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 ( 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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Inducibility of Metabolizing Enzymes

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

97

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

10

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

100

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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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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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11.1

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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

105

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

107

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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11.4

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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

109

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

Bibliography Appelt LC, Reicks MM (1999) Soy induces phase II enzymes but does not inhibit dimethylbenz[a] anthracene-induced carcinogenesis in female rats. J Nutr 129:1820–1826 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, Fahey JW, Wade KL et al (2007) Induction of the phase 2 response in mouse and human skin by sulforaphane-containing broccoli sprout extracts. Cancer Epidemiol Biomarkers Prev 16:847–851 Dulebohn RV, Yi W, Srivastava A et al (2008) Effects of blueberry (Vaccinium ashei) on DNA damage, lipid peroxidation, and phase II enzyme activities in rats. J Agric Food Chem 56:11700–11706 Feldman EB (1997) How grapefruit juice potentiates drug bioavailability. Nutr Rev 55:398–400 Hahn-Obercyger M, Stark AH, Madar Z (2005) Grapefruit and oroblanco enhance hepatic detoxification enzymes in rats: possible role in protection against chemical carcinogenesis. J Agric Food Chem 53:1828–1832 Higgins LG, Cavin C, Itoh K et al (2008) Induction of cancer chemopreventive enzymes by coffee is mediated by transcription factor Nrf2. Evidence that the coffee-specific diterpenes cafestol and kahweol confer protection against acrolein. Toxicol Appl Pharmacol 226:328–337 Huber WW, Parzefall W (2005) Modification of N-acetyltransferases and glutathione S-transferases by coffee components: possible relevance for cancer risk. Methods Enzymol 401:307–341 Joseph MA, Moysich KB, Freudenheim JL et al (2004) Cruciferous vegetables, genetic polymorphisms in glutathione S-transferases M1 and T1, and prostate cancer risk. Nutr Cancer 50:206–213 Kasimsetty SG, Bialonska D, Reddy MK et al (2009) Effects of pomegranate chemical constituents/intestinal microbial metabolites on CYP1B1 in 22Rv1 prostate cancer cells. J Agric Food Chem 57:10636–10644 Lam LK, Sparnins VL, Wattenberg LW (1987) Effects of derivatives of kahweol and cafestol on the activity of glutathione S-transferase in mice. J Med Chem 30:1399–1403 Leung HY, Yung LH, Shi G et al (2009) The red wine polyphenol resveratrol reduces polycyclic aromatic hydrocarbon-induced DNA damage in MCF-10A cells. Br J Nutr 102:1462–1468 Lhoste EF, Gloux K, De Waziers I et al (2004) The activities of several detoxication enzymes are differentially induced by juices of garden cress, water cress and mustard in human HepG2 cells. Chem Biol Interact 150:211–219 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 Maliakal PP, Wanwimolruk S (2001) Effect of herbal teas on hepatic drug metabolizing enzymes in rats. J Pharm Pharmacol 53:1323–1329 Moon YJ, Wang X, Morris ME (2006) Dietary flavonoids: effects on xenobiotic and carcinogen metabolism. Toxicol In Vitro 20:187–210 Morimitsu Y, Hayashi K, Nakagawa Y et al (2000) Antiplatelet and anticancer isothiocyanates in Japanese domestic horseradish, wasabi. Biofactors 13:271–276

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Munday R, Munday CM (2004) Induction of phase II enzymes by aliphatic sulfides derived from garlic and onions: an overview. Methods Enzymol 382:449–456 Nakamura Y, Yoshida C, Murakami A et al (2004) Zerumbone, a tropical ginger sesquiterpene, activates phase II drug metabolizing enzymes. FEBS Lett 572:245–250 Offord EA, Macé K, Avanti O et al (1997) Mechanisms involved in the chemoprotective effects of rosemary extract studied in human liver and bronchial cells. Cancer Lett 114:275–281 Sasaki K, Wada K, Tanaka Y (2005) Thyme (Thymus vulgaris L.) leaves and its constituents increase the activities of xenobiotic-metabolizing enzymes in mouse liver. J Med Food 8:184–189 Singletary KW, Jung KJ, Giusti M (2007) Anthocyanin-rich grape extract blocks breast cell DNA damage. J Med Food 10:244–251 Tanaka T, Kawabata K, Kakumoto M et al (1997) Citrus auraptene inhibits chemically induced colonic aberrant crypt foci in male F344 rats. Carcinogenesis 18:2155–2161 Teyssier C, Amiot MJ, Mondy N et al (2001) Effect of onion consumption by rats on hepatic drugmetabolizing enzymes. Food Chem Toxicol 39:981–987 Zhang Y, Munday R, Jobson HE et al (2006) Induction of GST and NQO1 in cultured bladder cells and in the urinary bladders of rats by an extract of broccoli (Brassica oleracea italica) sprouts. J Agric Food Chem 54:9370–9376

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

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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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Teyssier C, Amiot MJ, Mondy N et al (2001) Effect of onion consumption by rats on hepatic drug-metabolizing enzymes. Food Chem Toxicol 39:981–987 Wu L, Noyan Ashraf MH, Facci M et al (2004) Dietary approach to attenuate oxidative stress, hypertension, and inflammation in the cardiovascular system. Proc Natl Acad Sci USA 101: 7094–7099 Zhang Y, Talalay P (1998) Mechanism of differential potencies of isothiocyanates as inducers of anticarcinogenic phase 2 enzymes. Cancer Res 58:4632–4639 Zhou C, Poulton EJ, Grün F et al (2007) The dietary isothiocyanate sulforaphane is an antagonist of the human steroid and xenobiotic nuclear receptor. Mol Pharmacol 71:220–229

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

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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

126

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.

Bibliography Beedham C (1985) Molybdenum hydroxylases as drug-metabolizing enzymes. Drug Metab Rev 16:119–156 Beedham C (1998) Molybdenum hydroxylases. In: Gorrod JW et al (eds) Metabolism of xenobiotics. Taylor and Francis, London, New York Benedetti MS (2001) Biotransformation of xenobiotics by amine oxidases. Fundam Clin Pharmacol 15:75–84 Benedetti MS, Dostert P (1994) Contribution of amine oxidases to the metabolism of xenobiotics. Drug Metab Rev 26:507–535

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Cashman JR (1995) Structural and catalytic properties of the mammalian flavin-containing monooxygenase. Chem Res Toxicol 8:166–181 Cashman JR (1998) Stereoselectivity in S- and N-oxygenation by the mammalian flavin-containing and cytochrome P-450 monooxygenases. Drug Metab Rev 30:675–707 Cashman JR (2000) Human flavin-containing monooxygenase: substrate specificity and role in drug metabolism. Curr Drug Metab 1:181–191 Cashman JR, Zhang J (2006) Human flavin-containing monooxygenases. Annu Rev Pharmacol Toxicol 46:65–100 Christofferson A, Wilkie J (2009) Mechanism of CB1954 reduction by Escherichia colinitroreductase. Biochem Soc Trans 37:413–418 Dawson JH (1988) Probing structure–function relations in heme-containing oxygenases and peroxidases. Science 240:433–439 Edmondson DE, Mattevi A, Binda C et al (2004) Structure and mechanism of monoamine oxidase. Curr Med Chem 11:1983–1993 Guengerich FP (1991) Reactions and significance of cytochrome P-450 enzymes. J Biol Chem 266:10019–10022 Guengerich FP (1996) The chemistry of cytochrome P450 reactions. In: Ioannides C (ed) Cytochrome P450: metabolic and toxicological aspects. CRC Press, Boca Raton Guengerich FP, Shimada T (1991) Oxidation of toxic and carcinogenic chemicals by human cytochrome P-450 enzymes. Chem Res Toxicol 4:391–407 Guengerich FP, Peterson LA, Böcker RH (1988) Cytochrome P-450-catalyzed hydroxylation and carboxylic acid ester cleavage of Hantzsch pyridine esters. J Biol Chem 263:8176–8183 Heikkila RE, Kindt MV, Sonsalla PK et al (1988) Importance of monoamine oxidase A in the bioactivation of neurotoxic analogs of 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine. Proc Natl Acad Sci U S A 85:6172–6176 Hodgson E, Levi PE (1992) The role of the flavin-containing monooxygenase (EC1.14.13.8) in the metabolism and mode of action of agricultural chemicals. Xenobiotica 22:1175–1183 Ioannides C (2002) Xenobiotic metabolism: an overview. In: Ioannides C (ed) Enzymes systems that metabolise drugs and other xenobiotics. Wiley, New York Ito K, Nakanishi M, Lee WC et al (2008) Expansion of substrate specificity and catalytic mechanism of azoreductase by X-ray crystallography and site-directed mutagenesis. J Biol Chem 283:13889–13896 Koder RL, Haynes CA, Rodgers ME et al (2002) Flavin thermodynamics explain the oxygen insensitivity of enteric nitroreductases. Biochemistry 41:14197–14205 Krenitsky TA, Neil SM, Elion GB, Hitchings GH (1972) A comparison of the specificities of xanthine oxidase and aldehyde oxidase. Arch Biochem Biophys 150:585–599 Kulkarni AP, Hodgson E (1984) The metabolism of insecticides: the role of monooxygenase enzymes. Annu Rev Pharmacol Toxicol 24:19–42 Meunier B, de Visser SP, Shaik S (2004) Mechanism of oxidation reactions catalyzed by cytochrome p450 enzymes. Chem Rev 104:3947–3980 de Montellano O, Paul R, de Montellano O (2005) Cytochrome P450: structure, mechanism, and biochemistry. Kluwer Academic/Plenum Publishers, New York O’Brien PJ (2000) Peroxidases. Chem Biol Interact 129:113–139 Parkinson A, Ogilvie BW (2008) Biotransformation of Xenobiotics. In: Klaassen CD (ed) Casarett and Doull’s toxicology: the basic science of poisons. McGrawHill, New York Rafii F, Cerniglia CE (1995) Reduction of azo dyes and nitroaromatic compounds by bacterial enzymes from the human intestinal tract. Environ Health Perspect 103:17–19 Rajagopalan KV (1980) Xanthine oxidase and aldehyde oxidase. In: Jakoby WB (ed) Enzymatic basis of detoxication. Academic, New York Satoh T, Hosokawa M (1998) The mammalian carboxylesterases: from molecules to functions. Annu Rev Pharmacol Toxicol 38:257–288 Satoh T, Hosokawa M (2006) Structure, function and regulation of carboxylesterases. Chem Biol Interact 162:195–211

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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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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.

158

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