Genetic Polymorphisms of Human N-Acetyltransferase, Cytochrome ...

Critical Reviews in Toxicology, 29(1):59–124 (1999)

Genetic Polymorphisms of Human N-Acetyltransferase, Cytochrome P450, Glutathione-S-Transferase, and Epoxide Hydrolase Enzymes: Relevance to Xenobiotic Metabolism and Toxicity Lars W. Wormhoudt, Jan N. M. Commandeur, and Nico P. E. Vermeulen* Leiden Amsterdam Center for Drug Research, Vrije Universiteit, Division of Molecular Toxicology, Department of Pharmacochemistry, De Boelelaan 1083, 1081 HV, Amsterdam, The Netherlands *

Corresponding author. Tel: (+31.20)-4447590, Fax: (+31.20)-4447610, e-mail: [email protected]

TABLE OF CONTENTS I. Introduction ............................................................................................................................... 61 II. N-acetyltransferases (NAT; E.C. 2.3.1.5) ................................................................................ 66 A. Introduction .......................................................................................................................... 66 B. Xenobiotic Metabolism by NAT Enzymes ........................................................................ 66 C. NAT Genomics ..................................................................................................................... 67 1. NAT1 Gene ...................................................................................................................... 67 2. NAT2 Gene ...................................................................................................................... 69 D. Effects of the NAT Genetic Polymorphisms on Xenobiotic Metabolism and Toxicity ..................................................................................................... 69 1. Studies Using Heterologously Expressed NAT 2 Mutants ........................................ 69 a. 341NAT2, 481NAT2, and 803NAT2 Mutations .............................................................. 69 b. 282NAT2, 590NAT2, and 857NAT2 Mutations .............................................................. 72 c. 191NAT2 Mutation ....................................................................................................... 72 d. Conclusions ................................................................................................................. 72 2. Activity of Enzymes Encoded by Mutated NAT1 Alleles .......................................... 73 3. Activity of Enzymes Encoded by Mutated NAT2 Alleles .......................................... 73 a. In Vitro (Ex Vivo) Studies ......................................................................................... 73 b. In Vivo Studies ............................................................................................................ 74 4. Clinical Response and Incidence of Adverse Effects.................................................. 75 Abbreviations: Ah, aryl hydrocarbon; ANA, antinuclear antibodies; ARNT, Ah receptor nuclear translocator; AUC, area under the plasma concentration versus time curve; CAT, chloramphenicol acetyltransferase; EM, extensive metabolizer; GSH, glutathione; GST, glutathione-S-transferase (E.C. 2.5.1.18); HPRT, hypoxanthine-guanine phosphoribosyl transferase; mEH, microsomal epoxide hydrolase (E.C. 3.3.2.3); MR, metabolic ratio; NAT, N-acetyltransferase (E.C. 2.3.1.5); P450, cytochrome P450 (E.C. 1.14.14.1); PAH, polycyclic aromatic hydrocarbon; PM, poor metabolizer; RFLP, restriction fragment length polymorphism; SCE, sister chromatid exchanges; SLE, systemic lupus erythematosis; SSRI, selective serotonin reuptake inhibitor; UM, ultrarapid metabolizer

Copyright© 1999, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without the consent of the publisher is prohibited.

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5. DNA Adducts .................................................................................................................. 75 6. Cancer Risk ..................................................................................................................... 75 E. Conclusions ........................................................................................................................... 76 III. Cytochromes P450 (P450; E.C. 1.14.14.1) .............................................................................. 76 A. Introduction .......................................................................................................................... 76 B. Cytochrome P450 1A1 (CYP1A1) ...................................................................................... 77 1. Xenobiotic Metabolism by CYP1A1 ............................................................................. 77 2. CYP1A1 Genomics ......................................................................................................... 77 3. Effects of the CYP1A1 Genetic Polymorphism .......................................................... 77 a. Expression ................................................................................................................... 77 b. Inducibility .................................................................................................................. 77 c. Enzymatic Activity ..................................................................................................... 78 d. DNA Adducts .............................................................................................................. 79 e. Mutations in Cancer-Related Genes ........................................................................ 79 C. Cytochrome P450 2A6 (CYP2A6) ...................................................................................... 79 1. Xenobiotic Metabolism by CYP2A6 ............................................................................. 79 2. CYP2A6 Genomics ......................................................................................................... 79 3. Effects of the CYP2A6 Genetic Polymorphism .......................................................... 79 D. Cytochrome P450 2C9 (CYP2C9) ...................................................................................... 80 1. Xenobiotic Metabolism by CYP2C9 ............................................................................. 80 2. CYP2C9 Genomics ......................................................................................................... 80 3. Effects of the CYP2C9 Genetic Polymorphism .......................................................... 80 a. CYP2C9*2 Allele ......................................................................................................... 80 b. CYP2C9*3 Allele ......................................................................................................... 81 E. Cytochrome P450 2C18 (CYP2C18) .................................................................................. 81 F. Cytochrome P450 2C19 (CYP2C19) .................................................................................. 81 1. Xenobiotic Metabolism by CYP2C19 ........................................................................... 81 2. CYP2C19 Genomics ....................................................................................................... 82 3. Effects of the CYP2C19 Genetic Polymorphism ........................................................ 82 G. Cytochrome P450 2D6 (CYP2D6) ...................................................................................... 84 1. Xenobiotic Metabolism by CYP2D6 ............................................................................. 84 2. CYP2D6 Genomics ......................................................................................................... 84 3. Effects of the CYP2D6 Genetic Polymorphism .......................................................... 86 a. Transcription, Protein Structure, and Enzyme Activity ....................................... 86 b. Metabolism and Pharmacological Activity of CYP2D6 Substrates .................................................................................................................... 89 H. Cytochrome P450 2E1 (CYP2E1) ...................................................................................... 90 1. Xenobiotic Metabolism by CYP2E1 ............................................................................. 90 2. CYP2E1 Genomics ......................................................................................................... 91 3. Effects of the CYP2E1 Genetic Polymorphism ........................................................... 93 a. Pst I/Rsa I Polymorphisms ........................................................................................ 93 b. Dra I Polymorphism .................................................................................................. 94 I. Conclusions ........................................................................................................................... 94 IV. Glutathione-S-Transferases (GST; E.C. 2.5.1.18) .................................................................. 95 A. Introduction .......................................................................................................................... 95 B. Xenobiotic Metabolism by GSTs ........................................................................................ 95 C. Glutathione-S-Transferase Mu (GSTM1-1) ...................................................................... 95 1. GSTM1-1 Genomics ....................................................................................................... 95 2. Effects of the GSTM1-1 Genetic Polymorphism ........................................................ 95


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a. DNA Adducts .............................................................................................................. 96 b. Protein Adducts .......................................................................................................... 96 c. Cytogenetic Damage and Mutations in Cancer Related Genes ............................ 96 d. Linkage of the GSTM*1a Allele to the GSTM3-3 Gene and Lung Cancer Risk .............................................................................................. 96 D. Glutathione-S-Transferase Theta (GSTT1-1).................................................................... 97 1. GSTT1-1 Genomics ........................................................................................................ 97 2. Effects of the GSTT1-1 Genetic Polymorphism ......................................................... 97 E. Glutathione-S-Transferase Pi (GSTP1-1) .......................................................................... 98 1. GSTP1-1 Genomics ........................................................................................................ 98 2. Effects of the GSTP1-1 Genetic Polymorphism .......................................................... 98 F. Conclusions ........................................................................................................................... 98 V. Microsomal Epoxide Hydrolase (mEH; E.C. 3.3.2.3) ........................................................... 99 A. Xenobiotic Metabolism by Microsomal Epoxide Hydrolase ........................................... 99 B. Microsomal Epoxide Hydrolase Genomics ....................................................................... 99 C. Effects of Genetic Polymorphisms of Microsomal Epoxide Hydrolase ....................... 100 D. Conclusions ......................................................................................................................... 100 VI. Combinations of Genetic Polymorphisms of Biotransformation Enzymes ...................... 100 VII. Final Conclusions and Future Perspectives ......................................................................... 102 A. Individual Sensitivity to Genotoxic Effects and Cancer Risk ...................................... 102 B. Drug Development ............................................................................................................. 102

ABSTRACT: In this review, an overview is presented of the current knowledge of genetic polymorphisms of four of the most important enzyme families involved in the metabolism of xenobiotics, that is, the N-acetyltransferase (NAT), cytochrome P450 (P450), glutathione-S-transferase (GST), and microsomal epoxide hydrolase (mEH) enzymes. The emphasis is on two main topics, the molecular genetics of the polymorphisms and the consequences for xenobiotic metabolism and toxicity. Studies are described in which wild-type and mutant alleles of biotransformation enzymes have been expressed in heterologous systems to study the molecular genetics and the metabolism and pharmacological or toxicological effects of xenobiotics. Furthermore, studies are described that have investigated the effects of genetic polymorphisms of biotransformation enzymes on the metabolism of drugs in humans and on the metabolism of genotoxic compounds in vivo as well. The effects of the polymorphisms are highly dependent on the enzyme systems involved and the compounds being metabolized. Several polymorphisms are described that also clearly influence the metabolism and effects of drugs and toxic compounds, in vivo in humans. Future perspectives in studies on genetic polymorphisms of biotransformation enzymes are also discussed. It is concluded that genetic polymorphisms of biotransformation enzymes are in a number of cases a major factor involved in the interindividual variability in xenobiotic metabolism and toxicity. This may lead to interindividual variability in efficacy of drugs and disease susceptibility. KEY WORDS: genetic polymorphisms, biotransformation, enzymes, metabolism, toxicology, inter-individual variability.

I. INTRODUCTION Biotransformation enzymes play an important role in the metabolism of xenobiotics that thus may be either bioactivated or bioinactivated. A wide inter-individual variability in the levels and activities of biotransformation enzymes ex-

ists among humans, as has been shown for a number of enzymes, both ex vivo and in vivo.1–6 Examples of such interindividual variabilities include the metabolism of xenobiotics to which people have been exposed therapeutically, occupationally, or through the diet. For example, clear interindividual variations were observed in the


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urinary excretion of styrene metabolites in a group of 20 male workers in the polyester industry, with styrene-related mercapturic acids being only detectable in 1 out of the 20 urine samples.7 In a study on ethylene oxide-derived DNA damage in peripheral mononuclear blood cells of 97 male and female workers occupationally exposed to this compound, a “sensitive” subgroup of 67% was observed with 5-fold more DNA single strand breaks when compared with a “non-sensitive” group.8 Clear interindividual variability was also observed in the excretion of 1-hydroxypyreneglucuronide in urine and in the level of polycyclic aromatic hydrocarbon (PAH)-DNA adducts in white blood cells after consumption of charcoalbroiled beef.9 An increase in the urinary excretion of 1-hydroxypyrene-glucuronide was measured in all of 10 individuals with an 8-fold variability,

while an increase in DNA adducts was only measured in 4 out of these 10 individuals. Besides all kinds of environmental factors, genetic polymorphisms of biotransformation enzymes might be one of the major causes of these kinds of interindividual variabilities. With the growing interest in the genetic basis, epidemiology, and effects of genetic polymorphisms on metabolism of pharmaceuticals, this line of research has been called ‘pharmacogenetics’.10,11 The genetically determined interindividual variability in drug and xenobiotic metabolism is thus one important factor that determines the variability of xenobiotic-related toxicity,1,12–14 adverse drug reactions,15 as well as the efficacy of drugs16–21 (Figure 1A). Because biotransformation enzymes might also be involved in the bioactivation or bioinactivation of genotoxic

FIGURE 1. Schematic view of the effects of genetic polymorphisms of biotransformation enzymes on metabolism of (pro)drugs and (pro)toxins (A) or (pro)genotoxic compounds (B). Depending on whether the metabolic reaction involved is bioactivating or bioinactivating, genetic polymorphisms might increase or reduce drug efficacy or (geno)toxic effects. Also shown is the often investigated epidemiological relationship with cancer risk. This review focusses on two main topics: the molecular mechanisms of genetic polymorphisms of biotransformation enzymes and their effects on xenobiotic metabolism and toxicity. Copyright© 1999, CRC Press LLC — Files may be downloaded for personal use only. Reproduction of this material without the consent of the publisher is prohibited.

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compounds, genetic polymorphisms of these enzymes are thought to be one of the susceptibility factors in cancer risk (Figure 1B).22–28 Therefore, mutated genes encoding biotransformation enzymes are called susceptibility genes, which should be distinguished from (proto)oncogenes.29 One of the obvious differences between these two classes of genes is that susceptibility genes require exposure to certain compounds in order to have an effect on cancer risk, whereas (proto) oncogenes do not. A large number of epidemiological studies has been performed in which a relation between genetic polymorphisms of biotransformation enzymes and cancer risk (or risk of other diseases) has been investigated (for reviews see Refs. 30– 40). However, these epidemiological studies have sometimes been inconclusive or even contradictory, for which there may be a number of explanations. First, the occurrence of genetic polymorphisms has sometimes been measured in patient and matched control groups without measurement of the exposure to suspected genotoxic compounds. It has already been mentioned above that an essential feature of susceptibility genes is that there should be an exposure to a compound that is metabolized by the particular enzyme being studied. Furthermore, because genetic polymorphisms of biotransformation enzymes might be related to cancer risk only at specific levels of exposure,41,42 such studies might lead to inconsistent results if individual exposure differs. Second, environmental factors that might influence cancer risk as well (e.g., exposure to inducers or inhibitors of biotransformation enzymes) are usually not taken into account because this would require continuous monitoring. Third, studies do not always make a distinction between subtypes of cancer, while a correlation between cancer risk and genetic polymorphisms of biotransformation enzymes is sometimes restricted to specific subtypes of cancer.43 Fourth, because large interracial differences exist in the frequency of genetic polymorphisms,44–48 studies may be hampered when individuals from different ethnic origin participate in the same study. Fifth, because the ultimate mutagenic or carcinogenic effect(s) of suspected genotoxic compounds will, in a number of cases, be mediated by more than one biotransformation enzyme, it might be necessary to consider the consequences of combi-

nations of genetic polymorphisms of multiple biotransformation enzymes.41,43,49 However, for statistical reasons, often only a limited number of genotypes is considered. Finally, it is known that a substantial degree of variability exists in the capacity to repair DNA damage among humans,50 which may also influence the individual cancer risk. Recently, there has been a growing interest in the role of genetic polymorphisms of biotransformation enzymes in interindividual variability of xenobiotic metabolism, drug toxicity, drug efficacy, and especially in the field of cancer risk assessment. Instead of epidemiological studies in which the occurrence of a genetic polymorphism is related to an end point like cancer (Figure 1B), interest becomes more focussed on the molecular genetic mechanisms of genetic polymorphisms of biotransformation enzymes and the effects on the metabolism and toxicity of drugs and xenobiotics. A number of molecular genetic mechanisms causing genetic polymorphisms of biotransformation enzymes are known to exist (Figure 2). First, the complete gene encoding a biotransformation enzyme might be absent. Second, the expression of the gene may be lost or impaired due to mutations in regulatory parts of the gene. Third, due to mutations at intron-exon boundaries, the pre-mRNA might be spliced incorrectly. Fourth, noncritical amino acids in the protein may have been mutated, causing a change in activity of the enzyme. Fifth, critical amino acids in the protein may have been mutated, causing an inactive enzyme. A number of approaches are possible for investigating the effects of genetic polymorphisms of biotransformation enzymes on the metabolism and toxicity of drugs and xenobiotics (Table 1). Metabolism of xenobiotics can be measured in vitro/ex vivo, using, for example, cell lines or human tissue samples that express the wild-type or mutated genes encoding biotransformation enzymes. This approach is particularly useful with enzymes for which only toxic or carcinogenic substrates are known. Alternatively, metabolism can be measured in vivo using a panel of volunteers that have been either genotyped or phenotyped with respect to the genetic polymorphism of interest, in which case the compound of interest itself can be used or, alternatively, an


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FIGURE 2. Schematic representation of different molecular mechanisms of polymorphisms of biotransformation enzymes. In this scheme, introns are represented by open boxes, exons by filled boxes, and mutations by small shaded boxes. The regulatory region of the gene is represented by a line beside the gene. Several molecular mechanisms of genetic polymorphism are distinguished here. The gene encoding the biotransformation enzyme may be completely absent (1), causing a complete absence of protein. The regulatory part of the gene may be mutated, causing the formation of less (pre)mRNA and less protein (2). A mutation may be present at an intron-exon boundary (3), which causes an incorrect splicing of the pre-mRNA and the formation of an incomplete or inactive protein. A mutation may be present somewhere in an exon causing an amino acid change in the protein that may be one non-critical for enzymatic activity, but nevertheless causes the production of a protein with an altered enzymatic activity (4) or one that is critical for enzymatic activity, causing the production of an inactive protein (5).

isoenzyme-specific substrate for the isoenzyme of interest. In human studies, metabolites are often measured in urine because this is noninvasive. In the case of model compounds, the metabolic ratio (MR) (i.e., the ratio between metabolite and parent compound) is often measured. Usually, for a given substrate, a certain value of the MR, called the antimode, distinguishes so-called slow or poor metabolizers (PM) and fast or extensive metabolizers (EM). Useful metabolites in this case are mercapturic acids, the metabolic end-products of glutathione-S-transferase (GST) catalyzed metabolism that are excreted in the urine.51,52 Next to measuring the metabolism of xenobiotics, it is

also possible to measure a pharmacological or toxicological effect, either in vitro/ex vivo or in vivo in clinical studies. In the case of genotoxic xenobiotics, a number of parameters can be used. In ex vivo measurements using human tissue samples (often lymphocytes) DNA adducts and mutations might serve as dosimeters for the exposure to genotoxic compounds52–55 but might also reflect interindividual variability in metabolism. This issue was addressed addressed in a study where adducts of ethylene oxide to hemoglobin were shown to reflect an individual’s ethylene oxide-metabolizing capacity.56,57 When DNA adducts or mutations are measured ex vivo in blood


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TABLE 1 Methods for the Measurement of the Effects of Genetic Polymorphisms of Biotransformation Enzymes on Xenobiotic Metabolism and Toxicity Metabolism In vitro

Ex vivo In vivo

Toxicity/pharmacology In vitro/ex vivo

In vivo Genotoxicity Ex vivo

Ex vivo/in vitro

Cell lines expressing wild-type and mutated enzymes Assess metabolism of compounds of interest Assess metabolism of isoenzyme specific substrates Use human tissue samples to measure the metabolism of compounds of interest or of isoenzyme specific substrates Genotyped or phenotyped panel of human volunteers Measure, for example, urinary metabolites of the compound of interest (if it is nontoxic) or alternatively of isoenzymespecific substrates approved for human in vivo studies

Measurement of a toxicological or pharmacological effect (subcellular fractions or human tissue samples) Measurement of a toxicological or pharmacological effect in clinical studies

DNA adducts or mutations in, for example, white blood cells or exfoliated bladder cells from genotyped or phenotyped human volunteers Parameters reflecting genetic damage using human tissue samples or cell lines Structural abberations in chromosomes Micronuclei Aneuploidy Sister chromatid exchanges

cells, however, the question is whether these reflect the amount of DNA adducts or mutations in target organs where tumors appear.58,59 Instead of blood cells, exfoliated urothelial cells might also be used for the measurement of carcinogen-DNA adducts in the bladder.60 Some DNA adducts are excreted in urine after metabolism, and therefore these adducts might be measured in relation to genetic polymorphisms of biotransformation enzymes.61 In this case, the question again is whether these excreted and metabolized DNA adducts reflect the amount of DNA adducts in target organs or reflect the DNA repair in vivo. A number of other parameters have been developed in order to measure genotoxicity both in vitro and in (ex) vivo, for example, structural abberations in chromosomes, micronuclei, aneuploidy, and sister chromatid exchanges (SCE).62 Combining information obtained from studies into the mechanistic effects of genetic polymorphisms of biotransformation enzymes with the power of epidemiological studies (large num-

bers of study objects) improves the ultimate understanding of how genetic polymorphisms of biotransformation enzymes affect cancer risk.63 If it is possible to identify highly susceptible individuals on the basis of genetic polymorphisms of biotransformation enzymes, this might ultimately lead to a larger degree of prevention of the number of cancer cases, for example, by closely monitoring the exposure of highly susceptible individuals to potentially carcinogenic compounds.64 The aim of this review is to give an up-to-date overview of the current knowledge of genetic polymorphisms of four of the most important biotransformation enzymes, viz. the N-acetyltransferase (NAT), cytochrome P450 (P450), glutathione-S-Transferase (GST), and microsomal epoxide hydrolase (mEH) enzymes. Both the molecular genetics of these polymorphisms and the consequences of genetic polymorphisms on metabolism and toxicity are the subjects of this review. Other recent reviews in this field, which discuss some parts also described in this review, are following.65–67


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II. N-ACETYLTRANSFERASES (NAT; E.C. 2.3.1.5)* A. Introduction The NAT polymorphism was the first genetic polymorphism in drug metabolism already recognized more than 40 years ago [for reviews see Refs. 70,71]. The observed inter-individual variation in isoniazide metabolism provided an important indication for a polymorphism in this class of biotransformation enzymes.72 The N-acetylation of isoniazide appeared to be reduced in a significant number of individuals, and the human population could be divided in two groups, so-called slow and fast acetylators.73 Based initially on family studies, the polymorphism was found to be of a genetic origin. After isolation and characterization of enzymes with acetyltransferase activity, two different NAT proteins, NAT 1 and NAT 2, were found in humans.74,75 Both NAT 1 and NAT 2 enzymes are expressed in liver,75 while only NAT 1 is expressed in mononuclear leukocytes.76 It has also become clear that these two proteins are encoded by two different genes, NAT1 and NAT2 both on human chromosome 8.77,78 Mutations have been described in both of these genes. Mutations responsible for the NAT2 genetic polymorphism have been described first, while mutations responsible for the NAT1 genetic polymorphism were discovered only recently.

B. Xenobiotic Metabolism by NAT Enzymes The NAT enzymes catalyze the acetylation of a wide variety of amines, among which are both arylamines and heterocyclic aromatic amines.79 Historically, a distinction was made between two classes of NAT substrates, those that were affected by the NAT genetic polymorphism (“polymorphic substrates”, for example, sulfametazine and pro-cainamide) and those that, apparently, were not (“monomorphic substrates”, for example, p-aminobenzoic acid and p-aminosalicylic *

acid).75,80,81 The monomorphic substrates were found to be metabolized by NAT 1, while the polymorphic substrates were found to be metabolized by NAT 2, which was at that time the only NAT enzyme for which a genetic polymorphism was known. The distinction between monomorphic and polymorphic substrates recently has become less clear, however, because the NAT1 gene was also found to be polymorphic, and the metabolism of some monomorphic substrates has also been shown to be polymorphically distributed.82,83 Jenne and Orser already detected some individual variability in the N-acetylation of p-aminosalicylic acid (a monomorphic substrate) some 30 years ago,84 while also more recently, inter-individual variability in the activity toward p-aminosalicylic acid and p-aminobenzoic acid was found in humans.75,76,82,85–89 As described in this review, genetic polymorphisms are involved in causing these interindividual variabilities. Despite the fact that NAT 1 and NAT 2 have different substrate selectivities,90 some substrates (e.g., 2-aminofluorene) are metabolized by both NAT 1 and NAT 2 enzymes,80 and the NAT 1 protein has been shown to be involved in the metabolism of compounds that are primarily metabolized by NAT 2 in slow acetylators.91 The NAT enzymes are able to perform a number of different reactions, among which are both bioactivation and bioinactivation reactions (Figure 3). N-Acetylation of arylamine carcinogens, a bioinactivation reaction, and O-acetylation, a bioactivation reaction, are catalyzed by both NAT 1 and NAT 2 enzymes, while N,O-acetyltransferase is catalyzed predominantly by NAT 1 enzymes, which has been shown in a study with recombinant human enzymes.93 The relative contribution of NAT 1 and NAT 2 to the N- and O-acetylation of different substrates shows, however, a large variation.93,94 Based on experiments with heterologously expressed NAT 1 and NAT 2 enzymes in COS-1 cells, it has been suggested that NAT 2 is the major isoenzyme involved in the N-acetylation of heterocyclic amines as well as in the O-acetylation of the N-hydroxy metabolites of heterocyclic amine procarcinogens.

Nomenclature of NAT proteins, genes, and allelic variants is according to the guidelines for human gene nomenclature68 and the NAT nomenclature.69 Individual mutations are indicated by the nucleotide number (in superscript) followed by the gene symbol (e.g., 803NAT2). Individual (changes) in amino acids are indicated by the amino acid position (in superscript) followed by the amino acid (e.g.,64 arginine).


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FIGURE 3. Schematic view of the role of NAT enzymes in the metabolism of aromatic amines. This scheme shows the contributions of NAT enzymes to the metabolism of aromatic amines. N-acetylation might be a detoxification reaction in a number of cases; however, after N-hydroxylation of aromatic amines (e.g., by P450 enymes), NAT enzymes can bioactivate these intermediates by either O-acetylation or intramolecular N,O-acetyltransfer, leading to the formation of nitrenium-ions, which might react with DNA or alternatively be detoxified by, for example, GST enzymes. Importantly, it is shown that a number of other biotransformation enzymes are involved in the metabolism of aromatic amines as well. (Based on a figure from Ref. 92.)

However, NAT 1 was found to have a contribution to the metabolism of some substrates as well.95 It is important to realize that other biotransformation enzymes contribute to the metabolism of NAT substrates and their metabolites as well. For example, the N-hydroxylation by cytochrome P450 (P450) isoenzymes (toxification) is a major competitive reaction to the N-acetylation by NAT enzymes (detoxification) (Figure 3). Furthermore, NAT enzymes can O-acetylate the P450 derived N-hydroxy metabolites and thereby play a role in the bioactivation of these compounds (Figure 3).92,96,97 Therefore, the NAT enzymes play a

dual role in the metabolism of arylamines and heterocyclic aromatic amines with respect to bioactivation and bioinactivation.

C. NAT Genomics

1. NAT1 Gene Several mutations in the NAT1 gene have been described (Figure 4). One wild-type allele, NAT1*4, and five mutant alleles, NAT1*3, NAT1*5, NAT1*10, NAT1*11, and NAT1*17, are


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FIGURE 4. Mutations currently known to exist in the human NAT1 gene. The nucleotide changes in the mutant NAT1 alleles have been indicated. Nucleotide substitutions have been indicated by the wild-type and mutant nucleotides, separated by the position in the gene. ∆976 and ∆1105 in the NAT1*5 allele each represent one deleted nucleotide. ∆9 in the NAT1*11 and NAT1*17 alleles represents a deletion of nine nucleotides. The preferred nomenclature is indicated in boldface.

currently known. The NAT1*3 differs from the wild-type NAT1*4 at only one nucleotide in the 3′-nontranscribed region.77 NAT1*5 contains six nucleotide changes (in the coding region) and two deletions (in the 3′-nontranscribed region) when compared with NAT1*4.78,98 The NAT1*10 allele contains two mutations in the 3′-nontranscribed region, one of which changes the polyadenylation

signal at the 3′-nontranscribed region of the NAT1 gene.83 NAT1*11 contains two nucleotide substitutions in the 5′-nontranscribed region, two nucleotide substitutions in the coding region, and one nucleotide substitution and a deletion of 9 basepairs in the 3′-nontranscribed region.83 The recently described NAT1*17 allele is similar to the NAT1*11 allele except for a nucleotide substitu-


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tion at position 445 of the NAT1 gene causing an amino acid change of 149valine to isoleucine.99

2. NAT2 Gene The human NAT2 gene is an intronless gene containing 870 nucleotides encoding a protein of 290 amino acids.77,78,100 In addition, an exon is present in the 5′-flanking region that does not encode for amino acids.78 Nine point mutations have been identified in the human NAT2 gene, either occurring alone or in combination to yield 14 mutant alleles (Figure 5). 282NAT2 and 481NAT2 C → T mutations are silent mutations and do not change 94tyrosine and 161leucine in the NAT 2 protein, respectively.191 NAT2 and 590NAT2 G → A mutations change the charged 64arginine and 197arginine to the polar glutamine residues. A 341NAT2 T → C mutation changes 114isoleucine to a more polar threonine that has been shown to change the hydropathy profile of the protein.102 The 803NAT2 A → G mutation changes 268lysine to an arginine, which can be regarded as a conservative amino acid change. Finally, the 857NAT2 G → A mutation changes a 286glycine to a more polar glutamic acid. More than 95% of the NAT phenotypes can be predicted by genotyping.113 Methods are available that can detect all of the currently known NAT2 mutations.106 However, there is still some discrepancy between genotyping and phenotyping.114 Because some individuals are phenotyped as slow acetylators, but none of the known mutant alleles is found after genotyping, this suggests the presence of as yet unidentified mutations. Indeed, very recently a new mutation (759NAT2 C → T) was found to be present in a slow acetylator.115 Table 2 shows the interethnic variability in the percentage of slow acetylators. In addition to the percentage of slow acetylators, interethnic variability exists in the occurrence of the mutant alleles among genotyped slow acetylators. In a Caucasian population, the wild-type allele, NAT2*4, was present at an allelic frequency of 19.9% while the most common mutated alleles were NAT2*5B (44.8%) and NAT2*6A (30.2%).104 NAT2*5A, NAT2*5C, and NAT2*7B occurred at allelic frequencies of 1.0, 3.1 and 1.0%, respectively. In contrast, the NAT2*5B allele was al-

most absent in a Japanese population, while the NAT2*6A and NAT2*7B alleles were the most frequently occurring mutated alleles.107,108 The interethnic variability in the occurrence of the NAT2*5B allele might partly explain the observed difference in the frequency of the slow acetylator phenotype between Caucasian and Japanese populations.131 In a large number of studies, individual mutations are measured instead of all mutations that characterize one single allele. The occurrence of the 191NAT2 G → A mutation is low in a variety of populations,109,132 suggesting a low incidence of NAT2*14A and NAT2*14B alleles. The 481NAT2 C → T mutation, however, occurs much more in Caucasian populations, reflecting the interethnic difference in the occurrence of the NAT2*5B allele. No large interethnic variability is observed in the occurrence of the 590NAT2 G → A mutation, apart from a very low occurrence in two Amerindian populations of Panama and Columbia.132 The 857NAT2 G → A mutation occurs less in Caucasian populations and seems to be a mutation characteristic for Orientals, which is also frequent in the two Amerindian populations.109,132

D. Effects of the NAT Genetic Polymorphisms on Xenobiotic Metabolism and Toxicity

1. Studies Using Heterologously Expressed NAT 2 Mutants In order to investigate the effects of the NAT 2 mutations on the genes and proteins and the consequences of the NAT 2 genetic polymorphism on the metabolism of xenobiotics, a number of mutant NAT 2 proteins and protein chimeras have been expressed in both prokaryotic and eukaryotic expression systems and evaluated for their effects on acetylation capacity and protein stability.

a. 341NAT2, Mutations

481NAT2

, and

803NAT2

341

NAT2, 481NAT2, and 803NAT2 mutations are present in the mutant alleles NAT2*5A, NAT2*5B,


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FIGURE 5. Mutations currently known to exist in the human NAT2 gene.1 This list provides the designations used in literature to indicate the mutant alleles. The preferred nomenclature is indicated in boldface.2 This list provides a number of references that describe the mutant alleles but is not intended to list all references in which the mutant alleles are described.3 The 857NAT2 mutation has also been described in Ref. 98. It was later found to contain the 282NAT2 mutation as well and to be similar to the NAT2*7B allele.107

NAT2*5C, NAT2*12A, and NAT2*12B (Figure 5). Expression of a protein with the 341NAT2 and 481NAT2 mutations in COS-1 cells revealed that

the individual mutations did not lead to a lower acetylation activity toward sulfamethazine when compared with the wild-type protein.101 However, when both mutations were present, a 7-fold lower


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TABLE 2 Percentages of Slow NAT, CYP2C19 PM, CYP2D6 PM, GSTM1-1 Null, and GSTT1-1 Null Genotypes/Phenotypes in Several Populations Population European White Americans Black Americans African-Americans Japanese Chinese Korean

Slow NAT

CYP2C19 PM

59116 55112 — 41112 8113 20114 —

3.5117 2.6121 — — 22.5126 17.4126 12.6128

activity was observed compared with the wildtype gene. Furthermore, the amount of NAT protein was only reduced when both mutations were present, although the expression of mRNA was similar when compared with the wild-type allele. However, by expression of different protein chimeras containing the 341NAT2, 481NAT2, and 803 NAT2 mutations in COS cells and using p-phenetidine as a substrate, it has been found that the 341NAT2 mutation alone is responsible for the slow acetylator phenotype due to these alleles.103 NAT2 genes containing the individual 341NAT2, 481NAT2, and 803NAT2 mutations, as well as the combinations, 341/481NAT2; 341/803NAT2, 481/803NAT2, and 341/481/803NAT2, have also been expressed in Escherichia coli.133 The expressed amount of protein of these mutant genes was not different when compared with the wild type. The Km for the substrate 2-aminofluorene as well as for the cofactor acetylCOA were found not to be different from the wild-type protein for any of these mutations. However, the V max with 2-aminofluorene and 4-aminophenol as substrates was reduced 22-fold for 341NAT2, 20-fold for 341/481NAT2, 9-fold for 341/803NAT2, and 31-fold for 341/481/803NAT2. Therefore, it seems that the 341NAT2 mutation has the largest contribution to the slow acetylation activity of these proteins. In another study in which 341/803NAT2 has been expressed in Escherichia coli, it was found that both the Km and the Vmax of this mutant protein for the substrates 2-aminofluorene, 4-aminobiphenyl, 3,2′dimethyl-4-aminobiphenyl, and 2-aminodipyrido[1,2-a:3′,2′d]imidazole were significantly lower when compared with the wild-type protein.105 The mutant protein also appeared to be less stable when compared with the wild type, having a more than 2-fold higher inactivation

CYP2D6 PM 7.4117 7.7122 1.9122 — 0.5126 0.95128 —

GSTM1-1 null GSTT1-1 null 48.2118,119 52123 — 27123 48.6127 — —

10120 14.7124 24.1124 21.8125 — 61.2125,129 60.2125

rate. In a recent expression study in Escherichia coli, the 341NAT2 mutation resulted in a significant reduction in N-, O-, and N,O-acetyltransferase activity toward 2-aminofluorene (50-fold), n-hydroxy-2-amino-fluorene (7-fold), and n-hydroxy4-aminobiphenyl (35-fold) and n-hydroxy-2acetylaminofluorene (1.5-fold), respectively.134 The activity of NAT2 12A and NAT2 12B toward these substrates was not different when compared with the wild-type enzyme, suggesting that the 803NAT2 mutation has no significant effect on the NAT 2 activity.134 Similarly, the 481NAT2 mutation alone had no effect on these enzymatic activities in recombinant Escherichia coli. 134 NAT2*5A, NAT2*5B, and NAT2*5C alleles had a reduced ability to O-acetylate N-hydroxy-2aminofluorene of 32, 11, and 16% of the wildtype protein when expressed in Escherichia coli, respectively.94 No such effects were found with n-hydroxy-2-amino-3-methyl-imidazo[4,5f]quinoline, n-hydroxy-2-amino-3,4-dimethylimidazo[4,5-f]quinoxaline, and n-hydroxy-2amino-1-methyl-6-phenylimidazo[4,5-b] pyridine, however. The low acetylation activity of the proteins encoded by the NAT2*5A, NAT2*5B, and NAT2*5C alleles in vivo thus might be explained by the presence of the 341NAT2 mutation, which also causes a different hydrophobicity pattern of the proteins102 and is suggested to cause a decrease in translatability of the mRNA and/or an unstabile protein as well.103 It has to be noted, however, that the extent to which the enzymatic activity is reduced is largely dependent on the substrate used. This would point to a change in the structure of the active site of the enzyme as a cause of these reductions in enzymatic activity.


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b. 282NAT2, Mutations

590NAT2

, and

857NAT2

282

NAT2, 590NAT2, and 857NAT2 mutations are present in the mutant alleles NAT2*6A, NAT2*6B, NAT2*7A, NAT2*7B, NAT2*12B, NAT2*13, and NAT2*14B (Figure 5). After expression of the NAT2*6A allele (containing both 590NAT2 and 282NAT2) and the individual mutations, it appeared that the 590NAT2 mutation was sufficient to cause the slow acetylator genotype and a less stable protein. The silent 282NAT2 mutation had no effect on the activity of the protein toward sulfamethazine101 or on the activity of mutants expressed in CHO cells.107 These observations are in line with expression experiments in Escherichia coli, in which the 590NAT2 mutant had a 4-fold lower acetylation activity toward 2-aminofluorene, while the activity of the 282NAT2 mutant was not different from the wild-type protein.133 The 590NAT2 mutant also had a 3-fold higher inactivation rate when compared with the wild-type protein, and therefore appears to be less stable. Recently, both NAT2 6A and NAT2 6B enzymes were found to have reduced N-, O-, and N,O-acetyltransferase activity toward 2-aminofluorene, n-hydroxy-2aminofluorene and n-hydroxy-4-aminobiphenyl and n-hydroxy-2-acetylamino-fluorene of 76, 81, and 58% when compared with the wild-type enzyme, respectively.134 Because these activities were not different for the NAT2 13 enzyme when compared with the wild-type enzyme, it is concluded that the 282NAT2 mutation has no effect on these activities in recombinant Escherichia coli.134 The 857NAT2 mutation was not found to have any effect on the acetylation activity toward 2-aminofluorene, but was found to produce an 11-fold less stable enzyme when compared with the wildtype when expressed in Escherichia coli.133 In another expression study in Escherichia coli, this mutation was found to lower the Km for sulfametazine (10-fold) and dapsone (5-fold), while no change was observed in the Km values for 2-aminofluorene, p-anisidine, isoniazide, and procainamide, which would imply a changed structure of the active site.135 A lower acetylation activity of the 857NAT2 mutant toward 2-aminofluorene, procainamide, sulfametazine, p-phenetidine, and 5-methoxytryptamine was observed after expression of this mutant in CHO cells.98

Recently, both NAT2 7A and NAT2 7B enzymes were found to have reduced N-, O-, and N,Oacetyltransferase activity toward 2-aminofluorene, n-hydroxy-2-aminofluorene, and n-hydroxy-4aminobiphenyl and n-hydroxy-2-acetylaminofluorene of 30, 61, and 55% when compared with the wild-type enzyme, respectively.134 The NAT2 6A and NAT2 7B enzymes had a reduced ability to O-acetylate n-hydroxy-2aminofluorene of 16 and 37% of the wild-type protein when expressed in Escherichia coli, respectively.94 No such effects were found with nhydroxy-2-amino-3-methylimidazo[4,5-f] quinoline, n-hydroxy-2-amino-3,4-dimethylimidazo[4,5-f]quinoxaline, and N-hydroxy-2amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, however. Based on the experiments described above, the 590NAT2 mutation is probably responsible for the slow acetylation activity of the enzyme encoded by the NAT2*6A allele. Because the 590NAT2 mutation is also present in NAT2*6B, this allele is also expected to encode an enzyme with a lower activity. However, it has been reported that this allele is not associated with the slow acetylator phenotype.92 The 857NAT2 mutation might cause the slow acetylation phenotype of the enzyme encoded by allele NAT2*7B. Because the 857NAT2 mutation is also present in NAT2*7A, this allele might also be expected to be associated with the slow acetylation phenotype.

c.

191NAT2

Mutation

The 191NAT2 mutation was found recently in the mutant NAT2*14A and NAT2*14B alleles, which are also associated with the slow acetylation phenotype.105,112 After expression of the 191NAT2 mutant in Escherichia coli, the activity toward different substrates was 5- to 6-fold lower when compared with the wild-type protein, while its stability was found to be 14- to 20-fold lower.105,133 Recently, NAT2 14A and NAT2 14B enzymes were found to have reduced N-, O-, and N,O-acetyltransferase activity toward 2-aminofluorene, n-hydroxy-2-aminofluorene, and n-hydroxy-4-aminobiphenyl and n-hydroxy-2-acetylaminofluorene of 77, 92, and 64% when compared with the wild-type enzyme, respectively.134 The NAT2 14A enzyme had a reduced ability to


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O-acetylate n-hydroxy-2-aminofluorene of 53% of the wild-type protein when expressed in Escherichia coli, respectively.94 No such effects were found with n-hydroxy-2-amino-3-methyl-imidazo [4,5-f]quinoline, n-hydroxy-2-amino-3,4-dimethyl-imidazo[4,5-f]quinoxaline, and N-hydroxy2-amino-1-methyl-6-phenylimidazo-[4,5-b] pyridine, however. Similar to the 590NAT2 mutation, the 191NAT2 mutation causes an amino acid change from 64arginine to glutamine. However, the 191NAT2 mutation is very close to the codon that codes for 68cysteine, an important residue in the catalytic mechanism of NAT 2.136 Therefore, the amino acid change 64arginine to glutamine might affect the catalytic mechanism of the protein as well.

d. Conclusions Based on the heterologous expression studies described above, four mutations (341NAT2, 590NAT2, 857NAT2, and 191NAT2 ) seem to be most important with respect to the slow acetylation activity of proteins encoded by a number of the mutant alleles. The presence of these mutations might explain why the NAT2*5A, NAT2*5B, NAT2*5C, NAT2*6A, NAT2*7B, NAT2*14A, and NAT2*14B alleles encode enzymes with lower acetylation activities. Based on the presence of the 590NAT2 mutation and the 857NAT2 mutation in alleles NAT2*6B and NAT2*7A, respectively, these alleles are expected to encode proteins with lower acetylation activities as well. The NAT2*12A, NAT2*12B, and NAT2*13 alleles contain one or both of the 282NAT2 and 803NAT2 mutations that have shown not to have any effect in the heterologous expression studies described above. Therefore, these alleles are expected to encode fully active NAT proteins, which has been shown to be true for NAT2*12A.111 It has to be mentioned that the NAT2*13 allele has been found recently to encode an enzyme with reduced activity, despite the fact that only one mutation has up to now been identified that does not change an amino acid.116

2. Activity of Enzymes Encoded by Mutated NAT1 Alleles The NAT1 10 enzyme has been shown to have a 2-fold higher activity toward p-aminoben-

zoic acid in bladder and colon tissue when compared with the wild-type, NAT1 4, enzyme.137 Furthermore, a higher average activity of the NAT1 10 enzyme toward benzidine was observed when compared with the wild-type enzyme.138 Because the NAT1*10 allele contains an altered polyadenylation signal, it has been hypothesized that NAT 1 mRNA derived from the NAT1*10 allele might be more stable than the mRNA derived from the NAT1*4 (wild-type) allele.137 The 445NAT1 mutation, present in the NAT1*17 allele, resulted in the production of a protein with up to 2-fold higher activity than the wild-type enzyme in a prokaryotic expression system.99

3. Activity of Enzymes Encoded by Mutated NAT2 Alleles a. In Vitro (Ex Vivo) Studies In human liver samples with the NAT2*5A and/or NAT2*6A alleles, the acetylation activity toward sulfametazine was reduced, as was the amount of immunochemically detectable NAT protein.101 It is important to note that the amount of immunodetectable protein might have been underestimated if the antibodies used have different affinities for the mutated proteins. The amount of mRNA was not different for mutant when compared with the wild-type alleles, suggesting that defective translation of the mutant mRNA and/or a decreased stability of the mutant protein are responsible for the lower enzymatic activity. The NAT2*6A and NAT2*7B alleles have also been associated with the slow acetylator phenotype in human liver in in vitro studies.107 Again, the acetylation activity (toward p-phenetidine) and the amount of immunochemically determined NAT protein were reduced, especially in the case of homozygous mutant alleles, while the mRNA levels did not differ between the wild-type and mutant genotypes.107 From these in vitro studies, it is clear that the effects on acetylation activity and the amount of immunochemically detectable NAT protein are much larger in liver samples from individuals homozygous for NAT2 mutations than in liver material from individuals heterozygous for NAT2 mutations, suggesting a so-called gene dosage effect.


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b. In Vivo Studies Several studies have been performed in which individuals were both genotyped for NAT2 mutations as well as phenotyped for their in vivo acetylation activity (Table 3). It is clear that the reduction of acetylation activity in individuals homozygous for NAT2 mutations was larger when compared with individuals who were heterozygous for NAT2 mutations (gene dosage effect), comparable to the results from in vitro studies. The NAT2*5A, NAT2*5B, NAT2*5C, NAT2*6A, and NAT2*7B and NAT2*14B alleles have been

associated with the slow acetylator phenotype in vivo by phenotyping with different substrates in healthy volunteers who were heterozygous for one of these alleles in combination with a wildtype (NAT2*4) allele.104,112,116,131 Furthermore, the combination of these alleles in homozygous mutant individuals yielded a reduced acetylation activity in vivo as well. The NAT2*13 and NAT2*14 alleles (no distinction between NAT2*14A and NAT2*14B was made in the cited study) have been shown to lead to a reduced acetylation activity when present in individuals homozygous for these alleles (Table 3). Recently,

TABLE 3 In Vivo Acetylation Activity in Individuals with Different NAT2 Genotypes Genotype

NAT2*4/*5A NAT2*4/*5B NAT2*4/*5C NAT2*4/*6A NAT2*4/*7B NAT2*4/*14B NAT2*4′′*14 NAT2*5A/5A NAT2*5A/*5B NAT2*5A/*5C NAT2*5A/*6A NAT2*5A/*7B NAT2*5A′′*14 NAT2*5B/*5B NAT2*5B/*5C NAT2*5B/*6A NAT2*5B/*7B NAT2*5B/*13 NAT2*5C/*5C NAT2*5C/*6A NAT2*6A/*6A NAT2*6A/*7B NAT2*6A/*13 NAT2*13/*13 NAT′′*14/*14

Cascorbi et al. (1995)a

Hickman et al. (1992)b,d

Bell et al. (1993)c,d,e

85 (11) 64 (107) 78 (14) 66 (77) 48 (7) 59 (1) — 12 (2) 16 (16) 20 (3) 11 (4) 9 (1) — 16 (78) 18 (14) 13 (118) 6 (1) 9 (5) 12 (2) 11 (13) 11 (46) 8 (6) 11 (8) 9 (1) —

— 47 (5) — 52 (4) — — — — — — 8 (1) — — 14 (6) 11 (1) 11 (2) — — — 21 (1) 15 (1) 19 (1) — — —

68 (9) — — 62 (5) 36 (2) — 46 (1) 10 (3) — — 12 (8) 12 (1) 12 (3) — — — — — — — 9 (3) — — — 10 (1)

Note: The numbers in this table are the relative activity of the mutant genotypes, expressed as a percentage of the activity of the wild-type NAT2*4/*4 genotype (100% activity). Between brackets are the number of individuals with the corresponding genotype. a b c d

e

116: Phenotyping with caffeine; individuals with the wild-type allele NAT2*4/*4: 29. 104: Phenotyping with sulfametazine; individuals with the wild-type allele NAT2*4/*4: 1. 112: Phenotyping with caffeine; individuals with the wild-type allele NAT2*4/*4: 6. The data in these columns were obtained from a graphical representation of the data in the cited references. No distinction was made between the NAT2*14A and NAT2*14B alleles in this study.


74

the NAT2*12A allele was found to encode a fully active enzyme.111

such hypersensitivity reactions if this was the only risk factor involved.

5. DNA Adducts 4. Clinical Response and Incidence of Adverse Effects For a number of drugs (i.e., dapsone, hydralazine, isoniazid, and prizidilol), a larger therapeutic effect was obtained in slow acetylators when compared with fast acetylators.139–144 With some drugs (i.e., dapsone, hydralazine, isoniazide, and prizidilol), a higher dose was required to achieve a desired therapeutic effect in fast vs. slow acetylators.142,145–148 In the case of the experimental antineoplastic compound amonafide, a higher area under the plasma concentration vs. time curve (AUC) of the activated acetylated metabolite, and a greater myelosuppressive effect were found in fast versus slow acetylators.149 The occurrence of adverse effects (e.g., the development of antinuclear antibodies [ANA] and systemic lupus erythematosus [SLE]) has also been correlated to the slow acetylator genotype.150–161 The slow acetylator phenotype was also largely overrepresented (90%) in a group of patients experiencing hypersensitivity reactions towards sulfonamides when compared with a race-matched control group (55%).162 In a recent study among patients experiencing adverse drug reactions toward sulfonamides, it was found that 17 of 18 patients was either homo- or heterozygous mutant at the NAT2 locus with primarily the M1 (481NAT2) and M2 (590NAT2) mutations present.163 This suggests that individuals with a low acetylation capacity might be at higher risk for these adverse drug reactions that might result from a larger formation of toxic N-hydroxylated metabolites of the sulfonamides by P450 isoenzymes. Another recent study, however, found that the acetoxy-metabolite, which may be formed from the N-hydroxylated sulfonamide sulfamethoxazole, was nontoxic in human cell lines in vitro.164 It was pointed out recently that due to the low occurrence of severe hypersensitivity reactions to sulfonamides, the NAT2 genotype cannot be the only risk factor involved.165 In as much as the incidence of the slow acetylator genotype is more than 50% in some Caucasian populations, far more people would experience

The NAT genetic polymorphism was shown to affect the levels of DNA adducts in human tissues. Significantly higher levels of 4aminobiphenyl-hemoglobin adducts were found in slow acetylators with the M1 (481NAT2), M2 (590NAT2), M3 (857NAT2), or M4 (191NAT2) mutations.166 Furthermore, higher, but statistically not significant, levels of 4-aminobiphenyl-DNA adducts were found in exfoliated bladder cells.166 No effect was found of the NAT2 genotype on urothelial DNA adducts of benzidine.167 Furthermore, a 2-fold higher level of DNA adducts in urinary bladder mucosa has been found in individuals heterozygous for the NAT1*10 allele when compared with the homozygous wild-type allele.168 Based on these findings, it has been suggested that individuals with mutations in both NAT2 and NAT1 are at particularly high risk for bladder cancer.168

6. Cancer Risk The NAT genetic polymorphisms appear to play a role in interindividual variability in cancer risk. Epidemiological studies have shown that fast acetylators (NAT 2) are at larger risk for colon cancer when compared with slow acetylators.169–171 In contrast, fast acetylators (NAT 2) are at lower risk for bladder cancer when compared with slow acetylators.172–175 In a recent study, in which the occurrence of bladder cancer was investigated among people with several NAT 2 genotypes, it was found that especially the NAT2*5B allele is associated with a higher incidence of bladder cancer.176 The presence of the NAT1*10 allele has also been associated with a higher risk for colorectal cancer.177 Furthermore, long-time smokers who are homozygous mutant at the NAT1 locus have a 27-fold increased risk of bladder cancer.178 The variability in bladder and colon cancer risk might be related to the different metabolism of arylamines and heterocyclic amines in slow and fast acetylators.179,180 In a slow acetylator,


75

more N-hydroxy-arylamine metabolites are formed in the liver when compared with a fast acetylator as a consequence of the competition between NAT and P450 enzymes (Figure 3). These N-hydroxyarylamine metabolites might be transported to the bladder, either unconjugated or as a glucuronide conjugate. In the bladder, they might form a nitrenium ion and cause the formation of DNA adducts. This explains why slow acetylators are at higher risk for bladder cancer. The situation is different with colon cancer, however. Compounds most often implicated in the risk of colon cancer are heterocyclic amines,181 which are relatively poor substrates for the NAT enzymes in liver and therefore are also N-hydroxylated by P450 enzymes. The N-hydroxy-derivatives might be converted by NAT enzymes to carcinogenic O-acetylated metabolites in the colon after transport as a glucuronide conjugate via the bile. Therefore, fast acetylators might be at higher risk for colon cancer due to the formation of these reactive metabolites. However, interethnic differences have also been shown to exist.182 Moreover, NAT 1 may also contribute significantly to arylamine metabolism in human colon and thereby influence the risk for colon cancer,183 although a recent study found no association between the NAT1*10 allele and the risk for colorectal adenomas.184 The NAT2 genotype has also been suggested to be involved in the risk for other types of cancer, although evidence is still limited.185,186

E. Conclusions Genetic polymorphisms have been found for both NAT enzymes (NAT 1 and NAT 2) present in humans. The mutant NAT1 alleles, which were discovered relatively recently, seem to produce enzymes with higher activity when compared with the wild-type enzymes. A number of mutant NAT2 alleles have been described that contain one or more of nine well-established mutations. Based on studies in which individual mutations and combinations of them have been expressed in heterologous expression systems, four of these muta*

tions (341NAT2, 590NAT2, 857NAT2, and 191NAT2 ) have been found to be most important in reducing the activity of the NAT 2 enzymes produced. The fact that these alleles encode enzymes with reduced activity has been established both in vitro and also in vivo in humans. Furthermore, clear effects have been found on the metabolism and pharmacokinetics of several drugs that are substrates for NAT 2 enzymes. Both the pharmacological action and the occurrence of side effects is influenced by the NAT genetic polymorphisms. Also, the role of the NAT genetic polymorphisms as a risk factor for bladder and colon cancer has been well established. The variable metabolism of aromatic and heterocyclic amines, as a consequence of the NAT genetic polymorphisms, seems to provide a sound mechanistic basis for a correlation with the occurrence of bladder and colon cancer.

III. CYTOCHROMES P450 (P450; E.C. 1.14.14.1)* A. Introduction P450 isoenzymes constitute a superfamily of enzymes,189 which are important in the oxidative and reductive metabolism of numerous compounds.190,191 In a number of cases, P450 isoenzymes are also involved in the metabolism of (pro)carcinogenic and cytotoxic compounds.192–194 The P450 isoenzymes are divided into families based on their evolutionary relationship, which is determined by the degree of homology of the individual genes and thus amino acid structures of the proteins.195–197 A number of genetic polymorphisms have now been established for several isoenzymes of the P450 enzyme system (i.e., in the CYP1A1, CYP2A6, CYP2C9, CYP2C18, CYP2C19, CYP2D6, and CYP2E1 genes). Considering the fact that bioactivation by P450 enzymes may play an important role in human drug toxicity198 and human cancer,199 these polymorphisms may result in large interindividual variations in the metabolism and toxicity of xenobiotics.

Nomenclature of P450 proteins, genes, and allelic varients is according to the guidelines for human gene nomenclature68 and according to Ref. 187 for CYP2A6, CYP2C9, CYP219, and CYP2D6 and188 for CYP1A1. Individual mutations are indicated by the nucleotide nuimber (in superscript) followed by the gene symbol (e.g.,1934CYP2D6). Individuals (changes) in amino acids are indicated by the amino acid position (in superscript) followed by the amino acid (e.g., 462isoleucine).


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B. Cytochrome P450 1A1 (CYP1A1)

1. Xenobiotic Metabolism by CYP1A1 CYP1A1 mainly catalyzes the metabolism of relatively large flat-structured aromatic hydrocarbons. Examples include benzo[a]pyrene, dimethylbenz[a]anthracene, and 6-nitrochrysene.192,194 However, several smaller molecules are also metabolized by CYP1A1. Predictive computer models of the active site of CYP1A1 have been developed, in which both the relatively large, flat aromatic molecules as well as some smaller molecules can be accomodated (for a review see Ref. 200). Although a lot of CYP1A1 substrates are carcinogens or are metabolized to carcinogens by the action of CYP1A1, some noncarcinogenic substrates are also known (e.g., ethoxycoumarin and ethoxyresorufin).

mutation of 461threonine to asparagine in the CYP1A1 enzyme.188 This mutant allele is designated CYP1A1*4. Frequencies of CYP1A1 alleles and genotypes in different populations are presented in Table 4.188,212

3. Effects of the CYP1A1 Genetic Polymorphism a. Expression The expression of the CYP1A1*2A gene, as reflected by the CYP1A1 mRNA level was not different when compared to the wild-type allele in a study among 20 healthy Caucasian volunteers.213,214 In contrast, an effect on CYP1A1 expression has been found in individuals homozygous and heterozygous for the CYP1A1*2B allele, where mRNA levels were significantly higher when compared with the wild-type genotypes.215

2. CYP1A1 Genomics The gene encoding the human CYP1A1 isoenzyme has been localized at human chromosome 15 and contains seven exons of which the first is non-coding.201 The sequence of the complete gene spans 6311 base pairs.202 A polymorphism for the restriction endonuclease Msp I is present in the 3′ end of this gene, caused by a 6235CYP1A1 T → C (m1) mutation.203,204 This results in a mutant CYP1A1 allele designated CYP1A1*2A. This allele is reported to be in linkage disequilibrium with a polymorphism for Pst I.205 Furthermore, a polymorphism has been reported in exon 7 of the CYP1A1 gene, caused by a 4889CYP1A1 A → G (m2) mutation, resulting in the replacement of 462isoleucine by valine in the mutant form of the protein. 206 This mutant allele is designated CYP1A1*2B and is reported to be in linkage disequilibrium with the CYP1A1*2A allele.207 However, it appears that these alleles are not always linked.208–210 A third polymorphism, which is observed exclusively in African-Americans, is caused by a 5996CYP1A1 T → C (m3) mutation in the 3′ non-coding region of the gene and is designated CYP1A1*3.211 This allele is not linked to either the CYP1A1*2A or CYP1A1*2B alleles. Recently, a fourth mutation was detected at position 4887 in exon 7 of the human CYP1A1 gene, caused by a 4887CYP1A1 C → A (m4) mutation, resulting in a

b. Inducibility The occurrence of the CYP1A1*2A allele has originally been correlated with the inducibility of the encoded CYP1A1 enzyme in mitogen stimulated lymphocyte cultures in a study of a 3-generation family. In this study, the mutant allele resulted in a higher inducibility when compared with the normal allele.216 However, after reexamination of the samples, no correlation was found between the presence of the CYP1A1*2A allele and the inducibility of the CYP1A1 protein.209 Recently, however, the inducibility of CYP1A1 proteins encoded by the CYP1A1*2A allele appeared to be much higher when compared with both homozygous normal and heterozygous proteins in lymphocytes of Japanese individuals.217 Cosma et al. and Crofts et al. have also found a higher inducible 7-ethoxyresorufin O-dealkylation (EROD) activity in lymphocytes of individuals with the CYP1A1*2B allele when compared with wild-type proteins.208,215 The presence of both the CYP1A1*2A and the CYP1A1*2B alleles thus seems to result in the formation of a protein with a higher inducibility. A number of other proteins are involved in the induction of CYP1A1 as well.218 The arylhydrocarbon (Ah)-receptor is present in the cyto-


77

TABLE 4 Nomenclature of CYP1A1 Alleles and CYP1A1 Allele and Genotype Frequencies in Different Populations Old nomenclature

African–Americana (n = 148)

Africana (n = 59)

Caucasianb (n = 880)

Wild-type Msp I mutation Exon 7 mutation African-American mutation

67.6 22.0 2.7 7.8 —c

61.0 25.5 0.0 13.6 —

89.3 5.1 2.7 0.0 3.0

Wild-type; A; A/A; Ile/Ile Heterozygous AA Heterozygous variant; B A/G; Ile/Val

47.3 11.5 28.4 0.7 0.0 4.1 0.0 3.4 0.0 4.7 — — — —

35.6 18.6 32.2 0.0 0.0 8.5 0.0 5.1 0.0 0.0 — — — —

79.7 — 9.2 4.8 — — — 0.11 0.0 0.45 5.3 0.23 0.11 0.11

New nomenclature

Alleles CYP1A1*1 CYP1A1*2A CYP1A1*2B CYP1A1*3 CYP1A1*4 Genotypes CYP1A1*1/*1 CYP1A1*1/*3 CYP1A1*1/*2A CYP1A1*1/*2B CYP1A1*3/*3 CYP1A1*2A/*3 CYP1A1*2B/*3 CYP1A1*2A/*2A CYP1A1*2B/*2B CYP1A1*2A/*2B CYP1A1*1/*4 CYP1A1*2A/*4 CYP1A1*2B/*4 CYP1A1*4/*4 a b c

Homozygous variant; C G/G; Val/Val

Data from 212. Data from 188. No data reported.

plasm coupled to heat shock protein HSP90. After binding of an inducer, HSP90 dissociates from the inducer-Ah-receptor complex, which subsequently binds to the so-called Ah receptor nuclear translocator (ARNT) protein. The resulting complex translocates to the nucleus, where it binds to regulatory sequences associated with the CYP1A1 gene. Mutations in the genes encoding the various transcription factors might also result in an altered CYP1A1 inducibility.219 Interindividual variations in the expression levels of Ah mRNA and ARNT mRNA have been observed that were associated with the CYP1A1 expression.220,221 The Ah receptor exists in two different forms with an amino acid replacement of 554arginine by lysine. This polymorphism, however, appears not to be associated with the CYP1A1 inducibility.220,221 The exact role of genetic polymorphisms in the genes encoding the Ah receptor and the ARNT protein, in relation to the induction of CYP1A1, remains to be elucidated.

c. Enzymatic Activity With respect to the activity of mutant CYP1A1 enzymes, it has been reported that the CYP1A1 2B mutant (valine-type CYP1A1) has a higher enzymatic activity with benzo[a]pyrene in in vitro systems when compared with the wild-type (isoleucine-type) enzyme.199,217 In a recent study, however, the mutant valine-type CYP1A1 did not catalyze benzo[a]pyrene metabolism at a higher rate when compared to the wild-type enzyme.222 Furthermore, the metabolism of the CYP1A1specific substrate ethoxyresorufin by the mutant enzyme was only slightly, although significantly, higher when compared with the wild-type enzyme in that same study. Also, recently it was reported that there was no difference in Km or Vmax values for ethoxyresorufin O-dealkylation and benzo[a]pyrene 3-hydroxylation between the CYP1A1 2B mutant and the wild-type enzyme.223


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d. DNA Adducts No correlation has been observed between both the CYP1A1*2A and CYP1A1*2B alleles and DNA adducts in human lung.224,225 However, higher levels of DNA adducts have been found in total white blood cells of full-time chimney sweeps homozygous for the CYP1A1*2A allele when compared with heterozygous individuals.226

e. Mutations in Cancer-Related Genes Recently, it was found that smokers homozygous mutant for the CYP1A1*2A and CYP1A1*2B alleles had a higher frequency (4.5- to 5.5-fold) of mutations in the p53 and Ki-ras genes, two target genes in lung cancer.227 These effects were even more pronounced when individuals were also deficient for glutathione-S-transferase M1-1 (GSTM1-1) in addition to being homozygous mutant for CYP1A1. The higher frequency of mutations in the p53 and Ki-ras genes in smokers might be explained by a higher degree of bioactivation of cigarette smoke constituents by mutant CYP1A1 proteins, which have a higher inducibility and/or enzymatic activity, as discussed above. The synergistic interaction with the GSTM1-1 genotype may be explained by a decreased detoxification of reactive intermediates formed by CYP1A1 in individuals deficient in GSTM1-1 activity. C. Cytochrome P450 2A6 (CYP2A6)

1. Xenobiotic Metabolism by CYP2A6 CYP2A6 is involved in the metabolism of several carcinogenic compounds, such as afla-

toxin B1, N-nitrosodiethylamine, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).192,194 CYP2A6 also catalyzes the hydroxylation of coumarin and plays a role in the formation of cotinine from nicotine.228,229

2. CYP2A6 Genomics A cluster of genes encoding P450 enzymes from the CYP2A, CYP2B and CYP2F subfamilies have been localized at human chromosome 19.230 Subsequently, two polymorphisms were found to be present in the CYP2A6 gene.230,231 One CYP2A6 mutant allele, CYP2A6*2 (previously designated as CYP2A6v1), has a single base mutation (T to A) leading to a leucine to histidine change in exon 3, the other allele, CYP2A6*3 (previously designated as CYP2A6v2), contains parts of the CYP2A7 pseudogene in exons 3, 6 and 8 and therefore would be expected to encode for an inactive enzyme.231 Table 5 shows the frequency of the CYP2A6*1 wild-type and mutant alleles in different ethnic populations.

3. Effects of the CYP2A6 Genetic Polymorphism In a study among Caucasians, three individuals who had undetectable coumarin metabolism were homozygous for the CYP2A6*2 allele, while individuals who were heterozygous for the CYP2A6*2 allele had levels of coumarin metabolism ranging from 50 to 60% of the average value in the population.231 In contrast, in that same study three other individuals who were homozygous for

TABLE 5 Frequencies of CYP2A6 Wild-Type and Variant Alleles in Different Populationsa Population (n)

Finnish (13) English (29) Japanese (20) Taiwanese (89) African-American (20) a

CYP2A6*1 (wild-type)

CYP2A6*2 (CYP2A6*v1)

CYP2A6*3 (CYP2A6*v2 )

85 76 52 83 97.5

15 17 20 11 0

0 7 28 6 2.5

Data from Ref. 231.


79

the CYP2A6*2 allele had coumarin metabolism ratios similar to heterozygous subjects. So, the presence of the CYP2A6*2 allele does not explain the lower rate of metabolism of coumarin. As yet, no data have been published on the functionality of the CYP2A6*3 allele; however, it is expected to be inactive due to the fact that it contains large portions of the CYP2A7 pseudogene. An alternatively spliced product of the CYP2A7 gene has also been identified, although it is not known whether CYP2A7 contributes to coumarin metabolism in humans.232 D. Cytochrome P450 2C9 (CYP2C9)

to a change of 358tyrosine to cysteine, 1061CYP2C9 A → C leading to a change of 359isoleucine to leucine (CYP2C9*3 allele), and 1236CYP2C9 G → A leading to a change of 417glycine to asparagine. Because the initially reported 1059CYP2C9 A → G and 1236CYP2C9 G → A mutations were not found in subsequent population studies,241–244 they might be cloning artifacts or mutations that occur at extremely low frequencies. Because it is not certain that these are natural mutants, they have not been included in the nomenclature system yet (Dr. A. K. Daly, personal communication). The allele frequencies of the CYP2C9*1 (wild-type), CYP2C9*2, and CYP2C9*3 alleles in different populations are shown in Table 6.

1. Xenobiotic Metabolism by CYP2C9 CYP2C9 metabolizes a number of important clinically used drugs and is the main enzyme involved in the metabolism of S-warfarin, phenytoin, tolbutamide, tienilic acid, and a number of nonsteroidal antiinflammatory drugs (diclofenac, piroxicam, tenoxicam, ibuprofen, and acetylsalicylic acid).233 A number of CYP2C9 substrates exist as anions at physiological pH, and a small molecule model has been developed in which several CYP2C9 substrates were superimposed, and the anionic groups were in close distance to a (hypothetical) cationic interaction site within the CYP2C9 protein.234

2. CYP2C9 Genomics The cDNA of the CYP2C9 gene, present at human chromosome 10,235,236 has been cloned and subsequently four mutations have been identified.237–240 These mutations are 416CYP2C9 C → T causing a change of 144arginine to cysteine (CYP2C9*2 allele), 1059CYP2C9 A → G leading

3. Effects of the CYP2C9 Genetic Polymorphism Interindividual variability in the metabolism of both tolbutamide246–249 and phenytoin250–254 has been reported. The frequency of poor metabolizers (PMs) for these substrates was estimated to be relatively low (± 0.2%). Alleles occurring at such a low frequency would by definition not be caused by a genetic polymorphism. The metabolism of phenytoin and tolbutamide were closely correlated to each other but not to the mephenytoin metabolism in a study among Black Africans, showing that phenytoin and tolbutamide are metabolized by the same enzyme that is different from CYP2C19, which catalyzes the metabolism of mephenytoin.255

a. CYP2C9*2 Allele In a preliminary study, it was found that the presence of the allele containing the 416CYP2C9 mutation (CYP2C9*2 allele), encoding the mu-

TABLE 6 Allele Frequencies of Wild-Type and Mutant CYP2C9 Alleles in Different Populations Population (n) Caucasian (100) British (100) Chinese (135) Taiwanese (98) African-Americans (100)

CYP2C9*1 0.86 0.808 0.98 0.974 0.985

CYP2C9*2

CYP2C9*3

Ref.

0.08 0.192 0.0 0.0 0.01

0.06 — 0.02 0.026 0.005

243 245 242 243 243


80

tant enzyme with cysteine at position 144, resulted in a 5- to 10-fold higher expression of mRNA when compared with the wild-type form in two individual heterozygous for this mutation.241 This enzyme was also found to have a 3-fold higher activity for the metabolism of both tolbutamide and phenytoin.256 In contrast, the enzyme encoded by the CYP2C9*2 allele was found to have a 9-fold lower Vmax and a 5.6-fold lower intrinsic clearance for S-warfarin, when expressed in HepG2 cells, compared with the wild-type enzyme.257 This lower activity of the mutant enzyme was in line with a study in which individuals heterozygous for this mutant required a 20% lower warfarin maintanance dose when compared with individual homozygous for the wild-type enzyme.245

b. CYP2C9*3 Allele The enzyme encoded by the allele containing the 1061CYP2C9 mutation (CYP2C9*3 allele) was found to have an altered regio- and stereo-selectivity for warfarin metabolism. Instead of the 7-hydroxylation of (S)-warfarin, the mutant enzyme hydroxylates (R)-warfarin to its 4′-hydroxy metabolite.258 In contrast, in a more recent study no such effect was found on the stereoselectivity in the metabolism of warfarin by the enzyme encoded by the CYP2C9*3 allele.259 The mutant enzyme, however, exhibited a fivefold lower Vmax and a fivefold higher Km when compared with the wild-type enzyme. The tolbutamide hydroxylase activity of the enzyme encoded by the CYP2C9*3 allele was also reported to be 12-fold lower when compared with the wild-type enzyme.233 The Vmax for the metabolism of phenytoin in epilepsy patients was reported to be 40% lower in patients heterozygous for the CYP2C9*3 allele when compared with patients homozygous for the wild-type CYP2C9*1 allele.260 In fact, the CYP2C9*3 allele may be the most important in explaining the lower activity of mutant CYP2C9 enzymes toward tolbutamide in PMs.243

I restriction site.261 This allele is called CYP2C18*2 and its frequency is 21.4% in the Japanese population.261 This frequency is quite similar to that of the CYP2C19*2 mutant allele and suggests a possible linkage of the mutant alleles encoding CYP2C18 and CYP2C19 (both genes are present in the CYP2C locus at the same chromosome).262 Recently, two more mutations were found in the 5′-flanking region which might in fact represent the same allele as described by Tsuneoka et al.263 Another mutation was described recently in the CYP2C18 gene (CYP2C18*3 allele); a point mutation of T to A at position 204 in exon 2, causing the introduction of a stop codon at position 68, leads to the expression of a truncated 67 amino acids long protein lacking the heme-binding region.264 Among 40 unrelated Japanese subjects, 3 were homozygous for this mutation, 16 were heterozygous, and 21 were homozygous for the wild-type allele.264 As yet, little is known about the substrate specificity of CYP2C18. Therefore, the effects of the mutations in the gene encoding CYP2C18 on xenobiotic metabolism and toxicity are not well understood at this moment.

F. Cytochrome P450 2C19 (CYP2C19) The polymorphism in CYP2C19 is often referred to as the ‘mephenytoin polymorphism’ (for reviews see Refs. 265 to 267). The polymorphic metabolism of mephenytoin has now been observed in a number of human (family) studies and has a genetic origin.268–271 It was suggested that an absence or decreased activity of CYP2C19 was responsible for the decreased metabolism of (S)-mephenytoin.272,273 Later, CYP2C19 indeed appeared to be the main enzyme involved in the oxidative metabolism of the S-enantiomer of mephenytoin.274,275 The occurrence of PMs for CYP2C19 shows a large inter-ethnic variability (Table 2).

1. Xenobiotic Metabolism by CYP2C19 E. Cytochrome P450 2C18 (CYP2C18) A mutation has been found in the 5′-flanking region of the CYP2C18 gene that changed a Dde

CYP2C19 is involved in the metabolism of a number of important clinically used drugs, such as S-mephenytoin, omeprazole, diazepam, imipramine, propranolol, proguanil, and hexo-


81

barbital.233 Recently, in vitro experiments have shown that a number of the selective serotonin reuptake inhibitors (SSRI’s), being citalopram, fluoxetine, paroxetine, and sertraline, are potent inhibitors of the activity of CYP2C19, suggesting that they might be substrates of this enzyme.276

2. CYP2C19 Genomics The cDNA sequence of CYP2C19, which is present at human chromosome 10, 235,236 has been determined.277 Two genetic defects have been found that together explain all Oriental mephenytoin PMs and about 83% of Caucasian mephenytoin PMs. The major genetic defect, called CYP2C19*2, is a single base pair mutation (681CYP2C19 G → A) in exon 5 of the CYP2C19 gene, causing an abberrant splice-site.278 As a result of this mutation, the reading frame of the mRNA is altered and a stop codon is introduced leading to a truncated nonfunctional protein. Seven out of 10 Caucasian PMs and 10 out of 17 Japanese PMs were homozygous for the CYP2C19*2 allele. A second defect, called CYP2C19*3, was subsequently found that, together with CYP2C19*2, explains all of the Japanese mephenytoin PMs. The CYP2C19*3 allele, however, does not occur among Caucasians.279 The mutation in the CYP2C19*3 allele is a 636CYP2C19 G → A in exon 4 creating a premature stop codon. The CYP2C19*2 and CYP2C19*3 mutations have been shown to be inherited separately.280 A complete concordance was observed between genotyping for CYP2C19 mutations and phenotyping for CYP2C19 activity in a recent Japanese

study.281 The occurrence of the two mutant CYP2C19 alleles shows interethnic variability (Table 7). The CYP2C19*2 allele occurs predominantly in Oriental populations, although it also occurs in a black Ethiopean population.285 A particularly high occurrence of CYP2C19 mutant alleles was observed in a population of two islands of Vanuatu in Melanesia, where up to 70% of the population has the PM phenotype.286 On those islands, malaria is endemic and a number of antimalarial drugs are used there. Because the metabolism of, for example, proguanil, is determined by CYP2C19, this polymorphism may have major implications with respect to the efficacy of that drug in this specific population.

3. Effects of the CYP2C19 Genetic Polymorphism A number of compounds of which the metabolism and/or pharmacological or toxicological effects are influenced by the CYP2C19 polymorphism are listed in Table 8. Usually, mephenytoin has been used as the model substrate to study the effects of the genetic polymorphisms in this P450 isoenzyme. Recently, however, proguanil and omeprazole have been suggested as an alternative to mephenytoin in CYP2C19 phenotyping studies.303–305 As the proteins produced from all known mutant CYP2C19 alleles are completely nonfunctional, the occurrence of the mutations is clearly correlated to the impaired metabolism of mephenytoin.280,282,306–308

TABLE 7 Frequencies of Variant CYP2C19 Alleles in Different Populations Population (n) Japanese (233) Chinese (244) Chinese Taiwanese (118) Korean (103) Ethiopean (114) Filipinos (52) Saudi Arabians (97) European-Americans (105) African-Americans (108) Islands of Vanuatu (493)

CYP2C19*2

CYP2C19*3

Ref.

0.219 0.289 0.32 0.209 0.14 0.39 0.15 0.13 0.25 0.708

0.117 0.044 0.055 0.117 0.02 0.077 0 0 0 0.133

262 282 283 284 285 283 283 283 283 286


82

TABLE 8 Compounds of Which the Metabolism Co-Segregates with the Mephenytoin Polymorphism Amitriptyline Diazepam Hexobarbital Imipramine Mephobarbital Omeprazole Pantoprazole Proguanil (chloroguanide) Propranolol

a. Tricyclic Antidepressants In the case of the tricyclic antidepressants (amitriptyline and imipramine), CYP2C19 is not the only P450 enzyme involved in the metabolism. The genetic polymorphism in CYP2D6 is also important with those compounds because CYP2D6 is the major enzyme responsible for the hydroxylation of imipramine, a reaction not catalyzed by CYP2C19.309 PMs for CYP2D6 are not able to hydroxylate these compounds, and, consequently, they can only be N-demethylated. Because the N-demethylation is catalyzed by CYP2C19, this reaction is impaired in CYP2C19 PMs.294 In fact, an individual who was deficient in both CYP2D6 and CYP2C19 had the lowest imipramine oral clearance in a study among 22 individuals.293 Furthermore, a case report describing a patient with a combined deficiency of CYP2C19 and CYP2D6 who experienced severe cardiotoxicity after treatment with desipramine has been published. 310 In as much as the N-demethylated metabolites of tricyclic antidepressants are still pharmacologically active, the combined roles of both the CYP2D6 and the CYP2C19 polymorphisms are of interest.

b. Propranolol Also in the metabolism of propranolol, both CYP2D6 and CYP2C19 are involved. Similarly as with the tricyclic antidepressants, an individual who was deficient in both enzymes showed the lowest total propranolol clearance, indicating that

Tricyclic antidepressant287 Sedative288–290 Sedative291,292 Tricyclic antidepressant293,294 Sedative295,296 Gastric proton pump inhibitor297,298 Gastric proton pump inhibitor299 Antimalarial drug300,301 β-blocker302

both enzymes contribute significantly in the overall metabolism.302

c. Diazepam The N-demethylation of diazepam is catalyzed by CYP2C19, and thus its rate is influenced by the CYP2C19 polymorphism.289 Although it appeared that this was the case in both Swedish288 and Korean130 populations, in a Chinese population no effect of the CYP2C19 polymorphism on the diazepam metabolism was found.311 An explanation for that observation was given by the fact that the EM group in the Chinese population studied contained an overrepresentation of heterozygous EMs who had a slower metabolic rate when compared with homozygous EMs.312 The fact that homozygous EMs have higher metabolic capacities when compared with heterozygous EMs (a so-called gene dosage effect) was proven by genotyping studies.282

d. Proguanil Proguanil (chloroguanide) is a pro-drug that is metabolized to cycloguanil that possesses an antimalarial activity and is used in malaria prophylaxis. The metabolism of proguanil to cycloguanil has been shown to co-segregate with the mephenytoin polymorphism in vivo in humans.300,313 Because the formation of cycloguanil is catalyzed by CYP2C19, a deficiency in this enzymatic activity might be partially responsible


83

for a failure in malaria prophylaxis.301 In contrast, a recent in vitro study reported no effect of the CYP2C19 phenotype on the in vitro antimalarial activity.314

e. Omeprazole The metabolism of omeprazole, a so-called proton pump inhibitor used in the treatment of gastric ulcers, has also been shown to be dependent on the CYP2C19 genotype.297,298 The area under the plasma concentration vs. time curve (AUC) has been shown to be 12.3-fold higher in Caucasian PMs when compared with EMs because of the 3.3-fold higher elimination half life in PMs.315 The AUC was significantly higher in Chinese EMs when compared with Caucasian EMs, which might be a result of the gene dosage effect (higher percentage of heterozygous individuals in the Chinese population).315 It was suggested that not only the hydroxylation of omeprazole but also the formation or elimination of its sulfone metabolite might be influenced by the CYP2C19 genetic polymorphism.315 In a recent in vitro study, it was found that CYP2C19 was indeed the major omeprazole hydroxylase enzyme, although other CYP2C enzymes as well as CYP3A4 were able to catalyze this reaction.316 Recently, a polymorphism was also discovered in the metabolism of pantoprazole, another proton pump inhibitor,299 that might also be caused by the CYP2C19 genetic polymorphism.

G. Cytochrome P450 2D6 (CYP2D6) During the late 1970s and the early 1980s, it was discovered that a polymorphism existed in the metabolism of both debrisoquine317–319 and sparteine320,321 in the human population. Further studies showed that the polymorphic metabolism of these two compounds was linked to each other.322 The relative importance of environmental and genetic factors in the polymorphic metabolism of debrisoquine was analyzed, and it was found that this polymorphism was mainly determined by a genetic polymorphism and not by environmental factors.323 The polymorphic metabolism of debrisoquine was suggested to be due to the absence of hepatic CYP2D6 and mutant CYP2D6 mRNAs were found providing

evidence for the CYP2D6 genetic polymorphism.324,325 Because debrisoquine and sparteine were the first substrates known to be metabolized by the polymorphic CYP2D6, the CYP2D6 genetic polymorphism is often referred to as the ‘debrisoquine/sparteine polymorphism’ (for recent reviews on this polymorphism see Refs. 326 and 327). On the basis of phenotyping studies (e.g., with debrisoquine, sparteine, or dextrometh-orphan), populations can be divided into three groups, poor (PM), extensive (EM), and ultrarapid metabolizers (UMs). This distinction is generally made on the basis of the urinary MR between parent drug and metabolite, as discussed in the introduction to this review. For each substrate used for phenotyping, an antimode (MR = 12.6 for debrisoquine, MR = 20.0 for sparteine, and MR = 0.3 for dextromethorphan) is used to distinguish PMs from EMs. The percentage of PMs is known to be variable among different populations (Table 2). Among Europeans and White Americans, approximately 7.5% has the PM phenotype,117,122,328 while this percentage is much lower (0 to 2%) in Chinese, Japanese and also Black Americans.122,126,329,330

1. Xenobiotic Metabolism by CYP2D6 CYP2D6 is known to be involved in the metabolism of a large number of clinically used drugs, among which are antiarrythmics, antidepressants, and neuroleptics.16,331–333 Several predictive computer models have been published in which the distance between a basic nitrogen atom and the site of oxidation in the substrates determines whether a compound is metabolized by CYP2D6 (for a review see Ref. 200). An overview of a large number of substrates of which the metabolism is determined by the CYP2D6 polymorphism is given in Table 9.

2. CYP2D6 Genomics The human CYP2D6 is encoded at the CYP2D locus, which also contains two other genes, notably a related gene (CYP2D7), containing a mutation that disrupts the reading frame, and a pseudogene (CYP2D8P), which contains several mutations and does not encode a functional enzyme.398,399 The CYP2D locus has been localized


84

TABLE 9 Compounds for Which an Accumulation of Parent Compound or Active Metabolites or a Reduced Formation of Active Metabolites Occurs in CYP2D6 PMs Accumulation of parent compound Compound Alprenolol (β-blocker) Amiflamine (monoamine-oxidase inhibitor) Amitriptyline (tricyclic antidepressant) Aprindine (antiarrhytmic) Bufuralol (β-blocker) CGP 15 210 G (SSRI) Citalopram (SSRI) Clomipramine (tricyclic antidepressant) Desipramine (tricyclic antidepressant) Dexfenfluramine (anorectic) Dextromethorphan Flecainide (antiarrhytmic) Fluoxetine (SSRI) Fluvoxamine (SSRI) Guanoxan (antihypertensive) Haloperidol (neuroleptic) Imipramine (tricyclic antidepressant) Indoramin (α1-adrenoceptor antagonist) 4-Methoxyamphetamine Methoxyphenamine (β2-adrenergic stimulant) Metoprolol (β-blocker) Mexiletine (antiarrhytmic) Mianserine (antidepressant) Nicotine Nortriptyline (tricyclic antidepressant) Paroxetine (SSRI) Perhexiline (anti-anginal) Perphenazine (neuroleptic) Phenformin (anti-diabetic) Propafenone (antiarrhytmic) N-propylajmaline (antiarrhytmic) Propranolol (β-blocker) Thioridazine (neuroleptic) Timolol (β-blocker) Tomoxetine (antidepressant) Venlafaxine (SSRI) Zuclopenthixol

Ref. 334 335 336, 337, 338 339a 340, 341 342 343 344, 345 346, 310 347 348–350 351 352 353 354 355 357–359 360 361 362 363 364a 365 366 367–369 370, 371a 372–375 376–378 379, 380 381, 382 383 384 385–387 388, 389 390 391a 377, 392

Reduced formation of active metabolite(s) Compound Codeine (analgesic) Encainide (antiarrhytmic) Tramadol (analgesic) a

Ref. 393–395 396 397

In vitro studies.


85

at human chromosome 22.398,400,401 The CYP2D6 gene contains nine exons within 4378 base pairs.399 The mutated CYP2D6 alleles, which are currently known, including the most recent nomenclature187,402 and trivial names, are indicated in Table 10. Several mutant forms of CYP2D6 have also been detected among EMs. Alleles have been described that contain mutations causing a change of 296arginine to cysteine and of 486serine to threonine but still encode functional CYP2D6, the allele being referred to as CYP2D6*2.405,431,432 The CYP2D6*2 allele has also been associated with ultrarapid metabolism of debrisoquine.433 Subsequent studies demonstrated that only when the CYP2D6*2 allele is present in multiple copies, then called CYP2D6*2XN (due to inherited amplification), it is associated with ultrarapid metabolism of debrisoquine.404,407,434 Substantial interethnic differences in the occurrence of the mutant alleles exist. The major alleles associated with the PM phenotype are CYP2D6*3, CYP2D6*4, and CYP2D6*5 in Caucasian and White American populations. In contrast, the CYP2D6*3 and CYP2D6*4 alleles are rare among Chinese, Japanese, and Koreans, while the CYP2D6*10A, CYP2D6*10B, and CYP2D6*10C are far more common.435,436 The different distributions of the mutant CYP2D6 alleles, CYP2D6*3 and CYP2D6*4, among these populations explains why there is a lower percentage of PMs in Chinese, Japanese, and Korean populations when compared with Caucasians and White Americans. The fact that the CYP2D6*10A, CYP2D6*10B, and CYP2D6*10C alleles, which encode CYP2D6 enzymes with reduced activity, are more common in Chinese, Japanese, and Korean populations explains why the MRs in these populations are in general higher (caused by a slower metabolism of CYP2D6 substrates), while there are less PMs.

3. Effects of the CYP2D6 Genetic Polymorphism a. Transcription, Protein Structure and Enzyme Activity The effects of mutations in the CYP2D6 alleles on the gene and protein are listed in Table 10.

The CYP2D6*3 mutant allele was found to contain a frameshift mutation, due to a one base pair deletion, and therefore will not encode a functional enzyme.409 After expression of the wild type and chimeras of the mutant CYP2D6*4 allele in COS-1 cells, it was found that the 188CYP2D6 mutation, causing an amino acid change of 34proline to serine, abolished enzymatic activity toward bufuralol, while the 4268CYP2D6 mutation, causing an amino acid change of 486serine to threonine, had no measurable effect.409 However, the 1934CYP2D6 mutation is probably the major cause of the slow metabolizer phenotype due to this allele because of a disruption of a splice-site at the junction of the third intron and fourth exon.409,411 The CYP2D6*4 mutation is also present on the 16 + 9 kb Xba I restriction fragment length polymorphism (RFLP) fragments.437 The complete CYP2D6 gene is deleted at the CYP2D6*5 allele, and therefore this allele is associated with the PM phenotype.413 The CYP2D6*6 alleles encode for a truncated protein of 152 amino acids, due to a single base-pair deletion at position 1795, and yields an inactive CYP2D6 enzyme.415–417 The CYP2D6*7 allele contains one mutation that causes a 324histidine to proline amino acid change and has been found in an individual with a MR of 100 for sparteine, which is five times the antimode, and thus appears to reduce the CYP2D6 activity.418 In a study in which the CYP2D6*7 allele was expressed in insect cells by recombinant baculovirus, it was found that the mutant protein folded incorrectly and did not incorporate heme.438 The CYP2D6*8 allele contains a premature stop codon due to the 1846CYP2D6 mutation and will yield an inactive CYP2D6 protein.419,420 The CYP2D6*9 allele, in which one codon is deleted, leading to the absence of a lysine amino acid in the protein, did not have enzymatic characteristics different from the wild-type allele when expressed in HepG2 cells, while the activity of the protein was lower in human liver microsomes from an individual with this mutation.421 In vivo, however, the CYP2D6*9 allele did not cause the PM phenotype.422 The 188CYP2D6 mutation, causing an amino acid change of 34proline to serine, appears to be responsible for the reduced activity of CYP2D6 proteins encoded by CYP2D6*10 alleles. The 188CYP2D6 mutation has been found to reduce the 4-hydroxylation of propranolol significantly


86

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87

K29–1

CYP2D6*4C

A3023 G1749C; G1846T; G2064T; G4268C

CYP2D6E CYP2D6G

CYP2D6C

CYP2D6J CYP2D6Ch1

CYP2D6*7 CYP2D6*8

CYP2D6*9

CYP2D6*10A CYP2D6*10B

C188T; G1749; G4268C C188T; C1127T; G1749C; G4268C

A2701–A2703 or G2702– A2704 deleted

T1795 deleted

CYPD26*6B

CYP2D6*5 CYP2D6*6A CYPD6D CYP2D6T

CYP2D6B

CYP2D6*4B

CYP2D6*4D

CYP2D6B

CYP2D6*4A C188T; C1062A; A1072G; C1085G; G1749C; G1934A; G4268C C188T; C1062A; A1072G; C1085G; G1934A; G4268C C188T; G1749C; G1934A; T1975C; G4268C C188T; C1127T; G1749C; G1934A; G4268C CYP2D6 deleted T1795 deleted

A2637 deletion

CYP2D6A

CYP2D6*3

G1749C; C2938T; G4268C G1749C; C2938T; G4268C

CYP2D6L

CYP2D6*2

None G3916A

Mutation(s)a

CYPD6*2XN

Wild-type

Trivial names(s)

CYP2D6*1A CYP2D6*1B

Allele

P34S; S486T P34S; S486T

H324P Premature termination of translation Deletion of K281

Frameshift

Splicing defect No protein Frameshift

Splicing defect

Splicing defect

Splicing defect

R296C; S486T; Nc active genes Frameshift

None Silent Nucleotide Change R296C; S486T

Effect of mutation(s)b

TABLE 10 Nomenclature, Mutations, and the Effects of CYP2D6 Alleles

cDNA expression: Not significantly different from wild-type CYP2D6 toward sparteine, debrisoquine, and bufuralol; human liver in vitro: reduced activity toward sparteine and bufuralol Decreased toward sparteine in vivo Decreased toward debrisoquine in vivo and bufuralol in vitro

None toward debrisoquine and dextromethorphan in vivo None toward debrisoquine and sparteine in vivo None toward sparteine in vivo; MR = 100 None toward debrisoquine and sparteine in vivo

None toward debrisoquine and sparteine in vivo and bufuralol in vitro

Increased toward debrisoquine in vivo None toward debrisoquine and sparteine in vivo and bufuralol in vitro None toward debrisoquine and sparteine in vivo and bufuralol in vitro

403

Normal toward sparteine in vivo Decreased toward debrisoquine and dextromethorphan in vivo

Ref.

412 423

421, 422

416, 417 418 419, 420

415

403 413, 414

412

409

409–411

404, 407, 408 409

404–406

399

Normal in vitro and in vitro

Enzymatic activity



88 CYP2D6F

CYP2D6*11

430

429

428

424

427

426

425

423, 424

Ref.

b

N means the number of genes due to amplification.

Decreased toward debrisoquine in vivo and in vitro

None toward debrisoquine and dextromethorphan in vivo None toward debrisoquine in vivo

None toward debrisoquine in vivo

None toward debrisoquine and sparteine in vivo None toward sparteine in vivo; MR = 85.5 None toward dextromethorphan in vivo

Decreased toward debrisoquine in vivo and bufuralol in vitro

Enzymatic activity

c

T1071; R296S; S486T

Frameshift

P34S; G169R; R296C; S486T

G42R; R296C; S486T Frameshift

Splicing defect

P34S; S486T

Effect of mutation(s)b

The mutations are represented by the position of the mutated nucleotide in the CYP2D6 gene surrounded by the wild-type and mutated nucleotides, respectively. Amino acid changes are indicated by the position of the amino acid in the CYP2D6 protein surrounded by the one letter abbreviations for the wildtype and mutated amino acids, respectively. One letter abbreviations for the amino acids are: R, arginine; C, cysteine; S, serine; T, threonine; H, histidine; P, proline; and K, lysine.

CYPD6Z

CYP2D6*17

CYP2D7P/CYP2D6 hybrid C1111T; G1726C; C2938T; G4268C

C188T; C1127T; G1749C; G4268C; and gene conversion to CYP2D7P in exon 9 G971C; G1749C; C2938T; G4268C G212A; G1749C; C2938T; G4268C CYP2D7P/CYP2D6 hybrid; Exon 1 CYP2D7P, exons 2–9 CYP2D6 C188T; G1846A; C2938T; G4268C T226 insertion

Mutation(s)a

a

CYP2D6D2

CYP2D6*16

CYP2D6*15

CYP2D6*14

CYP2D6*13

CYP2D6*12

CYP2D6Ch2

Trivial names(s)

CYP2D6*10C

Allele

TABLE 10 (continued) Nomenclature, Mutations, and the Effects of CYP2D6 Alleles

in Chinese individuals. 439 The CYP2D6*10A allele has been associated with the PM phenotype in vivo.412 CYP2D6*10C was similar to CYP2D6*10B but contained a large portion of the CYP2D7 pseudogene and encoded a nonfunctional or unstable enzyme.423 Both the CYP2D6*10B and CYP2D6*10C alleles also contain mutations in the 5′ flanking region of the gene. These mutations, however, did not affect expression of the enzymes, as demonstrated in COS-1 cells.423 The CYP2D6*11 allele causes incorrect splicing of pre-mRNA due to the 971CYP2D6 mutation and thus yields an inactive enzyme.425 The CYP2D6*12 allele has been found in a PM with a MR for sparteine of 85.5, which is well above the antimode.426 Recently, a mutant CYP2D6 allele, now called CYP2D6*13, giving rise to the PM phenotype in vivo, was found that is characterized by a 9-Kb fragment after restriction with Xba I. This mutant was found to contain a chimeric CYP2D7/CYP2D6 structure containing exon 1 and part of intron 1 of the CYP2D7 gene, whereas the rest of the gene originates from CYP2D6.427

b. Metabolism and Pharmacological Activity of CYP2D6 Substrates There is a number of possible effects of the CYP2D6 polymorphism on the metabolism and pharmacological activity of CYP2D6 substrates. These effects depend on whether CYP2D6 is involved in the inactivation of a pharmacologically active parent compound, the formation of pharmacologically active metabolites, or the formation of toxic metabolites. In some cases, the treatment of patients with drugs that are metabolized by CYP2D6 might lead to treatment related toxicity in PMs, as shown for the antiarrhythmic drug propafenone.440 On the other hand, UMs might not reach therapeutically active plasma concentrations of certain drugs.441 In Table 9, a large number of drugs are listed that are known to be primarily metabolized by CYP2D6. These include neuroleptics, such as haloperidol, thioridazine, chlorpromazine and levomepromazine,442 antidepressants,443 antiarrhythmic drugs (e.g., encainide, flecainide, mexiletine, and propafenone), 444 and a number of psychoactive drugs.445 For most of these compounds, the effect

of the CYP2D6 genetic polymorphism is an accumulation of the parent compound. In addition to a reduced formation of pharmacologically active metabolites, other metabolic reactions may occur due to the increasing involvement of other P450s with lower affinity at high plasma concentrations.

i. Accumulation of Parent Compound or Metabolites In the case of the tricyclic antidepressants, which can be N-demethylated to active metabolites (e.g., amitriptyline, clomipramine, and imipramine), the consequence of the PM phenotype is an accumulation of the active N-demethylated metabolites. The reason for the accumulation of the active metabolites in PM individuals is that the metabolism (aromatic hydroxylation) of the parent compound is reduced, leading to an accumulation of the parent compound, but the N-demethylation (which is not catalyzed by CYP2D6) is still taking place. Although an accumulation of active metabolites is expected, a (preliminary) case report has been published that reports that PMs did not respond to imipramine treatment.359 Because no plasma levels of imipramine or its N-dealkylated metabolite desipramine were measured, it is not certain whether the N-dealkylation pathway occurred fully in those individuals. Recently, it has been reported that although the steady-state plasma levels of desipramine were influenced by the CYP2D6 phenotype, the antidepressive effect of desipramine was not.446 There has been some debate on whether CYP2D6 might be involved in the N-demethylation reactions mentioned above. In the case of amitriptyline, some evidence has been presented that CYP2D6 might be partially involved in its Ndemethylation in non-smokers,337 but, in general, there is no effect of the CYP2D6 polymorphism on these reactions, 338,339,347 CYP2C19 and CYP3A4/5 are generally thought to catalyze Ndemethylation reactions.338,391 Individuals with combined deficiencies of CYP2D6 and CYP2C19 have been described who had very low imipramine oral clearance293 and total propranolol clearance.302 Furthermore, a case report has been published on a patient with a combined deficiency of CYP2C19 and CYP2D6 who experienced severe cardiotoxicity after treatment with desipramine.310


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Recently, a class of compounds for which evidence has been found that CYP2D6 is involved in their metabolism is the selective serotonin reuptake inhibitors (SSRIs), which are used clinically as antidepressants.447,448 Higher concentrations of CGP 15 210 G have been found in CYP2D6 PMs when compared with EMs, suggesting that CYP2D6 is involved in the metabolism of this SSRI.342 The metabolism of paroxetine was also influenced by the CYP2D6 polymorphism; however, the effects of the polymorphism were less prominent at steady-state conditions of the drug than after a single dose, presumably due to saturation of CYP2D6 at the higher steadystate concentrations.370 PMs appeared to have 2-fold higher maximum plasma concentration, longer half-life and a 5-fold lower oral clearance of fluvoxamine when compared with EMs, although the CYP1A2 activity also plays a role in this variability.353 In fact, fluvoxamine is a potent inhibitor of CYP1A2 activity, resulting in a different role of the CYP2D6 polymorphism with respect to this compound when compared with other SSRIs.449 Recently, it was found that the metabolism of venlafaxine is also catalyzed by CYP2D6, in vitro.391 The clinical consequences for PMs, however, are not clear because the O-demethylated metabolite is pharmacologically similar to the parent compound.391 The pharmacokinetics of citalopram were altered in CYP2D6 PMs when compared with EMs, but the polymorphism in CYP2C19 was also important in the interindividual variability in the metabolism of this SSRI.343 CYP2D6 PMs were also found to accumulate fluoxetine, while there was no effect of the CYP2D6 phenotype on sertraline metabolism.352

ii. Reduced Formation of Pharmacologically Active Metabolites When CYP2D6 is the major enzyme catalyzing the formation of pharmacologically active metabolites, the absence of CYP2D6 activity might also lead to a reduced formation of these metabolites. In the case of the antiarrhythmic drug encainide, this also leads to a reduced pharmacological effect.396 The conversion of codeine to morphine is also catalyzed by CYP2D6; however, as this is only a small part of the total metabolism of

codeine, the consequences for PMs are probably less relevant and not yet completely understood.393,450,451 Recently, however, it was found that the analgesic effect of codeine in experimental pain models was lower and adverse effects were more pronounced in PMs when compared with EMs, compared with morphine for which no difference was observed.452 In a Chinese population, individuals who had the 188CYP2D6 mutation formed less morphine from codeine, measured as the amount of morphine and morphine glucuronide recovered in urine.394 Also, recently the effect of codeine on gastrointestinal motility was found to be dependent on the CYP2D6 genetic polymorphism.453 While pharmacokinetic parameters of codeine were the same in PMs and EMs, those of the metabolite morphine were found to be different. The peak serum concentration was 20-fold higher, AUC was 15-fold higher, and the total amount of morphine excreted in urine was 11-fold higher in EMs when compared with PMs.453 The delayed gastric emptying due to the morphine generated from the administered codeine was only observed in EMs. Another prodrug that is bioactivated by CYP2D6 is the analgesic drug tramadol.397 Tramadol has two mechanisms that are responsible for its analgesic activity, it enhances noradrenergic and serotonergic neurotransmission and it interacts with the µ-opioid receptor. This latter mechanism is probably due to a tramadol metabolite, (+)-O-desmethyl-tramadol (also called (+)M1) of which the formation is catalyzed by CYP2D6 (Figure 6). The formation of the (+)-M1 metabolite was found to be reduced largely in PMs. Consequently, tramadol showed a lower analgesic effect in PMs in experimental pain models.397

H. Cytochrome P450 2E1 (CYP2E1)

1. Xenobiotic Metabolism by CYP2E1 Recently, an updated overview of CYP2E1 substrates was published (Table 11).454 As can be seen, CYP2E1 catalyzes both oxidation and reduction reactions with a large number of substrates. Hydrophobic compounds of low molecular weight seem to be, especially good substrates


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FIGURE 6. Schematic representation of the metabolism of tramadol. The (+)-M1 metabolite of tramadol is mainly responsible for the hypoalgesic activity due to an interaction with the µ-opioid receptor. (Adapted from Ref. 397.)

for CYP2E1. The active site of CYP2E1 is probably shaped in such a way as to accomodate relatively small molecules. Several CYP2E1 substrates are also carcinogens or are metabolized to carcinogens by CYP2E1.

2. CYP2E1 genomics The gene encoding the human cytochrome P4502E1 (CYP2E1) isoenzyme has been localized at human chromosome 10.455 The gene spans 11,413 base pairs and contains nine exons.456,457 A number of RFLPs have been detected in this gene. RFLPs for the restriction enzymes Dra I and Rsa I have been found in a group of 26 unrelated individuals,458 a Msp I RFLP in intron 6 in 36 unrelated Japanese individuals,459 and a Taq I RFLP in intron 7 in a group of 39 unrelated North American Caucasians.455 Furthermore, RFPLs have been detected for Rsa I and Pst I in the 5′ upstream sequence of the CYP2E1 gene caused by –1259CYP2E1 G → C and –1019CYP2E1 C → T mutations.460 These RFLPs appeared to be in complete linkage disequilibrium with each other. Re-

cently, two additional mutations have been described, a 1017CYP2E1 A → G and a 1113CYP2E1 A → C mutation; however, these have as yet not been measured in a large population.461 Furthermore, two rare mutations have been found that also cause amino acid substitutions, a 1168CYP2E1 G → A and a 10059CYP2E1 G → A mutation, causing amino acid changes of 76arginine to histidine and 389valine to isoleucine, respectively.462 The 1168CYP2E1 and 10059CYP2E1 mutations were detected in 2 out of 78 Chinese subjects and 1 out of 42 Italian subjects, respectively.462 The best studied CYP2E1 genetic polymorphisms are the Pst I/Rsa I polymorphisms in the 5′ flanking region and the Dra I polymorphism in intron 6 of the CYP2E1 gene. A large interethnic variation of these polymorphisms exists (Table 12). The Pst I/Rsa I polymorphism occurs at a higher frequency in Japanese and Chinese when compared with Caucasians and (African) Americans.45 The Dra I polymorphism occurs at a higher frequency in Taiwanese when compared with European- and African-Americans.47 The Rsa I and Dra I polymorphisms appeared to be linked: all individuals with the Rsa I polymorphism also


91



92

Adapted from Ref. 454.

Acetoacetate Acetol

Isoniazid Phenol

a

(Halogenated) alkanes and alkenes

Arachidonic acid Lauric Acid

Fatty Acids

Diethylether Methyl t-butylether 1,1,2,3,3,3-Hexafluoropropylmethyl ether

Ethers

Pyridine p-Nitrophenol Pyrazole Styrene Tamoxifen Theophylline Toluene

Acetaminophen Aniline Benzene Bromobenzene Chlorzoxazone 3-Hydroxypyridine

Aromatic compounds

Acetaldehyde Butanol 2-Butanone Ethanol Glycerol Isopropanol Methanol Propanol Pentanol 1-Phenylethanol

Alcohols, aldehydes, ketones, and nitriles

TABLE 11 Recent Overview of CYP2E1 Substratesa

Hexane Methoxyflurane

Acetone Acetonitrile Acrylonitrile 1,3-Butadiene Carbon tetrachloride Chloroform Chloromethane Dibromoethane 1,1-Dichloro-2,2,2-trifluoroethane 2,2-Dichloro-1,1,1-trifluoroethane Dichloromethane 1,2-Dichloroethane 1,1-Dichloroethylene 1,2-Dichloropropane N,N-Dimethylacetamide N,N-Dimethylformamide Enflurane 1,2-epoxy-3,butene Ethane Ethylcarbamate Ethylene dichloride Halothane

N-Nitrosomethylbenzylamine N-Nitrosopyrrolidine

Azoxymethane N,N-Diethylnitrosamine N,N-Dimethylnitrosamine Methylazoxymethanol N-Nitroso-2,3dimethylmorpholine

Nitrosamines, azo compounds

Methyl formate Methylene chloride N-Methylformamide Pentane Seroflurane 1,1,1,2-Tetrafluoroethane 1,1,2,2-Tetrafluoro-1(2,2,2trifluoro-ethoxy)ethane Thioacetamide Tirapazamine 1,1,1-Trichloroethylene Vinyl chloride Vinyl bromide

TABLE 12 Frequencies of the Major Variant CYP2E1 Alleles and Genotypes Allele frequencies of the Pst I/Rsa I allele Population (n) Chinese (100) Japanese (202) Swedish (148) European-American (126) African-American (449)

Wild-type (c1)

Mutant (c2)

Ref.

0.750 0.807 0.950 0.990 0.960

0.250 0.193 0.050 0.010 0.040

463 460 464 47 47

Genotype frequencies of the Dra I polymorphism

Population (n) Taiwanese (119) African-American (142) European-American (114)

Wild-type (DD)

Heterozygous (CD)

Mutant (CC)

Ref.

0.55 0.80 0.84

0.43 0.19 0.16

0.03 0.01 0.01

47 47 47

exhibited the mutant Dra I allele. The reverse, however, is not always true.465 This complicates the nomenclature for the mutant alleles. In the remaining part, the CYP2E1 genetic polymorphisms are refered to as the Rsa I/ Pst I and the Dra I polymorphisms.

3. Effects of the CYP2E1 Genetic Polymorphism a. Pst I and Rsa I Polymorphisms i. Transcription The RFLPs for Pst I and Rsa I in the 5′ flanking region of the CYP2E1 gene appear to change the transcriptional activation of the gene.466 Experiments in which the 5′ flanking regions of the homozygous wild-type and mutant genotyes have been placed upstream of the chloramphenicol acetyltransferase (CAT) gene have shown that the enhancement of gene expression by the homozygous mutant genotype was 10 times higher when compared with the homozygous wild type.466 Further studies showed that the binding of a transcription factor is affected when the mutation at the Rsa I site is present and that this is mainly responsible for the different levels of expression seen in the CAT assay.467 However, whether these different genotypes will have an effect on the

expression of the gene in vivo remains to be established. Non-alcohol-consuming individuals with the heterozygous mutant genotype appeared to have a 1.7-fold higher mRNA expression in peripheral lymphocytes when compared with nonalcohol-consuming individuals with the homozygous wild-type genotype. Alcohol-consuming individuals with the heterozygous mutant genotype had a 2.0-fold higher mRNA expression in peripheral lymphocytes when compared with nonalcohol-consuming individuals with the homozygous wild-type genotype.467 Using liver biopsy material, it has been found that the mRNA expression was three times higher in individuals with the heterozygous mutant genotype when compared with the homozygous wild-type genotype, which is in line with the higher mRNA expression observed in peripheral lymphocytes.468,469 However, alcohol consumption was not taken into account in this study. Furthermore, the homozygous wild-type genotype was only found in healthy controls and not in individuals with alcoholic liver disease, while the heterozygous mutant genotype was found in both groups. Therefore, it is possible that healthy individuals have been underrepresented in the group of individuals with the heterozygous mutant genotype. As alcohol is known to induce CYP2E1 activity, also at the level of mRNA expression, this might have biased the results.


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ii. Enzymatic Activity In vitro, there was no correlation between the occurrence of the Rsa I polymorphism and the hydroxylation of chlorzoxazone.470 Studies toward the effects of the Rsa I/Pst I polymorphism on the in vivo CYP2E1 enzyme activity are limited due to the low occurrence of this polymorphism, especially in Caucasian populations.471–473 In a recent study, no relation was found between these genetic polymorphisms and the chlorzoxazone clearance by 6-hydroxylation between 65 homozygous normal individuals and 5 heterozygous mutants.474 In contrast, in a recent Japanese study it was found that the half-life of acetaminophen was shortest in individuals with the homozygous mutant genotype, and that the elimination rate in individuals with the homozygous mutant genotype was over twice that of individuals with the homozygous wild-type genotype, suggesting a higher activity of the mutant CYP2E1 protein in vivo.475 Furthermore, the elimination rate of ethanol was found to be higher in individuals with the homozygous mutant genotype when compared with individuals with the homozygous wild-type genotype, again suggesting that the mutant enzyme has a higher activity.476

b. Dra I Polymorphism The Dra I polymorphism is located in intron 6. Because this polymorphism does not give rise to any structural change in the CYP2E1 protein, any effects of this polymorphism have to be at the level of expression. However, studies in human liver autopsy material have not clearly proven any difference in mRNA expression due to this polymorphism.42 Furthermore, there was no difference in the hydroxylation of chlorzoxazone, which is almost exclusively metabolized by CYP2E1 in human liver, between individuals with the homozygous wild-type genotype and the hetero- and homozygous mutant genotypes, as assessed by the excretion of 6-hydroxychlorzoxazone.472,474 This confirms an in vitro study in which no effect of the occurrence of the Dra I polymorphism was found on the chlorzoxazone metabolism.470 In a study in which N 7-(m)ethylated guanine DNA bases were measured in 46 human lung samples, it appeared that N 7-ethyl-guanine levels correlated with the presence of the Dra I muta-

tion.471 Furthermore, N 7-methyl-guanine levels were found to be higher in samples with the Dra I mutation, especially at low levels of exposure to compounds responsible for the formation of these kinds of DNA adducts.477 Because compounds that might form such DNA adducts (e.g., di(m) ethylnitrosamines) are substrates for CYP2E1, variability in its enzymatic activity might be related to the formation of these kinds of DNA adducts.

I. CONCLUSIONS Genetic polymorphisms have been found for a number of the P450 isoenzymes known to exist in humans (i.e., CYP1A1, CYP2A6, CYP2C9, CYP2C18, CYP2C19, CYP2D6, and CYP2E1). At least three of these isoenzymes (i.e., CYP2C9, CYP2C19, and CYP2D6) are very important with respect to the metabolism of a large number of clinically used drugs. The genetic polymorphisms of these isoenzymes (in particular CYP2C19 and CYP2D6) thus have been shown to have clear effects on the metabolism, pharmacological action, and the occurrence of side effects of substrates of these isoenzymes. The genetic polymorphism of CYP2C9 has only been described relatively recently, and the consequences of this polymorphism for drug metabolism are not yet clear. The P450 isoenzymes CYP1A1, CYP2A6, and CYP2E1 are often involved in the metabolism of a number of potentially genotoxic compounds. In the case of CYP1A1, effects of the variant alleles on the transcription of the CYP1A1 gene have been described. However, the variable expression of CYP1A1 may also be caused by polymorphisms of other proteins involved in the induction of CYP1A1. In the case of CYP2E1, one of the variant alleles seems to have a higher rate of transcription in vitro. Some studies have also reported that such effects might occur in vivo. A problem with studies toward the CYP1A1 and CYP2E1 genetic polymorphisms is the relatively low occurrence of the mutant alleles, especially in Caucasian populations, which especially hampers human studies due to the large number of individuals required to find statistically significant effects. In studies toward the effects of genetic polymorphisms of biotransformation enzymes on the metabolism and effects of genotoxic


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compounds, occupationally exposed populations are often studied. In such studies it is important to monitor the (differences in) exposure of the individuals using biomarkers of exposure.

IV. GLUTATHIONE S-TRANSFERASES (GST; E.C. 2.5.1.18)* A. Introduction GSTs are dimeric enzymes that catalyze the conjugation of glutathione (GSH) to electrophilic xenobiotics in order to inactivate them and facilitate their excretion from the body.479–483 The isoenzymes are divided into at least seven classes, five of which are cytosolic (alpha, mu, pi, theta, and kappa) and two of which are membrane bound.484–486 Besides catalyzing the conjugation of electrophilic xenobiotics to GSH, the GSTs also have an intracellular transport function.487 As GSTs play an important role in the detoxification of potentially genotoxic compounds, they have an important protective role in chemical carcinogenesis.488,489 Three genetic polymorphisms thus far have been detected in this enzyme family in the human population, viz. in the genes encoding GSTM1-1, GSTT1-1, and GSTP1-1.

B. Xenobiotic Metabolism by GSTs GSTs are generally involved in the detoxification of reactive electrophilic compounds (for a review see Ref. 482). Several carcinogens, such as the reactive benzo[a]pyrene diol-epoxide, aflatoxin-2,3-epoxide, and reactive sulfates, are good substrates for GSTs. In addition to carcinogenic compounds, GSTs also metabolize several pesticides and environmental pollutants. Several chemotherapeutic compounds are also substrates for GSTs and in that case the conjugation of those compounds to GSH might lead to a lower effectiveness and the occurrence of cellular resistance to the effects of the compounds. An increased GSH conjugation is one of the mechanisms thought to be responsible for the occurrence of multidrug resistance observed during treatment with some *

specific chemotherapeutic compounds. In a number of cases, however, GSTs may be involved in the bioactivation of xenobiotics.490 This is, for example, the case with 1,2-dibromoethane, which is metabolized by GSTs to a reactive episulfonium ion that is considered to be the reactive genotoxic intermediate formed from this carcinogen.

C. Glutathione S-Transferase Mu (GSTM1-1)

1. GSTM1-1 Genomics The gene encoding the human mu class glutathione-S-transferase M1-1 (GSTM1-1) enzyme has been localized to chromosome 1.491–495 The GSTM1-1 polymorphism was first recognized as a polymorphic activity toward the substrate transstilbene oxide in leukocytes and appeared to be of a genetic origin.496 As can be seen from Table 2, there is a relatively small difference in the occurrence of the GSTM1-1 null genotype between Caucasian and Oriental populations. An AfricanAmerican population, however, displayed a significantly lower occurrence of the GSTM1-1 null genotype. Besides the GSTM1-1 null genotype, two mutant forms of GSTM1-1 exist in human populations, GSTM1a-1a and GSTM1b-1b (also called GSTµ and GSTψ, respectively), which differ in a single base pair (G or C at position 534).497 However, no differences in catalytic activity have as yet been observed for these two forms of class mu GSTs.498 As discussed below, a possible linkage of these variant alleles to genes encoding other GST mu enzymes may be involved in lung cancer risk.

2. Effects of the GSTM1-1 Genetic Polymorphism Because GSTM1-1 catalyzes the metabolism of a large number of potentially genotoxic compounds, many studies toward the effects of this genetic polymorphism on xenobiotic metabolism and toxicity have been directed to the formation

Nomenclature of GST proteins, genes, and allelic varients is according to the guidelines for human gene nomenclature68 and the GST nomenclature.478


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of DNA adducts, the occurrence of cytogenetic damage, and the risk of cancer.

a. DNA Adducts Significantly higher levels of PAH-DNA adducts have been found in lung samples of donors with the GSTM1-1 null genotype when compared with individuals with the GSTM1-1-positive genotype.225 Recently, the number of DNA adducts in human placenta was also found to be higher in a group of individuals with the GSTM1-1 null genotype when compared with a group of individuals with the GSTM1-1 positive genotype.499 In contrast, in other studies no significant influence of the GSTM1-1 genotype was observed on the levels of DNA adducts.500–504

b. Protein Adducts The level of 3- and 4-aminobiphenyl hemoglobin adducts was significantly higher in individuals with the GSTM1-1 null genotype. This level was even higher in combination with the slow acetylator (NAT) genotype.505 Higher levels of aflatoxin B1-albumin adducts were detected in individuals with the GSTM1-1 null genotype when compared with individuals with the GSTM1-1positive genotype.506 The higher levels of these protein adducts indicate the decreased detoxification of reactive intermediates in individuals with the GSTM1-1 null genotype.

c. Cytogenetic Damage and Mutations in Cancer-Related Genes Increased sister chromatid exchanges (SCEs) have been observed in lymphocytes of smokers with the GSTM1-1 null genotype at high exposure levels.507 Interestingly, G:C transversion mutations, which are often caused by PAH compounds, in p53 were significantly increased in lung tumor tissues of heavy smokers.508 The large inter-individual variability of hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutant frequency among nonsmoking individuals, however, was found to depend on factors such as age rather than GSTM1-1 null genotype frequency.509

A compound for which the effects of both the GSTM1-1 as well as the GSTT1-1 genetic polymorphisms have been studied is 1,3-butadiene. A number of epoxide metabolites can be formed after metabolism of 1,3-butadiene (Figure 7). The oxidation of 1,3-butadiene by cytochrome P450 (P450) enzymes initially leads to the formation of 1,2-epoxy-3-butene, which can be metabolized once more by P450 enzymes to 1,2:3,4diepoxybutane. Alternatively, it can be metabolized by epoxide hydrolase to 1,2-butenediol. 1,2Butenediol might be metabolized by P450 enzymes to 3,4-epoxy-1,2-butanediol. GST enzymes might be involved in the subsequent detoxification of the epoxide metabolites. It has been shown that the frequency of SCEs, after ex vivo treatment of lymphocytes from donors with the GSTM1-1 null genotype with 1,2-epoxy-3-butene, was higher when compared with lymphocytes from donors with the GSTM1-1-positive genotype.511 In contrast, there was no effect of the GSTM1-1 genotype on the SCE frequency induced by 3,4epoxy-1,2-butanediol.510 GSTT1-1 also appears to be involved in the detoxification of epoxide metabolites formed from 1,3-butadiene. The effects of the GSTT1-1 genetic polymorphism on the detoxification of these epoxides is described in the subsequent part of this review.

d. Linkage of the GSTM*1a Allele to the GSTM3-3 Gene and Lung Cancer Risk The GSTM1-1 genetic polymorphism was found to be a moderate risk factor (odds ratio 1.41) in a recent survey of 12 studies toward the effect of the GSTM1-1 genetic polymorphism on the susceptibility to lung cancer.512 The effect of GSTM1-1 genotype on lung cancer risk most likely depends on the metabolic detoxification of carcinogenic metabolites in the liver, in which GSTM11 is expressed.513 In GSTM1-1 null individuals, higher levels of bioactivated metabolites are thus able to reach the lung. Because of the low levels of GSTM1-1 in lung tissue, a deficiency of GSTM1-1 only has a limited effect on the capacity to detoxify carcinogens in the lungs.514,515 A more important role in determining the risk of lung cancer might be played by GSTM3-3, which is expressed in lung tissue.514,516 Recently, a polymorphism was found in the GSTM3-3 gene as


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FIGURE 7. Schematic representation of the metabolism of 1,3-butadiene. Three enzyme systems are involved in the metabolism of this compound. As discussed in the text, both GSTM1-1 and GSTT1-1 enzymes play a role in the metabolism of metabolites of 1,3-butadiene and their genetic polymorphisms have effects on the mutagenicity of these metabolites. (Adapted from Ref. 510.)

well, resulting from a three base-pair deletion in intron 6.517 This mutation appears to be linked to the GSTM*1a allele (encoding GSTµ) and thus might explain why GSTµ and GSTψ might play different roles in cancer susceptibility, although there are no known differences in catalytic activity between these two GSTM1-1 mutants. The linkage of these mutations also supports the view that GSTM3-3 plays a role in lung cancer susceptibility.518 However, it was reported recently that there was no relationship between GSTM3-3 activity and GSTM1-1 genotype in brain.519

D. Glutathione S-Transferase Theta (GSTT1-1)

1. GSTT1-1 Genomics The gene encoding human glutathione S-transferase T1-1 (GSTT1-1) has been localized to human chromosome 22.520 Genetic studies toward GSTT1-1 have revealed the existence of a genetic polymorphism, resulting from a gene deletion.521 The frequency of the occurrence of this gene

deletion in different populations is shown in Table 2. A relatively large interethnic variability is observed for the occurrence of this genetic polymorphism, with GSTT1-1 null frequencies ranging from 10% (European populations) to as high as 65% (Oriental populations).

2. Effects of the GSTT1-1 Genetic Polymorphism The GSTT1-1 genotype appears to determine the interindividual variability that has been observed in the GSH conjugation of compounds such as methyl bromide, methyl chloride, ethylene oxide, 1,2-dibromoethane, and dichloro-methane, especially in human erythrocytes, and populations can generally be divided into so-called conjugators and nonconjugators.522–526 The metabolism of methyl chloride in human erythrocytes showed a trimodal distribution, which is a result of the gene dosing effect of the GSTT11 genetic polymorphism.120 Furthermore, individuals who had no detectable activity toward 1,2epoxy-3-(p-nitrophenoxy)propane (EPNP), a


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standard theta-class GST substrate, did also not conjugate 1,2-dibromoethane to GSH.526 From other studies, it is known that GSTT1-1 increases mutagenicity in vitro caused by 1,2-dibromoethane.527 The rate of background SCE in lymphocytes, which was also known to show an interindividual variability,528 was found to be significantly higher in individuals with the GSTT11 null genotype when compared with individuals with the GSTT1-1-positive genotype as well.529 These background SCE levels might be caused by endogenous ethene and ethylene oxide, which are substrates of GSTT1-1.530,531 An interindividual variability in the sensitivity for the development of SCEs after exposure to the 1,3-butadiene epoxide metabolite 1,2:3,4diepoxybutane, in vitro, in human lymphocytes was observed.530,532 As GST theta enzymes are known to be important in the mutagenicity of 1,2:3,4-diepoxybutane,533 studies were conducted toward the effect of the GSTT1-1 genotype on the sensitivity toward 1,2:3,4-diepoxybutane. The frequency of SCEs in cultured lymphocytes of individuals with the GSTT1-1 negative genotype exposed in vitro to 1,2:3,4-diepoxybutane was found to be significantly higher when compared with individuals with the GSTT1-1-positive genotype. The GSTM1-1 genotype had no effect on the level of SCEs.534–536 Interestingly, the GSTT1-1 genetic polymorphism had no effect on the development of SCEs due to another 1,3-butadiene epoxide metabolite, 3,4-epoxy-1,2-butanediol.510 Taken together, the GSTM1-1 genetic polymorphism has an influence on the induction of SCEs by 1,2epoxy-3-butene, whereas the GSTT1-1 genetic polymorphism has an influence on the SCE induction by 1,2:3,4-diepoxybutane. The SCE induction by 3,4-epoxy-1,2-butanediol was not influenced by either the GSTM1-1 or the GSTT1-1 genetic polymorphisms. Thus, the GSTT1-1 genetic polymorphism seems to play a clear role in the variability of the metabolism of low-molecular-weight halogenated compounds and reactive epoxides. At least in vitro, it has been clearly shown that the presence of GSTT1-1 is either a protective or a risk factor for damage due to these kinds of compounds, depending on whether it is involved in bioactivation or bioinactivation reactions.

E. Glutathione S-Transferase Pi (GSTP1-1)

1. GSTP1-1 Genomics Recently, two different alleles have been identified coding for glutathione-S-transferase P1-1 (GSTP1-1), GSTP1a, and GSTP1b.537 These alleles differ only by one nucleotide at base pair 313, resulting in an amino acid replacement: 105valine in the GSTP1b mutant enzyme instead of 105isoleucine in the GSTP1a wild-type enzyme. In a population of 155 healthy volunteers from the Edinburgh area, the frequencies of GSTP1a and GSTP1b alleles were 0.72 and 0.28, respectively.537

2. Effects of the GSTP1-1 Genetic Polymorphism The GSTP1b allele has been reported to have an altered specific activity and affinity for electrophilic substrates and was reported to be associated with bladder and testicular cancer.537 Due to the very recent discovery of this genetic polymorphism, no other information was found in the literature up to now.

F. Conclusions GST enzymes play an important role in the detoxification of reactive intermediates that may be formed from a large number of compounds. Often, such reactive intermediates are involved in genotoxic processes. Consequently, a lot of studies have been performed to investigate the effect of genetic polymorphisms of GSTs on parameters that reflect genetic damage. Although some clear effects of the GSTM1-1 genetic polymorphism on parameters reflecting genotoxicity have been found, contradictory studies have also been reported. One of the reasons may be that sometimes individual exposure is not well monitored or not known at all. Another reason is that GSTs may also be involved in the formation, rather than detoxification, of reactive intermediates. Furthermore, variability of bioactivating enzymes (P450) will have a large impact on the genotoxicity of a


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compound. In those cases where both activating and inactivating enzymes show genetic polymorphisms, combinations of genetic polymorphisms of multiple biotransformation enzymes are important, as discussed below. The effects of the GSTT1-1 genetic polymorphism on the metabolism of small halogenated molecules is quite clear from several different studies. Because this GST isoenzyme is also expressed in human erythrocytes, it may play an especially important role in the metabolism of such compounds in blood.526

dihydrodiols by hydrolytic cleavage of the oxirane ring.538 Oxiranes with one or two hydrophobic substituents are among the best substrates of this enzyme. Although the conversion of reactive epoxides is often a bioinactivation reaction, in some cases mEH may be involved in a bioactivation pathway. An example of such a reaction is the formation of the 7,8-dihydrodiol-9,10-epoxide of benzo[a]pyrene in which mEH also plays a role (Figure 8).

B. Microsomal Epoxide Hydrolase Genomics V. MICROSOMAL EPOXIDE HYDROLASE (MEH; E.C. 3.3.2.3) A. Xenobiotic Metabolism by Microsomal Epoxide Hydrolase mEH metabolizes a large number of structurally diverse alkene and arene epoxides to

The gene encoding the human mEH has been localized at chromosome 6.539 Although several mutations have been described, only two were considered to represent genetic polymorphisms in a study with predominantly Caucasian DNA samples.540 These two polymorphisms affect the amino acids at positions 113 and 139 in the mEH

FIGURE 8. Schematic representation of the metabolism of benzo[a]pyrene into the carcinogenic 7,8-dihydrodiol 9,10-epoxide.


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protein. Among the Caucasian individuals, 36.2% were homozygous for 113tyrosine, 7.6% were homozygous for 113histidine, and 56.2% were heterozygous for these amino acids. Furthermore, 58.7% were homozygous for 139histidine, 4.6% were homozygous for 139arginine, and 36.7% were homozygous.

C. Effects of Genetic Polymorphisms of Microsomal Epoxide Hydrolase The amino acids mutations at positions 113 and 139 in the mutated mEH enzymes did not have an effect on transcription on the gene but did affect the amount of immunoreactive protein and catalytic activity, suggesting that they have an effect on protein stability.540 In a recent study it was demonstrated that the genetic polymorphisms in the mEH gene are not the only factors that determine the protein level and enzymatic activity.541 Although an effect of the mEH polymorphism was suspected on the occurrence of adverse drug reactions toward anticonvulsants, such as carbamazepine, no such effects have been reported as yet.542,543 A higher level of aflatoxin B1albumin adducts was detected among individuals who had mutant mEH alleles.506 In that same study, there also appeared to be a relationship between the occurrence of hepatocellular carcinoma and the presence of the aflatoxin B1-albumin adducts. However, the role of mEH in the detoxification of aflatoxin B1 is still controversial.544

D. Conclusions Although as yet relatively little is known about the genetic polymorphism of mEH, there is no doubt that these enzymes play an important role in the detoxification of potentially toxic or carcinogenic epoxides. Therefore, the genetic polymorphism in mEH enzymes has an effect on the metabolism and toxicity, especially in combination with genetic polymorphisms of other biotransformation enzymes.

VI. COMBINATIONS OF GENETIC POLYMORPHISMS OF BIOTRANSFORMATION ENZYMES In most of the studies discussed above, the consequences of only a single polymorphic biotransformation enzyme on the inactivation of a pharmacologically or toxicologically active parent compound, or in the formation of an active metabolite, are considered. However, there are several ways in which more than one polymorphic biotransformation enzyme may be involved in the metabolism of a particular compound. 1.

2.

3.

Multiple biotransformation enzymes may compete with each other for the metabolism of one compound, as is, for example, the case with N-acetyltransferase enzymes (NAT) and cytochrome P450 (P450) enzymes that compete for the metabolism of aromatic or heterocyclic amines. In this case, there is a direct competition between an enzyme that bioactivates the parent compound (in this case the P450 enzymes) and an enzyme that bioinactivates the parent compound (in this case the NAT enzymes) (Figure 3). Multiple biotransformation enzymes may be involved in the metabolism of a given compound, but not at the same stage of its metabolism. This is, for example, the case with NAT enzymes and glutathione-S-transferase (GST) enzymes that both bioinactivate aromatic or heterocyclic amines; however, NAT enzymes are active with the parent compound, whereas GST enzymes are active with the reactive intermediates generated at a later stage of metabolism (Figure 3). Multiple biotransformation enzymes may be involved in subsequent steps of the bioactivation of compounds. For example, benzo[a] pyrene is first metabolized into an epoxide by P450 enzymes, the epoxide is hydrolyzed by epoxide hydrolase (EH) enzymes, and again metabolized into an epoxide by P450 enzymes (Figure 8). The ultimate carcinogenic 7,8-dihydrodiol-9,10-epoxide may be detoxified by GST enzymes. A similar kind of situation occurs with aflatoxin B1; however, in


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4.

the first metabolic reaction, P450 enzymes may either bioactivate or bioinactivate aflatoxin B1 (Figure 9). In the case of aflatoxin B1, it has been suggested that the combination of genetic polymorphisms of GST and EH enzymes may determine the risk of hepatocellular carcinoma.506 Different isoenzymes of the same biotransformation enzyme may be involved in the metabolism of a given compound or its metabolites. This is, for example, the case with the metabolism of the tricyclic antidepressants, in which CYP2D6, CYP2C19, and CYP3A4 are involved. In this case, individuals with a combined deficiency of both CYP2D6 and CYP2C19 activity have very high plasma concentrations of the parent compound,293 which may result in toxicity.310

The above-mentioned examples illustrate that it may be important to investigate the combination of genetic polymorphisms of biotransformation enzymes, depending on the compounds involved. The fact that multiple biotransformation enzymes may be involved in the formation and inactivation of potentially carcinogenic reactive intermediates forms a rational basis for studies toward combinations of genetic polymorphisms of biotransformation enzymes and cancer risk. However, the number of individuals needed for such studies becomes larger as the number of studied enzymes increases. Especially when investigating the polymorphisms, which have a relatively low incidence (CYP1A1, CYP2A6, and CYP2E1), the number of individuals needed to obtain statistically significant results becomes very large. This may be the reason that such studies

FIGURE 9. Schematic representation of the metabolism of aflatoxin B1 (AFB1). AFM1, AFQ1, and the AFB1-endo8,9-oxide are noncarcinogenic products, while the AFB1-exo-8,9-oxide is reactive with DNA. The latter metabolite may be detoxified by either GST or EH enzymes. The hydrolysis reaction catalyzed by EH enzymes might also occur spontaneously, however. (Adapted from Ref. 545.)


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have not been performed on a large scale yet. Furthermore, the question is whether such studies would be relevant. Because the occurrence of some particular kind of combination of (sometimes rare) genetic polymorphisms, which may lead to a high susceptibility, is very low, very few individuals are affected. They might be relevant, however, in order to avoid exposure of individuals who are highly susceptible to specific compounds due to a combination of genetic polymorphisms of biotransformation enzymes.

VII. FINAL CONCLUSIONS AND FUTURE PERSPECTIVES In this review, a large number of genetic polymorphisms of biotransformation enzymes have been described. These polymorphisms were shown to be major factors in determining the variability in metabolism and toxicity of xenobiotics in a number of cases. Some conclusions regarding future research into the effects of these genetic polymorphisms, more specifically on sensitivity to genotoxic effects (cancer risk) and drug development, can be made.

A. Individual Sensitivity to Genotoxic Effects and Cancer Risk The relationship between genetic polymorphisms of biotransformation enzymes and the risk of cancer is of particular interest. The idea that these polymorphisms possibly determine the risk of cancer is based on the assumption that chemical exposure, subsequent metabolism, and some kind of interaction of a reactive metabolite and cell constituents are subsequent steps in the initiation of tumor formation. Thus, metabolism of (pro)genotoxic compounds is important in the process from chemical exposure to tumor formation. In attempts to study the relationship between genetic polymorphisms of biotransformation enzymes and the risk of cancer, a lot of epidemiological studies have been performed. These studies often measure the formation of tumors as an end-point, but also DNA adducts or cytogenetic damage are measured ex vivo, for example, in lymphocytes, as surrogate markers for geno-

toxicity. Populations used for such studies are often those that have been exposed occupationally, in combination with an unexposed control population. In such cases, a large interindividual variability in exposure might occur. This variability in exposure should be monitored closely and be taken into account in the interpretation of the results. This also indicates that studies that measure relations between exposure and the formation of tumors as an end-point are very difficult to perform, because the time between initiation and the final formation of tumors is believed to take several years. It is impossible to monitor the individual exposure over such a long time. Furthermore, if the effects of a specific compound are studied, one should realize that also exposure to other potential genotoxic compounds might occur during the development of a tumor. Epidemiological studies thus are useful when individual exposure to specific compounds is closely monitored. In that case, relationships between the exposure to a certain compound, the presence of a genetic polymorphism of a biotransformation enzyme, and some genotoxic effects can be studied. Furthermore, it would be advantageous when combinations of genetic polymorphisms of biotransformation enzymes are studied. However, a consequence of these multiparameter studies is that the number of individuals needed for such studies becomes very large for statistical reasons. Epidemiological studies should, however, be combined with mechanistic studies into the effects of biotransformation of specific compounds on genotoxicity. Although genetic polymorphisms of biotransformation enzymes might be important factors in the susceptibility toward genotoxic effects and the formation of tumors, it is clear that many other factors influence this susceptibility.

B. Drug Development A number of genetic polymorphisms of biotransformation enzymes have been shown to have significant effects on the pharmacological activity and the occurrence of side effects of drugs in humans. Therefore, the possible influence of genetic polymorphisms on drug metabolism should be taken into account in drug development studies. The fact that a genetically polymorphic


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enzyme is involved in the metabolism of a compound might even result in no further development of that compound as a drug for human use. Using either purified or heterologously expressed biotransformation enzymes, nowadays, it is easy to determine, in the preclinical stage of drug development, whether a compound will be metabolized by a genetically polymorphic enzyme. The involvement of the genetic polymorphism may be further substantiated by ex vivo studies with human tissues of known genotype. Although it is relatively easy to determine whether a compound is a substrate for a genetically polymorphic enzyme, the implications for the in vivo metabolism in humans are more difficult to determine. To this end, so-called bottom-up physiologically based pharmacokinetic (PBPK) models, which are based on individual isoenzyme activities, may be useful tools. Using such bottom-up PBPK models, as is done for the first time in the studies described in this thesis, interindividual variability in enzymatic activities can be modeled easily. Furthermore, a more rational prediction of the consequences of genetic polymorphisms of biotransformation enzymes for the in vivo metabolism of drugs and toxic compounds can be made.

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