminophen is metabolized by conjugation reaction to form water-soluble conjugate ready for elimination in the urine. However, in the case of a deficiency in conjugation enzyme UDP-glucuronosyl transferase, the toxic metabolite is capable of interacting with cellular proteins and hepatocyte membranes, causing hepatocellular damages. The hepatotoxicity associated with misuse or overdose of acetaminophen is well documented. Using 2-aminophenol as the substrate, UDP glucuronosyl transferase activity was reported to increase significantly following administration of green tea.

13.8.2

Xanthine

Xanthine (3,7-dihydro-purine-2,6-dione) and its derivatives are a group of alkaloids that are commonly used as mild stimulants and bronchodilators in treating the symptoms of asthma. Xanthine is a product of the pathway of purine degradation. Metabolic conversion of xanthine to uric acid is carried out by an enzymatic reaction catalyzed by xanthine oxidase, leading to subsequent excretion of xanthine from the body. In the case of a lack of sufficient xanthine oxidase, xanthine cannot be readily converted to uric acid and an accumulation of xanthine in the body occurs, which could result in xanthine-mediated oxidative stress. In cardiomyocyte studies, pretreatment with phase II enzyme inducers, such as resveratrol or 1,2 dithiole-3thione, was found to increase resistance to xanthine-mediated effects.

13.8.3

Terfenadine

Terfenadine, an antihistamine, is metabolized by CYP3A4 isozyme to its metabolite fexofenadine. Drugs (e.g., erythromycin) or food (e.g., grapefruit), can interfere with the metabolism of terfenadine, making it difficult to metabolize and remove terfenadine from the body. After continuous use of terfenadine, potential toxicity occurs as a result of interaction with other medications. An elevated level of terfenadine can lead to an adverse cardiac effect on the heart’s rhythm, although its metabolite has no such effect. Consequently, this drug was removed from the market in 1997.

132

13.9 13.9.1

13

Sources of Foreign Compounds

Household Products Benzene

Household products (e.g., lubricants, detergents, paints, and pesticides) contain a variety of organic solvents. Organic solvents are used to make resins, nylon, synthetic fibers, and plastics in industries. A typical example of organic solvent present in household products is benzene. Benzene is also present in unleaded gasoline and cigarette smoke. Long-term exposure of benzene can have harmful effects on the bone marrow and can cause a decrease in red blood cells, leading to anemia. Benzene was also found to affect the immune system, causing an increase in the chance for infection. The toxic effects of benzene in humans are attributed to hydroxylated metabolites (e.g., hydroquinone and phenol). Phenol is the primary metabolite of benzene. Phase II enzyme-catalyzed conjugation reactions are involved in detoxifying phenol. At low exposure concentrations of benzene, phenylsulfonate is the major conjugate of phenol in the blood. However, at high exposure concentrations, phenylglucuronide is the predominant conjugate. Reductions in spleen weight and white blood cell numbers were reported to correlate with the concentration of phenylsulfonate in the blood.

13.9.2

Di(2-ethylhexyl)phthalate

Di(2-ethylhexyl)phthalate (DEHP), an organic compound consisting of phthalate diesters with the branched-chain 2-ethylhexanol, is a plasticizer used to produce flexible PVC. It adds flexibility and softness to vinyl. DEHP represents about 40% of plasticizer production. Plasticizers are used primarily in durable applications (e.g., wire and cable insulation and coatings, roofing materials, and flooring). DEHP is colorless viscous liquid soluble in oil, but insoluble in water. Its chemical structure is shown in Fig. 13.6. DEHP could have reproductive toxicity in humans, which has lead to reduced use in the USA and Europe. It has been reported that most of adverse biological effects of DEHP are effects of metabolites. Metabolism of most diesters of phthalic acid in humans occurs by an initial phase I reaction in which phthalate monoesters are formed, followed by a phase II reaction in which phthalate monoesters react with glucuronic acid to form glucuronide conjugates. The phase II conjugation increases water solubility, facilitating urinary excretion of phthalate. Conjugation reaction reduces the potential biological activity of DEHP since the biologically active species is the monoester metabolite.

13.10

Environmental Chemicals

133

Fig. 13.6 Harmful compounds: household product and pollutant

13.10 13.10.1

Environmental Chemicals Diesel Exhausts

Diesel exhausts produced in combustion of diesel fuel are a mixture of gases and fine particles that contain harmful air contaminants such as polycyclic aromatic hydrocarbons. Diesel exhausts also include many potential cancer-causing substances such as benzene, arsenic, and formaldehyde as well as other toxic pollutants such as nitrogen oxides. Diesel exhaust particles can initiate and exacerbate airway allergic responses through enhanced IgE production. Exposure to diesel exhausts can cause inflammation in the lungs, aggravating chronic respiratory symptoms and increase the frequency or intensity of asthma attacks. Diesel harmful effects are especially pronounced in individuals whose phase II enzyme expression is impaired. Chemically active intermediates are believed to play a key role in cellular damage after exposure to diesel exhaust particles. Induction of phase II enzymes (e.g., quinone oxidoreductase) by sulforaphane was found to block the ability of diesel exhaust particles to enhance IgE production. Glutathione S-transferase was reported to involve in the detoxification of diesel exhaust particlemediated allergic inflammation.

13.10.2

Arsenic in Drinking and Underground Water

Besides organic solvents, humans are also exposed to nonvolatile chemicals such as arsenic compounds and polychlorinated biphenyls. Arsenic, a risk factor for cancer, is present in polluted drinking water and rice grown in polluted water. Ground water

134

13

Sources of Foreign Compounds

pollution by arsenic is a serious worldwide problem. Populations in South and East Asia and many other regions of the world are chronically exposed to arseniccontaminated drinking water. Paddy rice takes up arsenite readily from soaking soil. Arsenic is metabolized to monomethylarsonic acid, which is converted to dimethylarsinic acid by methyltransferase enzymes. Arsenic metabolite requires S-adenosyl-methionine as the methyl donating cofactor before excretion through urine. Drinking water from arsenic-tainted wells causes ailments marked by rough skin and often leads to serious diseases such as skin or bladder cancer. Arsenic was also reported to interfere with methyltransferases and inactivate tumor suppressor genes. Other studies reported that arsenic-induced malignant transformations are linked to DNA hypomethylation. A remarkable diversity in arsenic methyltransferase activity may account for the wide variability in the susceptibility of individuals to arsenic toxicity.

13.10.3

Polychlorinated Biphenyls

Polychlorinated biphenyls (PCBs), a major class of persistent organic pollutants, are metabolized to hydroxylated compounds. The chemical structure of PCB is presented in Fig. 13.6. Many of these metabolites are further converted to either the sulfonate or the glucuronic acid conjugate by phase II enzymes (sulfotransferase or uridine diphosphate (UDP) glucuronosyl transferase), thus facilitating their excretion from the body. But, some of hydroxylated PCBs persist in the body, which may reflect their inability to be conjugated. Glucuronidation studies of PCB metabolites have been carried out to include those excreted with relative ease and those retained in blood. PCBs were also reported to induce the activity of hepatic enzymes, mainly monooxygenases that catalyze the metabolism of PCBs, leading to formation of metabolites and to potential adverse health effects. The efficiency of glucuronidation was found to vary, depending on the structure of the PCB metabolites. Substitution of chlorine atoms on the nonhydroxylated ring significantly lowers the maximum velocity of UDP glucuronosyl transferase, while substitution in the meta and para positions is least favorable for the enzyme activity. In contrast, steric hindrance around the hydroxyl group by chlorines on adjacent carbon atoms does not play a major role in the efficiency of glucuronidation.

Bibliography Albro PW (1986) Absorption, metabolism, and excretion of di(2-ethylhexyl) phthalate by rats and mice. Environ Health Perspect 65:293–298 Balliet RM, Chen G, Dellinger RW et al (2010) UDP-glucuronosyltransferase 1A10:activity against the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol, and a potential role for a novel UGT1A10 promoter deletion polymorphism in cancer susceptibility. Drug Metab Dispos 38:484–490

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Bessems JG, Vermeulen NP (2001) Paracetamol (acetaminophen)-induced toxicity: molecular and biochemical mechanisms, analogues and protective approaches. Crit Rev Toxicol 31:55–138 Bu-Abbas A, Clifford MN, Ioannides C et al (1995) Stimulation of rat hepatic UDP-glucuronosyl transferase activity following treatment with green tea. Food Chem Toxicol 33:27–30 Cao Z, Li Y (2004) Potent induction of cellular antioxidants and phase 2 enzymes by resveratrol in cardiomyocytes: protection against oxidative and electrophilic injury. Eur J Pharmacol 489:39–48 Conaway CC, Wang CX, Pittman B et al (2005) Phenethyl isothiocyanate and sulforaphane and their N-acetylcysteine conjugates inhibit malignant progression of lung adenomas induced by tobacco carcinogens in A/J mice. Cancer Res 65:8548–8557 Dashwood RH, Xu M, Hernaez JF et al (1999) Cancer chemopreventive mechanisms of tea against heterocyclic amine mutagens from cooked meat. Proc Soc Exp Biol Med 220:239–243 Elbekai RH, El-Kadi AO (2004) Modulation of aryl hydrocarbon receptor-regulated gene expression by arsenite, cadmium, and chromium. Toxicology 202:249–269 Furst A (2002) Can nutrition affect chemical toxicity? Int J Toxicol 21:419–424 Gooderham NJ, Murray S, Lynch AM et al (2001) Food-derived heterocyclic aminemutagens: variable metabolism and significance to humans. Drug Metab Dispos 29:529–534 Kensler TW, Groopman JD, Eaton DL et al (1992) Potent inhibition of aflatoxin-inducedhepatic tumorigenesis by the monofunctional enzyme inducer 1,2-dithiole-3-thione. Carcinogenesis 13:95–100 Li X, Parkin S, Duffel MW et al (2010) An efficient approach to sulfate metabolites of polychlorinated biphenyls. Environ Int 36:843–848 Macé K, Aguilar F, Wang JS et al (1997) Aflatoxin B1-induced DNA adduct formation and p53 mutations in CYP450-expressing human liver cell lines. Carcinogenesis 18:1291–1297 Maliakal PP, Coville PF, Wanwimolruk S (2002) Decreased hepatic drug metabolising enzyme activity in rats with nitrosamine-induced tumours. Drug Metabol Drug Interact 19:13–27 Manson MM, Ball HW, Barrett MC et al (1997) Mechanism of action of dietary chemoprotective agents in rat liver: induction of phase I and II drug metabolizing enzymes and aflatoxin B1 metabolism. Carcinogenesis 18:1729–1738 Medinsky MA, Kenyon EM, Schlosser PM (1995) Benzene: a case study in parent chemical and metabolite interactions. Toxicology 105:225–233 Morse PM (2011) Phthalates face murky future. Chem Eng News 89:28–31 Pegram RA, Chou MW (1989) Effect of nitro-substitution of environmental polycyclic aromatic hydrocarbons on activities of hepatic phase II enzymes in rats. Drug Chem Toxicol 12:313–326 Pfeiffer E, Diwald TT, Metzler M (2005) Patulin reduces glutathione level and enzyme activities in rat liver slices. Mol Nutr Food Res 49:329–336 Pool-Zobel B, Veeriah S, Böhmer FD (2005) Modulation of xenobiotic metabolising enzymes by anticarcinogens – focus on glutathione S-transferases and their role as targets of dietary chemoprevention in colorectal carcinogenesis. Mutat Res 591:74–92 Shimada T (2006) Xenobiotic-metabolizing enzymes involved in activation and detoxification of carcinogenic polycyclic aromatic hydrocarbons. Drug Metab Pharmacokinet 21:257–276 Silva MJ, Barr DB, Reidy JA et al (2003) Glucuronidation patterns of common urinary and serum monoester phthalate metabolites. Arch Toxicol 77:561–567 Talalay P (1989) Mechanisms of induction of enzymes that protect against chemical carcinogenesis. Adv Enzyme Regul 28:237–250 Tampal N, Lehmler HJ, Espandiari P et al (2002) Glucuronidation of hydroxylated polychlorinated biphenyls (PCBs). Chem Res Toxicol 15:1259–1266 Thompson D, Oster G (1996) Terfenadine is indicated for the relief of symptoms associated with seasonal allergic rhinitis such as sneezing, rhinorrhea, pruritus, and lacrimation. J Am Med Assn 275:1339–1341 Vernhet L, Séité MP, Allain N et al (2001) Arsenic induces expression of the multidrug resistanceassociated protein 2 (MRP2) gene in primary rat and human hepatocytes. J Pharmacol Exp Ther 298:234–239

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Wang LQ, James MO (2006) Inhibition of sulfotransferases by xenobiotics. Curr Drug Metab 7:83–104 Wells MS, Nerland DE (1991) Hematotoxicity and concentration-dependent conjugation of phenol in mice following inhalation exposure to benzene. Toxicol Lett 56:159–166 Zhang H, Forman HJ (2009) Signaling pathways involved in phase II gene induction by alpha, beta-unsaturated aldehydes. Unsaturated aldehydes. Toxicol Ind Health 25:269–278

Chapter 14

Catalytic Reactions of Phase II Enzymes

Phase I activation enzymes catalyze oxidation, reduction, and hydrolysis reactions, which introduce functional groups to lipophilic foreign compounds. Chapter 3 presents the functional groups introduced by N-oxidation, S-oxidation, aromatic and aliphatic hydroxylation, O- and N-dealkylation, hydrolysis, and epoxidation reactions. Chapter 4 describes the functional properties of oxidases, reductases, and hydrolases, as well as the atoms and groups involved in oxidation, reduction, and hydrolysis reactions. This chapter discusses the chemical reactions catalyzed by major phase I enzymes including cytochrome P450, flavin monooxygenase, amine oxidase, nitroreductase, azoreductase, molybdenum hydroxylase, alcohol dehydrogenase, peroxidase, and carboxylesterase.

14.1

Cytochrome P450-Catalyzed Reactions

Cytochrome P450 family is a monooxygenase that incorporates one of two oxygen atoms into the substrate, while another oxygen atom participates in the formation of water. CYP450s play a prominent role in the biotransformation of a great variety of foreign compounds. CYP450s contribute to the clearance of drugs more than any other group of phase I enzymes. Many drugs are turned into more hydrophilic substances by hydroxylation during metabolic processes that facilitate drug excretion from the body. Besides the carbon hydroxylation in the metabolism of sterols and alkanes, CYP450s also involve a variety of other reactions such as the oxidation of olefins, acetylenes, and polyunsaturated fatty acids. Major reactions catalyzed by CYP450s consist of hydroxylation, epoxidation, dehydrogenation, heteroatom oxygenation, heteroatom dealkylation, and oxidation of aromatic rings.

C.-H. Chen, Activation and Detoxification Enzymes: Functions and Implications, DOI 10.1007/978-1-4614-1049-2_14, © Springer Science+Business Media, LLC 2012

137

138

14

14.1.1

Catalytic Reactions of Phase II Enzymes

Hydroxylation of Aliphatic or Aromatic Compound

Hydroxylation of aliphatic and aromatic compounds involves the oxidation of hydrogen in an alkyl or aromatic group, resulting in the formation of alcohol. Hydroxylation is a common process in the metabolism of sterols, alkanes, etc. Typical examples of CYP450-catalyzed hydroxylation reactions are represented as follows: R - CH3 + O2 + NADPH + H + ® R - CH 2 OH + H 2 O + NADP + F - H + O2 + NADPH + H + ® F - OH + H 2 O + NADP + where R and F denote aliphatic and aromatic derivatives, respectively. The reactions require NADPH and oxygen. Hydroxylation reactions are catalyzed by not only CYP450s but also other oxygenases.

14.1.2

Epoxidation of Ether

An epoxide is a cyclic ether with three-member ring that arises from oxidative metabolism of foreign compounds through enzymatic oxidation processes. Formation of epoxides can occur as CYP450 metabolites of unsaturated carbon– carbon bonds. The resultant epoxides are typically unstable and chemically reactive. A typical example of epoxidation reaction is shown below:

where R and R¢ represent aliphatic derivatives.

14.1.3

Dehydrogenation of Alcohol or Aldehyde

CYP450-catalyzed dehydrogenation reaction results in oxidizing either alcohol to form aldehyde or aldehyde to form carboxylic acid. Typical examples of dehydrogenation of alcohol and aldehyde are shown below: R - CH 2 OH + O2 + NADPH + H + ® RCH = O + 2H 2 O + NADP + R - CHO + O 2 + NADPH + H + ® RCOOH + H 2 O + NADP + where R denotes an aliphatic derivative.

14.1

Cytochrome P450-Catalyzed Reactions

14.1.4

139

Oxidation of N- or S-Compound

Oxygenation reactions involving N- or S-compounds are commonly seen with amines and sulfides. Amines are oxidized to hydroxyl amines, and thioethers are oxidized to sulfoxides. Typical examples of N- or S-oxidation reactions can be written as follows:

where F represents an aromatic derivative.

14.1.5

Dealkylation of Ether, Amide, or Carboxylic Acid

The cleavage of ether, amide, or carboxylic acid is a common CYP450-catalyzed reaction. Typical examples of dealkylation of ether, amide, or carboxylic acid are shown below: R - O - R' + O 2 + NADPH + H + ® ROOH + HOR' + NADP +

where R and R¢ denote aliphatic derivatives.

14.1.6

Oxidation of Carbon on Aromatic Ring

A typical example of CYP450-catalyzed oxidation of a carbon on aromatic ring is shown below, where X represents H, N, or S that attaches to a carbon on the aromatic ring and F denotes an aromatic derivative. F - X + O2 + NADPH + H + ® F - O - X + NADP + + H 2 O

140

14.2

14

Catalytic Reactions of Phase II Enzymes

Flavin Monooxygenase-Catalyzed Reactions

As oxidative enzymes, flavin-containing monooxygenases exhibit functions that overlap with CYP450s. Monooxygenases involve in the oxidation of numerous organic compounds that contain nitrogen, sulfur, or phosphorus to form oxides of nitrogen, sulfur, or phosphorus. Unlike CYP450s, monooxygenase-catalyzed reactions utilize flavin adenosine dinucleotide (FAD) as the coenzyme. A typical flavincontaining monooxygenase-catalyzed fatty acid oxidation reaction can be represented as follows: R - CH 2 - CH 2 - CO - S - CoA + FAD ® R - CH = CH - CO - S - CoA + FADH 2

where CH2–CH2 bond is oxidized to form CH=CH.

14.3

Amine Oxidase-Catalyzed Reactions

Amine oxidases catalyze the oxidation of amines in the metabolism of foreign compounds. The basic reaction is the oxidative cleavage of the a-H in aliphatic or aromatic amines. A typical example of amine oxidase-catalyzed reaction can be found below: RCH 2 - NR ¢R ¢¢ + H 2 O + O2 ® RCHO + H - NR ¢R ¢¢ + H 2 O 2 The reaction products, aldehyde, ammonia, and hydrogen peroxide, are potentially toxic. The resultant hydrogen peroxide is the source of hydroxyl radical (⋅OH). The produced aldehyde may be further metabolized by aldehyde oxidase or aldehyde reductase to form carboxylic acid or alcohol, according to the following reactions: RCHO + NAD + H 2 O ® RCOOH + NADH 2 RCHO + NADH 2 ® RCH 2 OH + NAD

14.4

Nitroreductase-Catalyzed Reactions

Nitroreductases catalyze the reduction of nitro compounds to form primary amine metabolites according to the following sequential reactions: RNO2 + NADPH + H + ® RNO + NADP + + H 2 O

14.6

Molybdenum Hydroxylase-Catalyzed Reactions

141

RNO + NADPH + H + ® RNHOH + NADP + RNHOH + NADPH + H + ® RNH 2 + NADP + + H 2 O where the nitro group (–NO2) is initially reduced to nitroso (–NO), then to hydroxylamine (–NHOH), and finally to primary amine (–NH2).

14.5

Azoreductase-Catalyzed Reactions

Azoreductases catalyze the reduction of azo compounds. Azo is initially reduced to hydrazo and finally to primary amine, according to the following reactions: F - N = N - F' + NADPH + H + ® F - NH - NH - F' + NADP + F - NH - NH - F' + NADPH + H + ® F - NH 2 + F' - NH 2 + NADP + where F and F¢denote aromatic derivatives, which may be replaced with R (an aliphatic derivative).

14.6

Molybdenum Hydroxylase-Catalyzed Reactions

Molybdenum hydroxylases exhibit oxidase activity toward a variety of heterocyclic compounds and aldehydes. Among the members of the molybdenum hydroxylase family are aldehyde oxidase and xanthine oxidase, which are important in the metabolism of drugs and other xenobiotics. Individual variation in aldehyde oxidase activity exists in humans. Xanthine oxidases play an important role in the catabolism of purines. The oxygen atom inserted into the substrate (foreign compound) is from water rather than molecular oxygen. Xanthine oxidases catalyze the oxidation of hypoxanthine to xanthine. The enzymes further catalyze the oxidation of xanthine to uric acid. The overall reaction is the oxidation of hypoxanthine to form uric acid as shown below.

142

14.7

14

Catalytic Reactions of Phase II Enzymes

Alcohol Dehydrogenase-Catalyzed Reactions

Alchol dehydrogenase catalyzes the conversion of primary or secondary alcohol to aldehyde or ketone. Alcohol dehydrogenase-catalyzed reaction requires NAD+ as a coenzyme. A typical example is illustrated below. RCH 2 OH + NAD + ® RCHO + NADH + H + The produced aldehyde is usually toxic and is further oxidized to acid (an important detoxification reaction) before excretion.

14.8

Peroxidase-Catalyzed Reactions

Peroxidases are a large family of enzymes (e.g., horseradish peroxidase and cytochrome c peroxidase) that catalyze the conversion of peroxides to form alochols. A typical peroxidase-catalyzed reaction is shown below: R - O - OR' + H + + NADH ® ROH + R'OH + NAD+ where RO–OR¢ represents hydrogen peroxide or organic hydroperoxides (e.g., lipid peroxides).

14.9

Carboxylesterase-Catalyzed Reactions

Carboxylesterase catalyzes the hydrolysis reaction, specifically involving the carboxylic ester bond. The reaction proceeds as follows: R - COOR' + H 2 O ® R'OH + RCOO - + H + where R–COOR¢ represents a carboxylic ester, and R and R¢ denote aliphatic derivatives. The reaction products are alcohol and carboxylate.

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Seitz HK, Oneta CM (1998) Gastrointestinal alcohol dehydrogenase. Nutr Rev 56:52–60 Senter PD, Marquardt H, Thomas BA et al (1996) The role of rat serum carboxylesterase in the activation of paclitaxel and camptothecin prodrugs. Cancer Res 56:1471–144 Shimada T, Martin MV, Pruess-Schwartz D et al (1989) Roles of individual human cytochrome P-450 enzymes in the bioactivation of benzo(a)pyrene, 7,8-dihydroxy-7,8- dihydrobenzo(a) pyrene, and other dihydrodiol derivatives of polycyclic aromatic hydrocarbons. Cancer Res 49:6304–6312 Strolin Benedetti M, Tipton KF (1998) Monoamine oxidases and related amine oxidases as phase I enzymes in the metabolism of xenobiotics. J Neural Transm Suppl 52:149–171 Uetrecht J (2003) Bioactivation. In: Lee JS, Obach S, Fisher MB (eds) Drug metabolizing enzymes. Marcel Dekker, New York Vasiliou V, Pappa A, Estey T (2004) Role of human aldehyde dehydrogenases in endobiotic and xenobiotic metabolism. Drug Metab Rev 36:279–299 Wong LL (1998) Cytochrome P450 monooxygenases. Curr Opin Chem Biol 2:263–268 Ziegler DM (1988) Flavin-containing monooxygenases: catalytic mechanism and substrate specificities. Drug Metab Rev 19:1–32

Chapter 15

Catalytic Reactions of Phase II Enzymes

After the functional groups catalyzed by phase I activation enzymes are introduced, lipophilic foreign compounds undergo phase II enzyme-catalyzed reactions, which result in greatly increasing the solubility of parent compounds, thus facilitating their excretion from the body. Foreign compounds that already contain such functional groups can proceed with phase II metabolism without undergoing functionalization reactions. Though phase II reactions are a major defense mechanism, in some cases, the formed conjugates undergo further reactions to yield unstable, reactive metabolites. Chapter 5 describes a variety of functional groups of foreign compounds (e.g., phenol, epoxide, polyphenol, carboxylic acid, and amino acid) that are targets of conjugation reactions. Such functional groups of substrates (foreign compounds) and their corresponding conjugation reactions are summarized in Table 15.1. This chapter discusses the chemical reactions catalyzed by major phase II enzymes, including conjugation enzymes (uridine 5¢-diphospho-glucuronosyl transferase (UGT), glutathione S-transferase (GST), sulfotransferase, acyltransferase, acetyltransferase, and methyltransferase) as well as nonconjugation enzymes that are also classified as phase II enzymes (quinone reductase and epoxide hydrolase). In conjugation reaction, the donor (often the cofactor of enzyme) transfers an ionic or nonionic group (e.g., glucuronic acid, glutathione, sulfonate, acyl, or methyl) to the functional group of the acceptor (an electrophilic or a nucleophilic group of a foreign compound). Figures 15.1 and 15.2 present the chemical structures of donor compounds.

15.1

UDP-Glucuronosyl Transferase-Catalyzed Conjugation Reactions

In phase II metabolism, nucleophilic metabolites converted by phase I enzymes (mainly CYP450) often undergo conjugation reactions catalyzed by UGT to form glucuronide conjugates. Glucuronidation forms a variety of O-, N-, S- and C- containing glucuronides which greatly increase the solubility of the parent compounds. C.-H. Chen, Activation and Detoxification Enzymes: Functions and Implications, DOI 10.1007/978-1-4614-1049-2_15, © Springer Science+Business Media, LLC 2012

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Catalytic Reactions of Phase II Enzymes

Table 15.1 Functional groups of foreign compounds in conjugation reactions Phase II enzyme Conjugation reaction Functional group of substrate UDP-glucuronyltransferase Glucuronidation –OH, SH, –NH2, CH2, –COOH, NHOH Glutathione S-transferase Glutathione Epoxide, halide, –NO2 Sulfotransferase Sulfonation –OH, –NH2 N-Acetyltransferase Acetylation –OH, –NH2, –SO2NH2 Methyltransferase Methylation –OH, –NH2, –SH

Donor compounds in conjugation reactions (1) Uridine-5'-diphospho-α-D-glucuronic acid (UDP-GA)

Glutathione

O COO-

COO-

H N

O O

OH

O

P

O

OH

O

OH

O-

O

P

CH N

CH2 O

O

O

O CH2

CH2

NH

C

NH

C

COO-

CH2

CH

NH2

CH2

O-

SH

OH OH

3'-phosphoadenosine-5'-phosphosulfate (PAPS)

S-Adenosylmethionine (SAM)

H2N H2N N N

-O

N N

O

O

N S O

O

P

O

CH2

N

H3C

-OOC

O

N CH

O-

(CH2)2

S+

CH2

N O

H2N

O

OH

= PO3

OH

OH

Fig. 15.1 Donor compounds in phase II conjugation reactions (part 1)

The excretion of glucuronide conjugates from the cells is carried out by ATPdependent export pumps such as multidrug resistant proteins (MRP). A schematic outline of activation, nucleophilic metabolite, glucuronidation, and excretion is described in Fig. 15.3. Glucuronidation of foreign compounds occurs in the liver, intestinal mucosa, and kidney. The produced glucuronide conjugates are usually excreted in urine and bile. Glucuronidation is an important step in the metabolism of aromatic amines, many of which are carcinogenic. UGT catalyzes the transfer of glucuronic acid (GA) from

15.1

UDP-Glucuronosyl Transferase-Catalyzed Conjugation Reactions

147

Donor compounds in conjugation reactions (2) Acetyl-coenzyme A

H2N N N O

O

CH3

O N

NH

C

CH2

CH

C

OH

CH3

CH2 O

P O-

O

P

CH2

O

N O

O-

CH2 C

O

O

NH

CH2 CH2 S

C

=

PO3

CH3

Glycine

Glutamine

-OOC

H

OH

O

O

-OOC

C

H +

NH3

CH H3N

CH2

CH2

C NH2

+

Fig. 15.2 Donor compounds in phase II conjugation reactions (part 2)

Phase I Phase II Lipophilic xenobiotic Nucleophilic metabolite Glucuronide conjugate CYP450 UGT MRP Phase III Excretion

Fig. 15.3 Excretion of nucleophilic metabolites via glucuronide conjugation

uridine 5'-diphospho-glucuronic acid (UDP-GA) to a substrate (foreign compound) that contains oxygen, nitrogen, sulfur, or carboxyl functional group. Typical examples of O-, N-, S-, and C-glucuronide conjugation reactions are shown as follows: R - OH + UDP - GA ® UDP + R - O - GA R - SH + UDP - GA ® UDP + R - S - GA R - NH 2 + UDP - GA ® UDP + R - NH - GA R - CHO + UDP - GA ® UDP + R - CO - GA

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Catalytic Reactions of Phase II Enzymes

R - COOH + UDP - GA ® UDP + R - CO(OGA) R - NHOH + UDP - GA ® UDP + R - N(OH)GA where R denotes an aliphatic derivative and may be replaced with F (an aromatic derivative). The functional group of a substrate is composed of hydroxyl (−OH), thiol (−SH), amine (−NH2), carbonyl (−C=O), carboxylic (−COOH), or hydroxylamine (−NHOH).

15.2

Glutathione S-Transferase-Catalyzed Conjugation Reactions

GST contributes to the metabolism of xenobiotics by catalyzing reactions that conjugate glutathione (GSH) with electrophilic metabolites derived from phase I metabolism, thus facilitating their excretion from the body. Conjugation with GSH occurs via a sulfhydryl group to the electrophilic center on the foreign compound. A schematic outline of activation, conjugation, and excretion of an electrophilic metabolite is described in Fig. 15.4. The family of GST isozymes catalyzes the conjugation of GSH with an electrophilic substrate to form a thioester bond between the sulfur atom of GSH and the substrate. Foreign compounds that undergo GST-catalyzed conjugation reactions include alkyl- and aryl-halides, epoxides, isothiocyanates, unsaturated carbonyls, and nitro compounds. Typical reactions involving glutathione conjugation with epoxide, aliphatic halide, or nitro compounds are shown as follows:

R - Cl + GSH ® GS - R + H + + Cl R - NO2 + GSH ® GS - R + H + + NO 2 where R denotes an aliphatic derivative and may be replaced with F (an aromatic derivative). GST plays an important role in the detoxification of a broad range of toxic foreign compounds, particularly those that may lead to cytotoxicity or mutagenic events (aflatoxin B1 and benzo[a]pyrene). The resulting conjugates generally are less Phase I Phase II Lipophic xenobiotic Electrophilic metabolite Glutathione conjugate GST CYP450 MRP Phase III Excretion

Fig. 15.4 Excretion of electrophilic metabolites via glutathione conjugation

15.4

Acyltransferase-Catalyzed Conjugation Reactions

149

reactive, more water-soluble and ready for excretion in urine or bile. GSH conjugation has the capacity to make harmful endogenous compounds less destructive. In contrast, GST activity may affect anticancer medication since cancer drugs can be detoxified by GST. Thus, an over-expression of GST activity in tumors could be a problem in chemotherapy.

15.3

Sulfotransferase-Catalyzed Conjugation Reactions

CYP450 catalyzes the functionalization reaction that introduces a functional group into a foreign compound to form electrophilic or nucleophilic metabolite. Sulfotransferase-catalyzed reaction conjugates the functional group of electrophilic metabolite with sulfo moiety. The sulfonate group is transferred from a donor molecule (cofactor of enzyme) to an acceptor molecule (e.g., alcohol or amine). The most common sulfonate donor is 3¢-phosphoadenosine-5¢-phosphosulfate (PAPS) which has a high concentration in the liver. Similar to Fig. 15.4 for GST, the addition of a sulfonate group also facilitates the excretion of a foreign compound from the cells, which is also carried out by MRP. A sulfonation reaction involves the transfer of sulfonate group (−SO3−) from PAPS to amine, hydroxylamine, or alcohol. Sulfonation conjugate formation is known to occur with aromatic or aliphatic amines, phenols, as well as primary, secondary, and tertiary alcohols. Sulfonate conjugates are excreted predominately in the urine. Typical sulfotransferase-catalyzed reactions are presented as follows for alcohols, phenols, and aliphatic and aromatic amines. R - OH + PAPS ® R - O - SO3 H + PAP R - NH 2 + PAPS ® R - NH - SO3 H + PAP where R denotes an aliphatic derivative and may be replaced with F (an aromatic derivative). PAP denotes 3-phosphoadenosine 5-phosphate. Sulfonate conjugation is the most important pathway in the metabolism of phenols.

15.4

Acyltransferase-Catalyzed Conjugation Reactions

Acyltransferase-catalyzed conjugation reaction acts upon the acyl group (R–C=O) in the carboxylic acid (R–COOH) of a foreign compound, leading to the formation of an amide, ester, or peptide bond between the acyl group of a foreign compound and the amino group of an endogenous compound. Amino acid conjugation is an important pathway in the metabolism of carboxyl acid-containing foreign compounds, for instances, benzoic acid with glycine, phenylacetic acid with glutamine, xanthurenic acid with serine, and 4-nitrobenzoic acid with arginine.

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Catalytic Reactions of Phase II Enzymes

Acyl conjugation reactions require an initial activation of xenobiotic to a CoA derivative, which is catalyzed by acyl-CoA ligase. The resulting acyl-CoA subsequently reacts with an amino acid, giving rise to acylated amino acid conjugate and CoA. Thus, acyl conjugation reaction occurs in two steps: the initial activation of the carboxyl group to yield reactive acyl-CoA thioester, followed by the transfer of acyl to the amino group of an amino acid. A typical example of acyltransferasecatalyzed conjugation reaction is shown below. Step 1:

F - COOH + Acyl - CoA ® F - CO (CoA - Acyl ) + H 2 O

Step 2:

Overall

where F denotes the aromatic portion of a foreign compound (e.g., benzoic, phenylacetic, or xanthurenic acid), and R represents the side chain portion of an amino acid (e.g., glycine, serine, or arginine). Amino acid conjugation is an alternative conjugation process for carboxylic acid-containing xenobiotics. It can occur in liver and kidney. The resulting amino acid conjugates are generally excreted from the body by urinary elimination.

15.5

N-Acetyltransferase-Catalyzed Conjugation Reactions

N-Acetyltransferase catalyzes the transfer of an acetyl group from acetyl CoA (AcCoA) to the terminal nitrogen of arylamine. Arylamine contains an aromatic hydrocarbon that has at least one amine group attached to it. Acetylation couples an amino group with the acetyl moiety, resulting in the formation of acetylated derivatives, which are generally less water soluble than the parent compound. N-Acetyltransferase has been shown to be important in the detoxification of drugs. Acetyltransferase-catalyzed reaction occurs in two steps: the enzyme is acetylated by AcCoA and then the acetyl group (the donor) is transferred to the acceptor (e.g., arylamine). A typical example of acetyl conjugation reactions are shown in the following: First step: Second step:

AcCoA + NAT ® NAT - AcCoA

15.7

Quinone Reductase-Catalyzed Reactions

151

Overall:

where NAT represents N-acetyltransferase and F-NH2 denotes arylamine, and Ac, CoA, and AcCoA denote acetyl group (−COCH3), coenzyme A, and acetyl CoA, respectively. Similarly, when the acceptor is arylhydroxylamine, the overall reaction is

15.6

Methyltransferase-Catalyzed Conjugation Reactions

Methyltransferase catalyzes the reaction that transfers the methyl group of the donor to the acceptor, the substrate (foreign compound). The methyl donor is the reactive methyl group bound to the sulfur in S-adenosyl methionine (SAM). SAM is the cofactor of methyltransferase. SAM-dependent methyltransferase acts on a wide variety of target molecules. For instances, methylation occurs on nucleic bases in DNA or amino acids in protein structures. Hydroxyl (−OH), amino (−NH2), and thiol (−SH) groups of foreign compounds may be metabolized through methylation. Typical examples of methyltransferase-catalyzed reactions that involve the transfer of a methyl group to N, O, and S nucleophiles are shown below:

F - OH + SAM ® F - O - CH 3 + SAH HS - CH 2 OH + SAM ® CH 3 - S - CH 2 OH + SAH where R and F denote aliphatic and aromatic portions of a foreign compound, respectively. SAM and SAH denote S-adenosyl methionine and S-adenosyl-lhomocysteine, respectively.

15.7

Quinone Reductase-Catalyzed Reactions

Quinones are among the toxic products of CYP450 oxidative metabolism of aromatic hydrocarbons. Quinone reductase is considered as a phase II nonconjugation enzyme that exhibits a broad specificity for structurally diversified quinones. The reduction of electrophilic quinones catalyzed by quinone reductase is an important detoxification pathway. The reaction usually utilizes NADH or NADPH as a source

152

15

Catalytic Reactions of Phase II Enzymes

of reductant. A typical example of quinone reductase-catalyzed reduction of quinone to form phenol containing two –OH groups is shown below: O = F = O + NADH + H + ® HO - F - OH + NAD + where O=F=O (e.g., p-benzoquinone) is reduced to O=F−OH (e.g., semiquinone) and then to HO−F−OH (e.g., hydroquinone). F denotes an aromatic derivative.

15.8

Epoxide Hydrolase-Catalyzed Reactions

Epoxides are ethers that contain a three-member ring, which gives them unusual reactivity. This unusual reactivity is due to the highly polarized oxygen–carbon bonds in addition to a highly strained ring. Some reactive epoxides are responsible for electrophilic reactions with critical biological targets (e.g., DNA and proteins), leading to toxic and carcinogenic effects. Epoxide hydrolase is also considered as a nonconjugation phase II enzyme. The addition of water to an epoxide (the substrate) catalyzed by epoxide hydrolases produces 1,2-diols. Water is the co-substrate and the reaction is energetically favorable. Epoxide hydrolase catalyzes the hydrolysis reaction of the epoxide ring in alkene or arene compound. A typical example of epoxide hydrolase-catalyzed reaction is shown as follow:

where F denotes an aromatic derivative which may be replaced with R (an aliphatic derivative). Examples of epoxide hydrolase-catalyzed reactions are the hydration of benzo[a]pyrene and allylbenzene oxide to form benzo[a]pyrene diol and allylbenzene diol, respectively.

Bibliography Armstrong RN (1987) Enzyme-catalyzed detoxication reactions: mechanisms and stereochemistry. CRC Crit Rev Biochem 22:39–88 Armstrong RN (1991) Glutathione S-transferases: reaction mechanism, structure, and function. Chem Res Toxicol 4:131–140 Baez S, Segura-Aguilar J, Widersten M et al (1997) Glutathione transferases catalyse the detoxication of oxidized metabolites (o-quinones) of catecholamines and may serve as an antioxidant system preventing degenerative cellular processes. Biochem J 324:25–28 Berhane K, Widersten M, Engström A et al (1994) Detoxication of base propenals and other alpha, beta-unsaturated aldehyde products of radical reactions and lipid peroxidation by human glutathione transferases. Proc Natl Acad Sci U S A 91:1480–1484 Butterworth M, Lau SS, Monks TJ (1996) 17 beta-Estradiol metabolism by hamster hepatic microsomes. Implications for the catechol-O-methyl transferase-mediated detoxication of catechol estrogens. Drug Metab Dispos 24:588–594

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McGurk KA, Brierley CH, Burchell B (1998) Drug glucuronidation by human renal UDPglucuronosyltransferases. Biochem Pharmacol 55:1005–1012 McLellan LI, Wolf CR, Hayes JD (1989) Human microsomal glutathione S-transferase. Its involvement in the conjugation of hexachlorobuta-1,3-diene with glutathione. Biochem J 258:87–93 Minchin RF, Hanna PE, Dupret JM et al (2007) Arylamine N-acetyltransferase I. Int J Biochem Cell Biol 39:1999–2005 Minchin RF (1995) Acetylation of p-aminobenzoylglutamate, a folic acid catabolite, by recombinant human arylamine N-acetyltransferase and U937 cells. Biochem J 307:1–3 Mulder GJ (ed) (1990) Conjugation reactions in drug metabolism. An integrated approach. Taylor and Francis, London Mulder GJ, Jakoby WB (1990) Sulfation. In: Mulder GJ (ed) Conjugation reactions in drug metabolism. An integrated approach. Taylor and Francis, London Negishi M, Pedersen LG, Petrotchenko E et al (2001) Structure and function of sulfotransferases. Arch Biochem Biophys 390:149–157 Orzechowski A, Schrenk D, Bock-Hennig BS et al (1994) Glucuronidation of carcinogenic arylamines and their N-hydroxy derivatives by rat and human phenol UDP-glucuronosyltransferase of the UGT1 gene complex. Carcinogenesis 15:1549–1553 Parker MH, McCann DJ, Mangold JB (1994) Sulfation of di- and tricyclic phenols by rat liver aryl sulfotransferase isozymes. Arch Biochem Biophys 310:325–331 Ritter JK (2000) Roles of glucuronidation and UDP-glucuronosyltransferases in xenobiotic bioactivation reactions. Chem Biol Interact 129:171–193 Servin AL, Wicek D, Oryszczyn MP et al (1987) Metabolism of 6,7-dimethoxy 4-(4¢-chlorobenzyl)isoquinoline. II. Role of liver catechol O-methyltransferase and glutathione. Xenobiotica 17:1381–1391 Temellini A, Mogavero S, Giulianotti PC et al (1993) Conjugation of benzoic acid with glycine in human liver and kidney: a study on the interindividual variability. Xenobiotica 23:1427–1433 Weber WW, Vatsis KP (1993) Individual variability in p-aminobenzoic acid N-acetylation by human N-acetyltransferase (NAT1) of peripheral blood. Pharmacogenetics 3:209–212 Weisiger RA, Pinkus LM, Jakoby WB (1980) Thiol S-methyltransferase: suggested role in detoxication of intestinal hydrogen sulfide. Biochem Pharmacol 29:2885–2887 Zeldin DC, Wei S, Falck JR et al (1995) Metabolism of epoxyeicosatrienoic acids by cytosolic epoxide hydrolase: substrate structural determinants of asymmetric catalysis. Arch Biochem Biophys 316:443–451 Zeldin DC, Kobayashi J, Falck JR et al (1993) Regio- and enantiofacial selectivity of epoxyeicosatrienoic acid hydration by cytosolic epoxide hydrolase. J Biol Chem 268:6402–6407

Chapter 16

Diversified Classes of Enzyme Modulators

Enzymatic catalyses are mediated by functional groups present in amino acid side chains. The amino acid side chains of enzymes that are frequently directly involved in enzyme catalytic processes include histidine, serine, cystein, lysine, glutamate, and aspartate. The active site of an enzyme is usually larger than the substrate, since in most cases the substrate is partially surrounded by the active site. The structure of an enzyme is required to stabilize the conformation of the active site for achieving enzymatic function. In a metabolic reaction that involves a chemical change in the parent compound, enzymatic catalysis could not be brought about only by the functional group present in the amino acid side chain alone. In such a catalytic reaction, the enzyme acts in cooperation with a small molecule called coenzyme that possesses the physicochemical property which is not found in the polypeptide chains of the enzyme. As in the cases of phase II enzyme-catalyzed reactions, the enzyme and the coenzyme provide a greater variety of functional groups than is provided by the amino acid side chains of the enzyme alone. An efficient condition for an enzyme catalytic action would be to have the activity of the enzyme proportional to its need at any particular time. In the circumstance of the accumulation of a large quantity of foreign compounds, the cells must be able to detoxify them by appropriate metabolizing enzymes to avoid their potential toxic effects. An important feature of foreign compound-metabolizing enzymes is their ability to be induced by a variety of chemical compounds referred to as enzyme modulators. A modulator is a small molecule that binds to the enzyme, either covalently or noncovalently, thereby changing the interaction or conformation of the enzyme in a manner that the activity of the enzyme is either increased or decreased. Prior to exerting its action on the enzyme present in the cells, an enzyme modulator must be able to penetrate across biomembranes. Extensive research indicates that substantial protection against foreign compound-mediated toxic effects or chemical carcinogenesis may be achieved by the modulation of the enzymes concerned with the metabolism of carcinogens or other foreign compounds.

C.-H. Chen, Activation and Detoxification Enzymes: Functions and Implications, DOI 10.1007/978-1-4614-1049-2_16, © Springer Science+Business Media, LLC 2012

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16.1

16

Diversified Classes of Enzyme Modulators

Substrate–Enzyme Interactions

Foreign compounds or their metabolites are the substrates for xenobiotic metabolizing enzyme-catalyzed reactions. These substrates are either electrophiles or nucleophiles. Electrophiles are electron-deficient substances that interact with electron-rich species and are considered to be Lewis acids. Electrophilic metabolites contain either positively charged ions (e.g., H+ and NO+) or molecules carrying a partial positive charge (e.g., polarized neutral molecules such as alkyl halides, acyl halides, and carbonyl compounds). Metabolite intermediates generated through metabolic activation catalyzed by phase I enzymes are substrates for phase II enzymes. These substrates often share a common feature that contains electrophilic atoms and most of chemically active intermediates are electrophiles. Electrophilc metabolites prefer to interact with nucleophilic groups of enzymes. There are a number of nucleophilic groups present in phase II enzymes, such as serine, tyrosine hydroxyl, aspartate or glutamate carboxylase, histidine imidazole, and cysteine sulfhydryl groups (Table 16.1). Electrophilic metabolites generated by phase I enzymes are often detoxified by glutathione S-transferases (GST) (Fig. 16.1). Detoxification of electrophilic quinones by quinone reductase is also an important pathway. In contrast, nucleophilic compounds are electron-rich substances that interact with electron-deficient species. Having an excess in electrons, nucleophiles are

Table 16.1 Typical electrophilic and nucleophilic atoms or groups Substrates Enzymes Electrophiles Nucleophiles Electrophiles H+ O atom –NH3+ + NO N atom Mg2+ Alkyl halides S atom Mn2+ Acyl halides –OH group Fe3+ Carbonyl compounds –NH2 group –SH group –COOH group

Electrophilic metabolites

GST

Nucleophiles (group) Serine Tyrosine hydroxyl Aspartate carboxylase Glutamate carboxylase Histidine imidazole Cysteine sulfhydryl

Conjugates

Phase I Xenobiotics enzymes Nucleophilic metabolites Conjugates UDP–GT / ST Abbreviations: GST: glutathione S-transferases; ST: sulfotransferases UDP–GT: uridine-diphosphate –glucuronosyltransferases

Fig. 16.1 Conjugation of electrophilic and nucleophilic metabolites

16.2

Modulator–Enzyme Interactions

157

considered as Lewis bases. Electron-rich nucleophilic metabolites include many compounds that contain O, N, or S atoms as well as functional groups such as –OH, –NH2, –SH, and –COOH. A list of typical electrophilic atoms or groups in foreign compounds and their metabolites (substrates) is also shown in Table 16.1. In nucleophilic catalysis, the roles of catalyst and substrate are the reverse of those defined by electrophilic catalysis. Most reactive metabolites consist of electrophiles and the detoxification of electrophiles is an important event. However, it becomes recognized that the detoxification of nucleophiles is as important as that of electrophiles, since many nucleophiles can be converted to electrophiles. Nucleophilic metabolites prefer to interact with electrophilic groups in the amino acid side chains of foreign compound metabolizing enzymes. There are a number of potentially electrophilic groups in these enzymes (e.g., –NH3+ and metal ions Mg2+, Mn2+, or Fe3+) (Table 16.1). Nucleophilic metabolites such as phenols are often detoxified by UDP-glucuronosyltransferases and sulfotransferases (Fig. 16.1). Glucoronidation is a primary metabolic reaction for many compounds containing nucleophilic functional groups (e.g. –OH, –COOH, –SH, and –NH2). In UDPglucuronosyltransferase-catalyzed reactions, the site of glucuronidation is generally an electron-rich nucleophilic O, N, or S atom. Sulfonation conjugation reactions involve the transfer of a sulfonate group (–SO3−) from the cofactor (3-phosphoadenosine 5-phosphosulfate) as the donor to a nucleophilic group of a metabolite intermediate as the acceptor.

16.2

Modulator–Enzyme Interactions

Foreign compound metabolisms require the substrate–enzyme interactions involving the reactive group of the metabolite and the functional group in the amino side chains of the enzyme. Through either covalent or noncovalent interactions, a modulator molecule is able to affect the activity of a metabolizing enzyme by interfering with substrate–enzyme interactions. Investigations of substrate–enzyme interactions in the presence of an enzyme modulator are essential to the elucidation of the mechanism underlying the effects of modulator on the enzyme activity. As a result of this, the activity of the enzyme may be increased or decreased, depending on whether the interference leads to more or less favorable substrate–enzyme interactions. An enzyme modulator may also be capable of affecting the activity of a metabolizing enzyme by altering its conformation. The conformation of an enzyme is crucial for its function. Studies of the conformation of a metabolizing enzyme in the presence of an enzyme modulator are also crucial for the elucidation of the mechanism underlying the effects of modulator on the enzyme activity. Fluorescence spectroscopy is useful for investigating the conformation of an enzyme upon the binding of a small molecule. Stop-flow kinetics is valuable in the elucidation of small molecule–enzyme interactions. Future research is needed in the applications of fluorescence spectroscopy and stop-flow kinetics to evaluate the effects of enzyme modulators on substrate–enzyme interactions and the conformation of metabolizing enzymes.

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16.3

16

Diversified Classes of Enzyme Modulators

Michael Acceptor Functionalities

A diversity of small molecules of naturally occurring or synthetic origins has been found to be effective inducers of phase II detoxification enzymes and have the potential to protect organisms against foreign compound-mediated toxic effects. Induction of phase II enzymes has been proposed as a major strategy for reducing the susceptibility of living cells to toxic and carcinogenic effects. Evaluation of the chemical structures of modulator molecules reveals that enzyme modulators belong to a variety of chemical classes with few common properties, except their ability to modify sulfhydryl group of cysteine residue of the enzymes. Extensive research has revealed that, besides their ability to modify sulfhydryl group of a cysteine residue, many modulators of phase II enzymes contain Michael acceptor functionalities (olefins or acetylenes conjugated to electron withdrawing groups). Olefins are unsaturated hydrocarbons that contain one or more pairs of carbon atoms linked by a double bond. Acetylenes are also unsaturated hydrocarbons, but consist of two carbon atoms linked by a triple bond. Either the carbon– carbon double bond in olefins or the carbon–carbon triple bond in acetylene serves as a source of electrons, whose availability is determined by the groups attached to it. An electron-withdrawing group attached to the carbon–carbon double or the carbon–carbon triple bond destabilizes the transition state of Michael acceptor by intensifying the positive charge, which activates the double or triple bond toward the reagents that are electron-rich (Michael donor). The potency of enzyme modulators was found to parallel their reactivity as Michael acceptors. Functional groups of Michael reaction receptors include a,b-unsaturated double bond attached to aldehydes, ketone, quinone, thioketone, sulfone, ester, nitrile, and nitro groups attached. A list of enzyme modulators that contain Michael acceptor functionalities is shown in Table 16.2. The C=O, –COOH, –COOR, and –CN groups Table 16.2 A list of enzyme modulators containing Michael acceptors Classes of modulators Compounds Isothiocyanate and derivatives Sulforaphane Phenylethyl isothiocyanate 1,2-Dithiole-3-thione and derivatives 1,2-Dithiole-3-thione (D3T) 4-Methyl-5-pyrazinyl-D3T (OPZ) Indole-3-carbinol Indole-3-carbinol Flavonoids Catechin, Epicatechin Epigallocatechin, Leucocyanidin Myricetin, Quercetin Fisetin, Isoliquiritigenin Diosmin, Hesperidin Isoflavones Genistein, Daidzein Phenols and polyphenols Resveratrol, Curcumin Gallic acid, Rosmarinic acid Carnosic acid, Tannin Ellagic acid, Protocatechuic acid (continued)

16.4

Diversities of Enzyme Inducers

Table 16.2 (continued) Classes of modulators Organosulfur Terpenes and terpenoids

Quinoline Others

159

Compounds Diallyl sulfide, Diallyl disulfide Diallyl trisulfide, Alliin Beta-carotene, Lycopene Canthaxanthin, Astaxanthin Zerumbone, Limonene Ethoxyquin Nivalenol

attached to the carbon–carbon double bond are powerful electron-withdrawing groups. Therefore, the carbon–carbon double bond of an a,b-unsaturated ketone, acid, ester, or nitrile is susceptible to nucleophilic attack, leading to Michael addition or reaction. Michael reaction is the addition of nucleophile (electron-rich Michael donor) to an electrophilic unsaturated carbonyl compound (electrondeficient Michael acceptor).

16.4

Diversities of Enzyme Inducers

Substantial evidence indicates that a significant protection against chemical carcinogenesis and inflammatory conditions can be achieved by the induction of enzymes responsible for the detoxification of carcinogens and other xenobiotics. It has been proposed that selective induction of phase II enzymes is a sufficient condition for chemoprotection. Elucidation of structural features of enzyme inducers has a significant impact on the understanding of the protective role of enzyme inducers. There are diversified classes of phase II enzyme inducers. Many of them contain Michael acceptor functionalities. To evaluate the functional characteristics of enzyme inducers, the chemical structures of a variety of enzyme inducers are shown in Figs. 16.2–16.9. In addition to Michael acceptor functionalities, some inducers of phase II enzymes (e.g., flavonoid and curcuminoid analogues) also contain phenolic hydroxyl groups. It has been reported that such phenol hydroxyl groups are able to scavenge oxygen- and nitrogen-centered reactive species directly. These findings suggest that enzyme inducers that contain phenolic hydroxyl groups in addition to Michael acceptor centers play not only an indirect protective role by inducing phase II enzymes, but also a direct protective role by scavenging hazardous oxidants. Such enzyme inducers therefore may be designated as bifunctional antioxidants. Ortho-hydroxyl groups also have a significant impact on the protective role of phase II enzyme inducers. Introduction of ortho-hydroxyl groups on the aromatic

Isothiocyanate and derivatives Phenylethyl isothiocyanate

Sulforaphane N

C

S N C S

O

S CH3

1,2-dithiole-3-thione and derivatives 1,2-dithiole-3-thione (D3T) 4-methyl-5-pyrazinyl-D3T (Oltipraz) S S

S

N

S

N

S

S CH3

Indole-3-carbinol NH

OH

Fig. 16.2 Chemical structures of phase II enzyme inducers (Part a) Flavonoids (1) Catechin

Epicatechin OH

OH

OH

OH HO

HO

O

O

OH

OH OH

OH

Epigallocatechin

Leucocyanidin OH

OH OH

HO

OH HO

O

O

OH OH

OH OH

OH

Myricetin

OH

Quercetin OH

OH OH

HO

O

OH HO

O

OH OH OH

O

OH OH

O

Fig. 16.3 Chemical structures of phase II enzyme inducers (Part b)

Flavonoids (2) Fisetin

Isoliquiritigenin OH OH

OH HO

HO

O

OH O

O

OH

Diosmin OH OH

OCH3

O

O

O

CH3 HO

OH

O

O

OH OH OH

OH

O

Hesperidin OH

O

O

CH3 HO

OCH3

O OH

OH

O

O

OH

OH OH OH

O

Fig. 16.4 Chemical structures of phase II enzyme inducers (Part c) Isoflavones

Genistein

Daidzein

O

HO

OH

O

O

HO

OH

O

OH

Phenols and polyphenols (1) Curcumin

Resveratrol OCH3

OH

OH

HO

OCH3

HO OH

O

Gallic acid HO

O

Rosmarinic acid

O OH

O

HO

OH

O HO

OH

OH OH

Fig. 16.5 Chemical structures of phase II enzyme inducers (Part d)

O

OH

162

16

Diversified Classes of Enzyme Modulators

Phenols and polyphenols (2) Carnosic acid OH

Tannic acid OH

CH3

OH

HO

HO HO2C

CH3

OH

O HO OH

O

O

O

HO CH3

H CH3

O O

OH

O

HO

OH

O OH

HO

OH

O

Ellagic acid

Protocatechuic acid

O HO

O

O

HO OH

HO

OH HO

O

OH

O Fig. 16.6 Chemical structures of phase II enzyme inducers (Part e)

Organosulfur compounds Diallyl sulfide

Diallyl disulfide S

S S

Diallyl trisulfide

Alliin O

S

S S

NH2 OH

S O

Fig. 16.7 Chemical structures of phase II enzyme inducers (Part f)

16.4

Diversities of Enzyme Inducers

163

Terpenes and terpenoids (1) Beta-carotene CH3

CH3

CH3

CH3

CH3

CH3 CH3

CH3

CH3

CH3

Lycopene CH3

CH3

CH3

CH3 CH3

CH3 CH3

CH3

CH3

CH3

Canthaxanthin CH3

CH3

CH3

CH3

CH3

O

O

CH3 CH3

CH3

CH3

CH3

Astaxanthin CH3

CH3

CH3

CH3

OH

CH3

O

O

CH3 HO

CH3

CH3

CH3

CH3

Fig. 16.8 Chemical structures of phase II enzyme inducers (Part g)

Terpenes and terpenoids (2) Zerumbone

Limonene

CH3 O

CH3 CH3 CH3 CH3

CH3 CH3

Quinoline Ethoxyquin H CH3

N

CH3 O

CH3

CH3

Fig. 16.9 Chemical structures of phase II enzyme inducers (Part h)

164

16

Diversified Classes of Enzyme Modulators

Table 16.3 Modulators containing ortho-hydroxyl group on the aromatic ring Class of compound Chemical modulator Flavonoids Catechin, Epicatechin Epigallocatechin, Leucocyanidin Myricetin, Quercetin Fisetin, Diosmin Phenols and polyphenols Curcumin, Gallic acid Carnosic acid Tannin, Ellagic acid Protocatechuic acid

rings of phenylpropenoids was found to dramatically enhance their potencies not only as inducers of quinone reductase, but also as quenchers of superoxide. The presence of an ortho-hydroxyl group on the aromatic ring was also reported to profoundly increase the induction potency of benzylidene-alkanones and -cycloalkanones. A list of enzyme inducers that contain ortho-hydroxyl groups on the aromatic rings is shown in Table 16.3. In addition to Michael acceptor functionalities, the potencies of quinone reductase induction by a series of bis(benzylidene)cycloalkanones appear to be correlated with their ability to quench superoxide radicals. The involvement of both Michael reaction reactivity and radical quenching mechanisms suggests that bis(benzylidene)-cycloalkanones are also bifunctional antioxidants.

Bibliography Bock KW, Lilienblum W, Fischer G et al (1987) The role of conjugation reactions in detoxication. Arch Toxicol 60:22–29 Bolton JL, Trush MA, Penning TM et al (2000) Role of quinones in toxicology. Chem Res Toxicol 13:135–160 Chen CH, Battaglioli G, Martin DL et al (2003) Distinctive interactions in the holoenzyme formation for two isoforms of glutamate decarboxylase. Biochim Biophys Acta 1645:63–71 Ciaccio PJ, Jaiswal AK, Tew KD (1994) Regulation of human dihydrodiol dehydrogenase by Michael acceptor xenobiotics. J Biol Chem 269:15558–15562 Cuendet M, Oteham CP, Moon RC et al (2006) Quinone reductase induction as a biomarker for cancer chemoprevention. J Nat Prod 69:460–463 Dinkova-Kostova AT, Abeygunawardana C, Talalay P (1998) Chemoprotective properties of phenylpropenoids, bis(benzylidene)cycloalkanones, and related Michael reaction acceptors: correlation of potencies as phase 2 enzyme inducers and radical scavengers. J Med Chem 41:5287–5296 Dinkova-Kostova AT, Cheah J, Samouilov A et al (2007) Phenolic Michael reaction acceptors: combined direct and indirect antioxidant defenses against electrophiles and oxidants. Med Chem 3:261–268 Dinkova-Kostova AT, Holtzclaw WD, Cole RN et al (2002) Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants. Proc Natl Acad Sci U S A 99:11908–11913

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Dinkova-Kostova AT, Massiah MA, Bozak RE et al (2001) Potency of Michael reaction acceptors as inducers of enzymes that protect against carcinogenesis depends on their reactivity with sulfhydryl groups. Proc Natl Acad Sci U S A 98:3404–3409 Prochaska HJ, Talalay P (1988) Regulatory mechanisms of monofunctional and bifunctional anticarcinogenic enzyme inducers in murine liver. Cancer Res 48:4776–4782 Rinaldi R, Eliasson E, Swedmark S et al (2002) Reactive intermediates and the dynamics of glutathione transferases. Drug Metab Dispos 30:1053–1058 Schultz TW, Yarbrough JW, Hunter RS et al (2007) Verification of the structural alerts for Michael acceptors. Chem Res Toxicol 20:1359–1363 Talalay P (1989) Mechanisms of induction of enzymes that protect against chemical carcinogenesis. Adv Enzyme Regul 28:237–250 Talalay P, De Long MJ, Prochaska HJ (1988) Identification of a common chemical signal regulating the induction of enzymes that protect against chemical carcinogenesis. Proc Natl Acad Sci USA 85:8261–5 Zhang F, Thottananiyil M, Martin DL, Chen CH (1999) Conformational alteration in serum albumin as a carrier for pyridoxal phosphate: a distinction from pyridoxal phosphate-dependent glutamate decarboxylase. Arch Biochem Biophys 364:195–202

Conclusion

This book incorporates many advances made toward the understanding of functions and implications of activation enzymes and detoxification enzymes. The body protects against potentially toxic effects of foreign compounds, to which humans are exposed including food, drugs, cigarette smoke, and environmental chemicals, by minimizing their exposure and speedily removing them. Prior to their elimination, lipophilic foreign compounds undergo metabolic processes that involve two distinctive steps (phase I metabolism and phase II metabolism) catalyzed by two characteristic enzyme systems. In phase I metabolism, activation enzymes catalyze functionalization reactions that introduce a functional group to the structure of a lipophilic foreign compound, moderately increasing its water solubility. The resulting compound then undergoes phase II metabolism which conjugates the introduced functional group with a chemical group of a small molecule (as the donor). Conjugation reactions catalyzed by transferase enzymes, lead to greatly increased solubility, inactivation of metabolic intermediates, and excretory potential of foreign compounds. Nonconjugation reactions also occur in phase II metabolism. Catalytic reactions of phase I enzymes and phase II enzymes represent critical elements of foreign compound metabolisms. They are essential for subsequent investigations of metabolic intermediates and metabolites generated in metabolic processes. Foreign compound-metabolizing enzymes catalyze a broad spectrum of reactions (e.g., oxidation, hydrolysis, reduction, and conjugation). Many foreign compounds become toxic after their conversion to reactive intermediates or metabolites catalyzed mainly by phase I activation enzymes. In certain cases, phase II enzymes are also involved. Reactive groups of foreign compounds or their metabolites are either electrophiles or nucleophiles. To exert toxic effects, most foreign compounds require activation to electrophilic intermediates catalyzed by phase I enzymes (mainly CYP450). A significant number of generated reactive intermediates have the potential to react with oxygen to form reactive oxygen species including free radicals. Reactive intermediates and reactive oxygen species are capable of interacting with cellular components (proteins, DNA, and lipids), leading to various conditions for diseases (e.g., cancer, cardiovascular disease, and neurological disorders). Knowledge about C.-H. Chen, Activation and Detoxification Enzymes: Functions and Implications, DOI 10.1007/978-1-4614-1049-2, © Springer Science+Business Media, LLC 2012

167

168

Conclusion

reactive intermediates and their underlying mechanisms is critical to the understanding of foreign compound-mediated toxic effects. Despite its importance, elucidation of metabolic intermediates is still lacking for many foreign compounds that humans are exposed to. Foreign compound-metabolizing enzymes that function correctly appear to be a vital means of preventing toxic effects mediated by foreign compounds. To minimize their exposure, it is essential to maintain metabolic intermediates or metabolites at minimum levels. Such maintenance requires a delicate balance between activation enzymes and detoxification enzymes. This fine balance is dependent on relative efficiencies of these two enzyme systems. The rate of generating reactive intermediate or metabolite comparable with that of detoxification reactions is essential to achieve this goal. The expression of activation enzymes and detoxification enzymes may vary among individuals. Genetic polymorphisms are an important factor in contributing to individual variations in the efficacies of these two enzyme systems, particularly, the family of CYP450 and glutathione S-transferases. Variations in genetic polymorphisms of either activation enzymes or detoxification enzymes can affect an individual’s susceptibility to foreign compound-mediated toxic effects. An unusual high expression of activation enzymes may give rise to an overload of reactive intermediates or metabolites. An extraordinary low efficacy of detoxification enzyme may result in abnormal low efficiency in detoxifying foreign compounds. Importantly, individual life styles (e.g., cigarette smoking and alcohol) can also contribute to variations in the expression of foreign compound-metabolizing enzymes. Extensive investigations in the past decades have discovered a variety of chemical compounds that are capable of acting as inducers or inhibitors for foreign compoundmetabolizing enzymes. A broad list of modulation compounds is included in this book. Two types of enzyme inducers have been characterized: monofunctional and bifunctional inducers. Monofunctional inducers raise phase II enzyme activity without significantly elevating phase I enzyme activity. In contrast, bifunctional inducers raise the activities of both phase I and phase II enzymes. The transcription factor Nrf2 binding to the antioxidant response element (ARE) has been reported to play an important role in the induction of phase II enzymes. The discovery of enzyme inducers and inhibitors has led to the proposal of modulating activation or detoxification enzymes as a useful approach for reducing foreign compound-mediated toxic effects. Such a proposal has been a subject of intense interest. Two important hypotheses have been postulated. One hypothesis is to inhibit activation enzymes low enough so that even those with less effective detoxification enzymes still can deal reactive intermediates. Another hypothesis proposes that the induction of detoxification enzymes alone is enough to provide a measure against carcinogenesis and other forms of toxicity. These two hypotheses have been examined essentially using animal model systems. Extension of such investigations to humans is needed in the forthcoming research. Many chemical compounds capable of acting as enzyme modulators are present in the daily human diet. In the past decades, advances in the understanding of

Conclusion

169

mechanisms that govern the detoxification of foreign compounds have revealed that diets can have significant impacts on the efficacies of activation enzymes and detoxification enzymes. Diets rich in vegetables and fruits that contain metabolizing enzyme modulators have received much attention, in particular, those that are rich in uridine-diphosphate-glucuronosyl-transferases, glutathione S-transferases, and quinine reductase. There is substantial evidence that supports the hypothesis that intaking a diet rich in phase II detoxification enzyme inducers is a promising proposal to minimize foreign compound-mediated toxic effects. Extensive lists of vegetables and fruits rich in such enzyme inducers are also available in this book. Inducers of phase II enzymes consist of a variety of chemical classes with few common properties. Nevertheless, structural investigations of enzyme modulators have revealed that many inducers of phase II detoxification enzymes contain Michael acceptor functionalities (olefins or acetylenes conjugated to electron withdrawing groups), and the potency of inducers parallel their reactivity as Michael acceptors. These findings provide important insights into future developments of new enzyme inducers that exhibit potential health benefits. A modulator molecule may affect the activity of a foreign compound-metabolizing enzyme by changing the enzyme conformation or by interfering with substrate–enzyme interactions. Elucidation of a possible change in the enzyme conformation or substrate–enzyme interaction upon inducer binding requires forthcoming investigation.

Index

A Acetaminophen, 131 Acetaminophen toxicity, 62, 131 Acetyl-coenzyme A (Acetyl-CoA), 21, 41, 45 Acetylenes, 137, 158 Acetyltransferase conjugation reaction, 149–150 Acetyltransferases, 38 Activation and detoxification enzymes, 2 Activators, 26 Active transport, 9, 10, 13 Acyltransferase conjugation reaction, 149–150 Acyltransferases, 42–43 Aflatoxin B1, 53, 78 Aflatoxin toxicity, 77–78 Alcohol, 86 Alcohol dehydrogenase (ADH), 29–30 Alcohol dehydrogenase-catlyzed reactions, 142 Alcoholism, 72, 75 Aldehyde dehydrogenase (ALDH), 29, 32, 72 Aldehyde oxidase, 30 Allyl sulfides, 119–120 Amine oxidase, 28–29 Amine oxidase-catalyzed reactions, 140 Antioxidant, 3, 23, 55, 56, 67, 90, 113 Antioxidant enzymes, 57 Antioxidant response element (ARE), 88–89, 115–116 Arsenic in drinking and underground water, 133–134 ATP binding cassette transporters (ABC transporters), 9 Azo dyes, 128 Azoreductase, 31 Azoreductase-catalyzed reactions, 141

B Benzene, 132 Benzo[a]pyrene, 8, 40, 53, 75, 88, 92, 94, 116, 124, 127 Benzo[a]pyrene toxicity, 94, 127 Beverages, 109–110 Bifunctional inducers. See Monofunctional inducers Bioactivation, 41, 49, 68, 127 Black tea, 108–110, 125

C Carbocation, 78, 79 Carbonium ion, 23, 49, 53, 54, 75, 76 Carboxylesterase, 31–32 Carboxylesterase-catalyzed reactions, 142 Carcinogen, 12, 40, 50, 55, 68, 71, 73, 78, 86, 88, 96, 98, 99, 104, 118, 119, 159 Catalytic reactions phase I enzymes alcohol dehydrogenase, 142 amine oxidase, 140 azoreductases, 141 carboxylesterase, 142 CYP450, 137–139 flavin monooxygenas, 140 molybdenum hydroxylases, 141 nitroreductases, 140–141 peroxidase, 142 phase II enzymes acyltransferase, 149–150 epoxide hydrolase, 152 GST, 148–149 methyltransferase, 151 N-acetyltransferase, 150–151

171

172 Catalytic reactions (cont.) quinone reductas, 151–152 sulfotransferase, 149 UDP-glucuronosyl transferase, 145–148 metabolic enzymes foreign compounds, 5 Phase I and II enzymes, 4 Cellular components, reactive intermediate formation DNA adducts, 54–55 lipid peroxidation, 55 protein adducts, 54 toxic effects, 55 Channels and transporters, 10 Chemical carcinogenesis, 68 Chemoprevention, 92–94, 114, 115 Cigarette, 86 Cinnamate, 87, 115, 116 Coffee, 109, 110 Conjugation enzymes acyltransferase, 42–43 GST, 39–40 methyltransferase, 42 NAT, 41–42 sulfotransferases, 40–41 UGTs, 38–39 Coumarin, 87, 102, 116 CYP1A1, 74–75, 86, 95, 98, 99, 109, 119, 127 CYP2A6, 27, 73–74 CYP450-catalyzed reactions dealkylation, 139 dehydrogenation, 138 epoxidation, 138 hydroxylation, 138 oxidation of N-or S-compounds, 139 oxoidation of carbon on aromatic ring, 139 CYP2E1, 62, 67, 75–76, 86, 98, 99, 108, 118 Cytochrome P450 (CYP450), 26–28, 72–76, 137

D Defense against foreign compounds detoxification processes, 2 hydrophilic substances, 1 lipophilic, 1–2 xenobiotics removal, 2 Defense against potential toxicities, 91–92 Defense against reactive intermediates antioxidant enzymes, 57 conjugation reactions, 56 glutathione, 56–57 Dibenz[a,h]anthracene, 127 Diesel exhausts, 133

Index Dietary contribution to enzyme modulators, 104 Dietary inducers, detoxification enzymes, 117–120 Di(2-ethylhexyl)phthalate (DEHP), 132–133 1,2-Dithiole–3-thiones (D3Ts), 93–95 DNA adducts, 54–55

E Electrophile, 40, 49–51, 56, 113, 114, 118, 156, 157 Electrophilic metabolite, 10, 26, 27, 38–39, 54, 93, 148, 156 Environmental chemicals arsenic in drinking and underground water, 133–134 diesel exhausts, 133 PCBs, 134 Environmental factors, 1, 24, 58, 113 Enzymatic functions, 66–67 Enzyme conformation, 157 Enzyme inducers alkyl sulfides, 116 azo dyes, 116 beta-naphthoflavone, 116 butylated hydroxytoluene, 116 canthaxanthin, 116, 163 catechol, 116 curcuminoid, 116, 159 daidzein, 116, 161 ethoxyquin, 116, 163 flavonoids, 116, 164 genistein, 116, 161 indole–3-carbinol, 116 phenethyl isothiocyanate, 116 phenylflavonoids, 116 quercetin, 116, 160, 164 resveratrol, 116, 161 Enzyme inducibility. See Inducibility Enzyme inhibitors, 4, 93, 103, 104, 106 Enzyme modulation as defense mechanism, 114–115 dietary effects, 4 foreign compound-mediated toxic effects, 4 against toxic effects, 89–90 Enzyme modulators beverages, 109–110 dietary contribution, 104 fruits, 106–107 herbs, 108 vegetables, 104–106 Epoxide hydrolase, 32, 43–44 Epoxide hydrolase-catsalyzed reactions, 152

Index Excretion, foreign compounds hepatic, 12–13 metabolic processes, 11 reabsorption in kidney, 13 renal, 12 urinary and biliary systems, 11 Excretors, 37–38

F Facilitated diffusion, 9, 13 Flavin-containing monooxygenase (FMO), 28 Flavin-monooxygenase, 26 Flavin-monooxygenase-catalyzed reactions, 140 Flavonoids anthocyanins, 96, 97 beta-naphthoflavone, 97 4’-bromoflavone, 97 catechin, 96, 97 epigallocatechin, 97 and isoflavones, 96–97 isoliquiritigenin, 97 leucocyanidin, 97 myricetin, 97 quercetin, 97 Free radicals, 3 Fruits blueberry, 106, 107 citrus fruit, 106, 107 grape, 106, 107 grapefruit, 106, 107 musa x paradisiacal, 106, 107 oroblanco, 106, 107 pomegranate, 107 Fuctionalization functional group, 2, 14, 19, 26, 149

G Genetic polymorphisms CYP450 CYP1A1, 74–75 CYP2A6, 73–74 CYP2E1, 75–76 interindividual and interethnic variability, 73 and enzymes inducibility, 3–4 GST, 76 role in alcoholism, 72 Genetic variations in metabolizing enzymes foreign compounds, 71 genetic polymorphisms, 72–76 Glucuronic acid (GA), 20, 21, 38, 44–46, 146

173 Glutathione, 56–57 Glutathione S-transferase (GST), 39–40, 76 Glutathione S-transferase conjugation reaction, 148–149 Green tea, 109, 125, 131 GST polymorphisms, 73

H Health benefits dietary inducers, detoxification enzymes, 117–120 metabolizing enzyme modulation, 114–115 monofunctional and bifunctional inducers, 115 Hepatic excretion, 12–13 Herbs dandelion tea, 108 rosemary, 108 sage tea, 108 thyme, 108 Heterocyclic amines, 125 Household products benzene, 132 DEHP, 132–133 Hydrolytic enzymes carboxylesterase, 31–32 epoxide hydrolase, 32 epoxide hydrolases, 31 Hydrolytic reactions carboxylesterase, 35 hydrolysis of amide, 36 of ester, 35 Hydrophilic compounds, 2, 7, 9, 13 Hydroquinone, 21, 47, 132, 152 4-Hydroxynonenal, 128, 129

I Immune suppression and stimulation, 67 Indole–3-carbinol, 95–96, 119 Inducibility activation vs. detoxification, 87–88 antioxidant response element, 88–89 enzyme modulation against toxic effects, 89–90 life style modification, 86 modulation activation enzymes, 84 detoxification enzymes, 84–85 monofunctional and bifunctional inducers, 86–87

174 Induction of toxicity, 62 Intermediate formation. See Reactive intermediate formation Intrinsic toxicity, 61 Ion transporters, 65–66 4-Ipomeanol, 53, 79–80 4-Ipomeanol toxicity, 79–80 Isoflavones daidzein, 97 genistein, 97 soy isoflavones, 97 Isoliquiritigenin, 97, 104, 116, 158, 161 Isothiocyanate, 92–94, 117–118

L Life style modification alcohol, 86 cigarette smoke, 86 Lipid peroxidation, 55, 64–65 Lipophiles nonpolar/weakly polar compounds, 8 solubility, typical lipophilic foreign compounds, 8 water molecule, polar species, 7 Lipophilic foreign compounds channels and transporters, 10 excretion (see Excretion, foreign compounds) lipid bilayers, 7 lipid solubility, 8–9 lipophiles (see Lipophiles) phase I and phase II metabolisms, 13–14 phase III metabolism, 15 sites of action (see Sites of action) transport mechanisms, 9–10 uptake, hydrophilic compounds, 9 Lipoxygenase, 29

M Metabolic conversion phase I enzyme, 17–19 phase II enzyme conjugation reactions, 19–21 nonconjugation reactions, 21–22 toxication vs. detoxification activation, 22–23 activation vs. deactivation, 23–24 deactivation, 23 Metabolic intermediates detoxification, foreign compound, 2–3 free radicals, 3 functioning detoxification system, 3 reactive oxygen species, 3

Index Methyltransferase conjugation reaction, 151 Methyltransferases, 42 Michael acceptor, 158–159 Michael donor, 158, 159 Michael reaction, 158, 159, 164 Mitochondria functions, 65 Modulation of phase I enzymes, 84 Modulation of phase II enzymes, 84–85 Modulator–enzyme interactions, 157 Molybdenum hydroxylase, 137, 141 Molybdenum hydroxylase-catalyzed reactions, 141 Monofunctional inducers cinnamate, 87, 116 coumarin, 87, 116 1,2-dithiol–3-thione, 87, 116 isoliquiritigenin, 116 isothiocyanate, 116–118 phenol antioxidant, 87, 116 resveratrol, 116 thiocarbamate, 87 Multidrug resistant proteins (MRP), 146 Mycotoxins, 129

N N-Acetyltransferases (NAT), 41–42 N-Acetyltransferases conjugation reactions, 150–151 Nicotine, 61, 73–74, 124 Nitrenium ion, 49, 51, 54 Nitroreductase, 31 Nitroreductase-catalyzed reactions, 140–141 Nitrosamines 4-(methylnitrosamino)–1-(3-pyridyl)–1butanone, 126 N-nitrosobutyl(4-hydroxybutyl)amine), 126 N-nitrosodibutylamine, 126 N’-nitrosonornicotine, 126 Non-conjugation enzymes epoxide hydrolases, 43–44 quinone reductase, 43 Nuclear transcription factor (Nrf2), 88 Nucleophile, 40, 49, 51, 156, 157, 159 Nucleophilic metabolite, 38–39, 145–147, 156–157

O Olefins, 137, 158 Oltipraz, 84, 85, 94, 95, 114 Organosulfur compounds alliin, 98, 99 diallyl disulfide, 98, 99

Index diallyl sulfide, 98, 99 diallyl trisulfide, 98, 99 Ortho-hydroxyl groups, 159–160, 164 Orthonitroaniline orange, 128 Overdose of drugs acetaminophen, 131 active ingredients, 130 terfenadine, 131 xanthine, 131 Oxidative DNA damage, 64 Oxidative enzymes ADH, 29–30 aldehyde oxidase, 30 amine oxidase, 28–29 cytochrome P450, 27–28 flavin-containing monooxygenase, 28 lipoxygenase, 29 peroxidase, 30 xanthine oxidase, 30 Oxidative protein damage, 63 Oxidative reactions at carbon atom, 32–33 at nitrogen atom, 33–34 of unsaturated hydrocarbon, 34 Oxidative stress detoxification reactions, 62–63 DNA damage, 63 reactive oxygen species, 62

P Passive diffusion, 9 Patulin, 128, 129 Peroxidase, 30 Peroxidase-catalyzed reactions, 142 Pharmaceuticals, 124 Phase I enzymes activators, 26 catalytic actions hydrolytic reactions, 35–36 oxidative reactions, 32–34 reductive reactions, 34–35 chemical reactions, 25 hydrolases, 25 hydrolytic enzymes (see Hydrolytic enzymes) oxidative enzymes (see Oxidative enzymes) reductive enzymes (see Reductive enzymes) Phase I metabolism aliphatic hydroxylation, 18 aromatic hydroxylation, 18 epoxidation, 19

175 hydrolysis, 18–19 N-dealkylation, 18 N-oxidation, 18 O-dealkylation, 18 S-oxidation, 18 Phase II enzymes catalytic actions conjugation at C atom, 46 conjugation at N atom, 45 conjugation at O atom, 44–45 conjugation at S atom, 46 conjugation of carboxylic acid, 46 nonconjugation reactions, 47 conjugation enzymes acyltransferase, 42–43 GST, 39–40 methyltransferase, 42 NAT, 41–42 sulfotransferases, 40–41 UGTs, 38–39 excretors, 37–38 nonconjugation enzymes epoxide hydrolases, 43–44 quinone reductase, 43 Phase II metabolism conjugation reactions amino acid, 20 formation, 19 glucuronide, 20 glutathione, 20 methyl, 21 N-acetyl, 20–21 sulfonate, 20 nonconjugation reactions epoxide hydrolase, 22 quinone reductase, 21 Phase III metabolism, 15 Phenol, 30, 38, 44, 87, 116, 132, 164 Phenolic hydroxyl groups, 159 3-Phosphoadenosine 5-phosphate, 44, 149 3-Phosphoadenosine 5-phosphosulfate, 44 Polychlorinated biphenyls (PCBs), 134 Polycyclic aromatic hydrocarbons, 127 Polyphenols carnosic acid, 99 carnosol, 99 curcumin, 98, 99 ellagic acid, 99 gallic acid, 99 protocatechuic acid, 99 resveratrol, 98, 99 tannic acid, 99 turmeric, 98, 99 Prostaglandin H synthase, 30, 74

176 Protein adducts, 54 Pyridine, 125 Pyrimidine, 125

Q Quinone, 8, 21, 43, 47, 87, 151 Quinone reductase-catalyzed reactions, 151–152 Quinone reductases (QR), 43, 87

R Re-absorption in the kidney, 13 Reactive intermediate formation affecting factors, xenobiotic toxicity, 57–58 CYP450, 49 defense against, 56–57 enzyme-catalyzed, 51–52 functionalization reactions, 50 interactions with cellular components, 52–55 lipophilic compounds, 49 oxygen species, 51 phase I and II enzymes, 49–50 Reactive intermediates mediated by phase I enzymes, 52, 53 Reactive intermediates mediated by phase II enzymes, 52, 54 Reactive oxygen species, 51 Red wine, 109–110 Reductive enzymes azoreductase, 31 enzymatic reduction reactions, 30 nitro-compounds, 31 nitroreductase, 31 Reductive reactions reducing agents, 34 reduction at nitrogen atom, 35 reduction of carbonyl group, 35 Renal excretion, 12 Resveratrol, 98, 99, 109, 116, 158, 161

S S-adenosyl-L-homocysteine, 45 S-adenosylmethionine (SAM), 44–45, 146 Semiquinone, 21, 43, 53, 152 Sites of action hepatocytes, 10–11 liver as metabolic site, 10 metabolites, 11 Solubility, 2, 7–9, 13, 14, 17, 19, 21, 38, 41, 42, 56, 132, 145

Index Solute carrier, 10 Sources of foreign compounds azo dyes, 128 environmental chemicals arsenic in drinking and underground water, 133–134 diesel exhausts, 133 polychlorinated biphenyls (PCBs), 134 heterocyclic amines, 125 household products benzene, 132 di(2-ethylhexyl)phthalate, 132–133 humans exposure environmental chemicals, 125 food, 123 household products, 124 pharmaceuticals, 124 smoking, 124 mycotoxins, 129 nitrosamines, 126–127 overdose of drugs, 130–131 polycyclic aromatic hydrocarbons, 127 a,b-unsaturated aldehydes, 129 Species difference 4-ipomeanol toxicity, humans vs. rodents, 79–80 resistance to tamoxifen toxicity, 78–79 susceptibility to aflatoxin toxicity, 77–78 Substrate–enzyme interactions, 156–157 Sulforaphane, 92–93, 118–119 Sulfotransferase conjugation reaction, 149 Sulfotransferases, 40–41

T Tamoxifen, 54, 68, 77, 79 Tamoxifen toxicity, 78–79 Terfenadine, 131 Terpenes and terpenoids astaxanthin, 100 canthaxanthin, 100 ßcarotene, 100 lycopene, 100 zerumbone, 100 Tetrodotoxin, 66 Thiocarbamate, 87, 115, 116 Tobacco-specific carcinogen, 85 Toxic reactive metabolites, 62 Toxication vs. detoxification, metabolic conversion activation, 22–23 activation vs. deactivation, 23–24 deactivation, 23

Index Transporters channels and, 10 mechanisms, 9–10

U UDP-glucuronosyl-transferase conjugation reaction, 145–148 a,b-Unsaturated aldehydes, 129 Uridine-diphosphate-glucuronosyl-transferases (UGT), 38–39

V Vegetables broccoli, 104–106 Brussels sprouts, 105, 106 cabbage, 105

177 cauliflower, 105 cruciferous vegetables, 104 garden cress, 105 garlic, 104, 105 ginger, 105 green leaf vegetables, 105 horseradish, 105 mustard, 105, 106 onion, 104–106 soy, 105 tonka bean, 104, 105 water cress, 104, 105

X Xanthine, 131 Xanthine oxidase, 30 Xenobiotic toxicity, 57–58