Molecular genetics of the human cytochrome P450 ... - Toxicology

x e n o b io t ic a

, 1998, v o l . 28, n o . 12, 1129± 1165

M olecular genetics of the hum an cytochrom e P450 m onooxygenase superfam ily G. SM ITH, M . J. STUBBINS, L. W . HARR IES and C. R. W OLF* Biomedical Research Centre, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK

In trodu ction

Cytochrome P450 genes (P450s) encode a multigene superfamily of mixedfunction monooxygenases responsible for the phase I oxidative metabolism of a wide range of structurally diverse substrates. The superfamily is subdivided into a number of families and subfamilies, based on nucleotide sequence homology (N elson et al. 1996), where genes within a family have a minimum of 40 % sequence identity. Cytochrome P450 proteins catalyse many important endogenous biochemical processes within the mammalian cell, including steroid hormone, prostaglandin and leukotriene biosynthesis. In contrast, P450 enzymes in gene families 1± 4 (table 1) are located in the endoplasmic reticulum where, along with their redox partner NADPH-450 reductase, they are primarily active in foreign compound metabolism. Indeed, m etabolism by one or more P450 isozyme is often the ® rst line of defence of an organism against the toxic or mutagenic eå ects of administered drugs or environm ental pollutants. P450-catalysed reactions increase substrate hydrophilicity by introducing a reactive centre into the substrate molecule, which can then be further conjugated by reaction with phase 2 enzymes such as the glutathione S-transferases (GSTs) or Nacetyl transferases (NATs) (Gibson and Skett, 1994). Although P450-mediated reactions are primarily detoxi® cation processes, certain substrates are metabolically activated following P450 metabolism, resulting in the generation of reaction products with increased toxicity or mutagenicity. As a result, inter-individual variation in P450 expression can have profound toxicological consequences (Tucker 1994). P450 proteins are predominantly expressed in the liver, although speci® c isozymes are also expressed in many extrahepatic tissues, including the lung, kidney and gastrointestinal tract (Raunio et al. 1995 and references therein). Expression of speci® c P450 isozymes is often restricted to a particular cell type, and can therefore result in tissue and cell-speci® c toxicities as a consequence of P450-mediated activation of toxic or mutagenic substrates. Individual P450 isozymes have distinct substrate speci® cities, active site structures and mechanisms of regulation (G onzalez 1990). These will be discussed in detail below. There is some degree of overlap in substrate speci® city, however, particularly between members of the same subfamilyÐ this ¯ exibility in active site structure allows the P450 superfamily to accommodate a wide range of structurally diverse substrates. There are numerous examples of compounds which are * Author for correspondence. 0049± 8254} 98 $12 . 00 ’

1998 Taylor & Francis Ltd

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G. Smith et al. Table 1. Gene family

Chromosomal localization

Polymorphism identi® ed ?

References

CYP1A1 CYP1A2 CYP1B1 CYP2A6 CYP2A7 CYP2A13 CYP2B6 CYP2C8 CYP2C9 CYP2C18 CYP2C19 CYP2D6 CYP2E1 CYP2F1 CYP2J2 CYP3A4 CYP3A5 CYP3A7 CYP4A9 CYP4A11 CYP4B1 CYP4F2 CYP4F3

15q22-q24 Ð 2 19q13.1-q13 .2 Ð Ð 19q12-q13 .2 10q24.1 10q24.1 10q24.1 10q24.1 22q13.1 10q24.3-qter 19q13.2 Ð 7q22-qter Ð Ð Ð 1 1p12-p34 Ð Ð

yes no no yes

Jaiswal et al. (1985) Quattrochi et al. (1985) Sutter et al. (1994) Phillips et al. (1985)

Table 2. Gene family CYP1A1 CYP1A2 CYP2A6 CYP2C9 CYP2D6 CYP2E1 CYP3A3} 4

Human xenobiotic-metabolizing cytochrome P450 genes.

no no yes no yes yes yes no no no no no no no no no

Nelson et al. (1996) Miles et al. (1988) Kimura et al. (1987) Shimada et al. (1986) Romkes et al. (1991) Romkes et al. (1991) Gonzalez et al. (1988) Song et al. (1986) Nhamburo et al. (1990) Nelson et al. (1996) Beaune et al. (1986) Aoyama et al. (1989) Kitada et al. (1987) Kawashima et al. (1994) Kawashima et al. (1992) Nhamburo et al. (1989) Kikuta et al. (1994) Kikuta et al. (1993)

P450 substrates and inhibitors used as metabolic probes. Substrate caå eine, phenacetin caå eine, phenacetin R-warfarin coumarin tolbutamide, torasemide, S-warfarin, phenytoin debrisoquine , codeine, dextrometh orphan, sparteine chlorzoxazone caå eine, dapsone, erythromycin, lignocaine, midazolam

Inhibitor a -naphtho¯ avone furafylline, a -naphtho¯ avone

sulphamethazole quinidine diethyldithiocarbamate gestodene, triacetyloleandomycin

substrates for more than one P450 isozyme. The steroid testosterone, for example, can be hydroxylated at eight distinct ring positions to stereo- and regiospeci® c reaction products by diå erent human P450 isozymes (Arlotto et al. 1991). Interindividual variation in P450 expression has been the focus of intense study in recent years. A number of human P450 genes are now known to be polymorphically expressed (table 1) and, for the majority of these, the genetic basis for the variation in enzyme activity is known and rapid DNA-based methods of determining an individual’ s drug-metabolizing capability are available. There is a number of P450 substrates or inhibitors which can be administered as in vivo probes of isozyme-speci® c metabolic activity (table 2). Following administration of an appropriate test compound, the metabolic ratio, i.e. the ratio of excreted metabolite to unm etabolised substrate can be determined, and an estimate made of enzyme function. Such phenotypic assessment has certain limitations, how ever. For

M olecular genetics of CYP P450

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example, many compounds can be metabolized by more than one metabolic pathway and, for these, it is diæ cult to attribute interindividual variations in the metabolic ratio to the relative levels of a speci® c P450 enzyme. This is further compounded by the possibility of drug± drug interactions due to other medications being taken concommitantly, or by the eå ects of smoking or alcohol. Impaired renal clearance and altered liver function can also aå ect in vivo drug clearance and thus metabolic ratio (Tucker 1994). A more accurate assessment of metabolic capacity can be made by DNA-based ` genotyping ’ assays, in which the presence of speci® c DNA mutations can be detected. Currently, most mutations are detected using one of the following techniques (Spurr et al. 1991) : (1) Ampli® cation of a region of genomic DNA ¯ anking the mutation site using the polymerase chain reaction (PCR), followed by the use of sequence-speci® c restriction endonuclease digestion, direct sequencing or single stranded conformational polymorphism (SSCP) techniques to con® rm the presence } absence of the mutation of interest. (2) Use of allele-speci® c PCR using a series of speci® c primers, each of which anneals to only one allelic variant. (3) Restriction fragment length polymorphism (RFLP) analysis, where genomic DNA is digested with a restriction endonuclease which has a recognition site at the polymorphic region of interest. Individual DN A samples are therefore cleaved or are resistant to digestion, dependent on the speci® c DN A sequence possessed by each individual. The digested DNA is then Southern blotted and hybridized with a gene-speci® c radiolabelled DNA probe. Using these techniques it is now possible to accurately predict the genetic variability in individual drug-metabolizing enzymes and to estimate allele frequencies within any given population. Allele frequencies for human drugmetabolizing enzymes can vary widely between populations from diå erent ethnic origins. For example, the most common mutations in CYP 2D6 are present at approxim ately 7 % in the Caucasian population (Eichelbaum et al. 1979), but have a frequency of ! 1 % in Orientals (Lou et al. 1987). For a number of drug-metabolizing enzymes, mutations have been identi® ed by DNA-based methods which can be correlated with a particular metabolic phenotype. For example, there are several mutations within the CYP 2D6 gene which lead to a ` poor metabolizer ’ phenotype, where no functional protein is produced. Aå ected individuals are unable to metabolise compounds which are CYP 2D 6 substrates (Smith et al. 1995 and references therein). Human P450s metabolize many compounds which are mutagenic and } or carcinogenic (Guengerich 1991, 1994)Ð altered substrate pharmacokinetics arising from speci® c mutations can therefore lead to altered toxicity or mutagenicity in aå ected individuals. As a consequence, mutations within the human drugmetabolizing enzymes have been associated with disease susceptibility. The incidence of certain cancers is often dependent on exposure to a speci® c initiating carcinogen. Exposure to the genotoxic difuranocoum arin mycotoxin a¯ atoxin B1 (AFB1), a product of the mould Aspergillus ¯ avus, has been associated with the development of hepatocellular carcinoma in communities which have consumed AFB1-contaminated diets (Hall and W ild 1994). It is therefore important accurately to determine mutation frequencies within the exposed population and to correlate the results obtained with appropriate controls. A¯ atoxin B1 can be

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metabolized to a reactive epoxide by a number of distinct P450 isozymes within the CY P1A, CYP2A and CYP 3A subfamilies (Forrester et al. 1990, Aoyama et al. 1990). Individuals expressing mutant forms of these proteins m ay therefore have an increased ability to activate AFB1 to its toxic form. M utations have been detected in human P450 genes, the functional consequences of which are presently unclear and which have not yet been associated with a speci® c metabolic phenotype. It is of obvious importance in such studies to be able to associate allelic diå erences, particularly those outw ith the coding region of a gene, e.g. within the promoter, within introns or at hypervariable microsatellite loci, with a phenotype. This is particularly important when attempting to establish a rationale for apparent associations with disease susceptibility. Reports of genetic polymorphism in speci® c P450 genes are described below.

C ytochrom e P 450 CY P 1

Two human CYP1A genes have been described, CYP 1A1 and CY P1A2. Both are located on human chromosome 15 (Hildebrand et al. 1985 ; table 1). CYP 1A1 is predominantly expressed in extrahepatic tissues including lung (Antilla et al. 1992), where it metabolizes, and is inducible by polycyclic aromatic hydrocarbons such as benzo(a)pyrene (Shimada et al. 1989). In contrast, CYP 1A2 is expressed almost exclusively in the liver, although low levels of expression have been detected in brain (Farin and Omiecinski 1993), duodenum (McDonnell et al. 1992) and umbilical vein endothelia (Farin et al. 1994). CY P1A2 preferentially metabolizes nitrosamines and arylamines (Wakabayashi et al. 1992) and is one of the isozymes which catalyses the hydroxylation of a¯ atoxin B1 (Galagher et al. 1994). CYP1A2 expression is inducible on exposure to cigarette smoke (Sesardic et al. 1988) and following consumption of charbroiled foods and cruciferous vegetables (Guengerich 1994). In situ hybridization analysis for CYP 1A mRNA expression in a variety of tissues has demonstrated considerable heterogeneity of expressionÐ in the liver, for example, the highest level of CY P1A2 mRNA is in hepatocytes surrounding terminal hepatic venules and intercalated veins (McKinnon et al. 1991). The expression of both CYP 1A1 and CYP 1A2 is regulated at the level of transcription by a process which is initiated by the binding of an appropriate inducing agent to the cytosolic Ah or dioxin receptor (Poland et al. 1976 ; ® gure 1). The human Ah receptor has been cloned and localized to chromosome 7p21 by ¯ uorescence in situ hybridization (Ema et al. 1994). The Ah receptor protein is a 484 amino acid helix-loop-helix transcription factor with partial sequence homology to the developmental genes sim and per from Drosophila melanogaster (Burbach et al. 1992). Transgenic mouse lines, in which the gene encoding the Ah receptor is disrupted by homologous recombination, have now been generated in two laboratories (Fernandez-Salguero et al. 1995, Schmidt et al. 1996). The ® rst of these (FernandezSalguero et al. 1995), in which the Ah receptor gene is disrupted by a deletion in exon 1, produced animals which were completely unresponsive to a range of CY P1A1-inducing agents, including TCDD. These animals also exhibited high neonatal m ortality and lethality, a marked decrease in liver size and a signi® cant increase in lymphocyte in® ltration of lung, intestine and urinary tract, suggesting that the Ah receptor may play some role in the developm ent of both the liver and the immune system. The second transgenic line (Schmidt et al. 1996), generated by a


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Figure 1. Ah receptor-me diated transcriptional regulation of CYP1A genes. CYP1A1 transcription is activated following binding of a complex containing ligand-bound Ah receptor and Arnt protein to a speci® c XRE in the 5 ´ promoter of the gene. Unliganded Ah receptor exists in the cytoplasm where it is complexed with two molecules of Hsp90. Hsp90 is dissociated following ligand binding and the resulting Ah receptor± ligand complex is further conjugated with Arnt, the aryl hydrocarbon nuclear translocator, either in the cytoplasm or nucleus.

deletion in exon 2 of the Ah receptor gene also showed a decrease in liver size, but were viable, fertile and showed no evidence of lymphocyte in® ltration of major organs. The reasons for the phenotypic diå erences between the two mouse lines remain unclear, but may be due to subtle diå erences in genetic background, environm ental factors or the creation of a ` partial ’ knockout, resulting in the generation of Ah receptor allelic variants with altered activities. The unliganded Ah receptor exists in the cell cytosol as a 280 kD com plex with two molecules of Hsp90 (Denis et al. 1988, Fukunaga et al. 1995). Following ligand binding, Hsp90 is dissociated from this complex (Pongratz et al. 1992) and the ligand-bound Ah receptor associates with ARN T, the aryl hydrocarbon nuclear translocator protein (Reyes et al. 1992). Binding of the resulting Ah receptor } ARNT dimer activates CYP 1A1 transcription by binding to the cis-acting xenobiotic response element (XRE ; consensus binding sequence 5 ´ TCAC GC 3 ´ in the 5 ´ promoter region of the gene (® gure 1)). Expression of unbound ARN T protein has been detected in both the cytoplasm and nucleus of the cellÐ it has not yet been unequivocally demonstrated whether the association of AR NT with the Ah receptor occurs before or following nuclear translocation. The liganded Ah receptor } ARN T com plex requires phosphorylation, almost certainly catalysed by protein kinase C (PK C), before it can bind to the XRE (Pongratz et al. 1991, Carrier et al. 1992). It is not yet known, how ever, at which stage of the translocation process, or at which subcellular localization the phosphorylation event occurs.

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Cytochrome P450 CY P1A1 CYP 1A1, the human aryl hydrocarbon hydroxylase, has been localized to human chromosome 15q22-24 (Jaiswal et al. 1985). In addition to the XRE element, which mediates induction by polycyclic aromatic hydrocarbons, the CYP1A1 promoter contains a number of negative regulatory elements (Boucher et al. 1993, 1995) which have been identi® ed by DNA footprint analysis of heterologously expressed promoter constructs. The transcription factors that bind to these regulatory sequences, and the mechanism of suppression of transcription, remain to be identi® ed. CY P1A1 expression is also induced on exposure to retinoic acid (Vecchini et al. 1994). A functional retinoic acid responsive element (5 ´ CTTAGGTCACCA CGGGGCA 3 ´), present in the CYP 1A1 prom oter, has been shown to mediate this induction. Individual variation in CYP1A1 inducibility, determined by measuring AHH activity, is well documented (Kouri et al. 1982, Petersen et al. 1991). Functional variability in AHH activity can arise for a variety of reasons, including polym orphic expression of the CY P1A1 gene itself or genetic diå erences in the Ah receptor or AR NT genes. Indeed, individual variation in the expression of both the Ah receptor and ARN T have been detected by RT -PCR analysis on mRNA extracted from human liver, lung and blood samples (Hayashi et al. 1994). Using SSCP analysis, Kawajiri et al. (1995) have identi® ed two distinct human Ah receptor sequences which diå er by a single amino acid (Arg ! Lys substitution) at position 554. The lysine variant, which had an allele frequency of 43 % in the Japanese population studied, was not, however, associated with any alteration in AHH inducibility. Three allelic variants have been described for the CY P1A1 gene. The ® rst of these, which has been associated with increased inducibility, involves a T ! C transition in the 3 ´ non-coding region of the gene (Kawajiri et al. 1985, Petersen et al. 1991). The T ! C transition introduces an M sp 1 restriction site and can therefore be detected by conventional RFLP analysis (Spurr et al. 1987). The allele frequency of this polymorphism is considerably higher in Oriental populations (31 % in Japanese ; Kawajiri et al. 1990) than in the Caucasian population (12 % in North Americans ; Petersen et al. 1991). A second mutation, an isoleucine to valine amino acid substitution in exon 7, resulting from an A ! G nucleotide substitution at position 4889 within the conserved heme-binding region of CYP 1A1 is linked to the M sp I polymorphism and can be detected by a PCR-based assay (Hayashi et al. 1991). Expression of the valine allele in yeast indicated that it has a 2-fold increase in AHH activity compared with the parental enzym e. The third CY P1A1 allele, which can also be detected by M sp I RFLP analysis, has only been detected in the African-American population, where it is present at an allele frequency of 17 % (Crofts et al. 1993, Taioli et al. 1995). The phenotypic consequences of this allele, a second T ! C substitution in the 3 ´ non-coding region of the gene, are not known.

Cytochrome P450 CY P1A2 The CY P1A2 gene spans approximately 7 .8 kb of genomic DNA (Ikeya et al. 1989) and shares approximately 70 % nucleotide identity with the coding region of CY P1A1. The corresponding mRNA contains an initial non-coding exon and encodes a 515 amino acid protein with a predicted molecular weight of 58 294. Consensus sequences for several putative regulatory motifs are present im mediately


1135

upstream of the transcriptional start site in CY P1A2Ð these include a TAT A box and SP1 recognition sequence (Eaton et al. 1995) in addition to an XRE element. Genetic polym orphism at this locus has been noted (Kalow and Tang, 1991, Kalow and Bertilsson 1994) but, to date, a genetic basis for the individuality in CY P1A2 expression has not been described. CYP 1A2 protein levels have been shown to vary by " 40-fold in panels of human liver samples (Butler et al. 1989, Forrester et al. 1992). A splice variant of CY P1A2, in which exon 4 is deleted, has been isolated by RT-PCR from an individual who had no immunodetectable CY P1A2 protein on W estern blot analysis. Further analysis, how ever, revealed that all individuals appear to possess low levels of this incorrectly spliced transcript, in addition to the full-length, correctly spiced CYP 1A2 mRNA (Schweikl et al. 1993). In the absence of a de® ned genetic polymorphism in CYP 1A2, individual CY P1A2 activities are presently measured by phenotypic analysis. Caå eine, which is metabolized to paraxanthine by CYP 1A2, is the most commonly administered metabolic probe of enzyme function (Butler et al. 1989, Kalow and Tang, 1991). CY P1A2 levels are also estimated using the analgesic drug phenacetin (Distelrath et al. 1985) and theophylline, which is produced from the 7-demethylation of caå eine (Robson et al. 1988). The interpretation of such studies can be complicated, however. For example, caå eine is also metabolized by CYP 2E1 and CYP 3A4 (K adlubar et al. 1990, Butler et al. 1992). The expression of both these proteins is also subject to marked interindividual variation.

Cytochrome P450 CY P1B A novel 5 .1 kb cDNA for a P450, now designated CYP 1B1, has been isolated from a human keratinocyte cell line, by selecting individual clones which were highly inducible by TCDD (Sutter et al. 1994). The CYP 1B1 gene is localized on human chromosome 2 and encodes a 543 amino acid protein. Like the CYP1A genes, the CYP1B1 promoter contains an XRE and is regulated by the Ah receptor. Southern blot analysis indicates that the CYP1B subfamily contains only a single gene, which is expressed in at least 15 human tissues. Levels of CYP1B1 mRNA is particularly high in the kidney, but this observation was based on a tissue panel generated from a single individual and therefore requires further con® rmation. It is interesting that in rodents CYP 1B1 is expressed in many steroidegenic tissues and, intriguingly, it has been detected in hum an breast and prostatic tumours (McKay et al. 1995, M urray et al. 1995). CY P1B1 has been shown to catalyse the 4hydroxylation of estradiol (Spink et al. 1994), an activity which is induced in human breast and endometrial tumours (Liehr et al. 1995). It will therefore be of particular interest to establish the speci® c metabolic role played by this enzyme.


Cytochrome P450 CY P2A The human CYP 2A genes are located on chromosome 19q13.2 in a cluster with members of the CYP 2B and CYP2F gene families (Miles et al. 1989a, Hoå m an et al. 1995). The human CY P2A gene family consists of three functional genesÐ CYP2A6, CY P2A7 and CY P2A13 and two tandemly arranged pseudogenes which are identical

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Figure 2. Structural organization of the CYP2 family genes on human chromosome 19. The arrangement of CYP2 family genes on a 350 kb region of human chromosome 19 is illustrated. The direction of transcription is illustrated by arrows (redrawn from Fernandez-Salguero et al. 1995).

(® gure 2). The two pseudogenes, CY P2A7P(T) and CYP2A7P(C), are highly homologous to CY P2A7, but contain two single nucleotide deletions in exons 3 and 4 which lead to frameshift mutations and the production of truncated versions of CY P2A7, lacking a large part of the coding sequence from the 3 ´ end of exon 5 (Fernandez-Salguero et al. 1995). As a consequence, these genes do not possess the conserved hem e-binding domain and are inactive. CYP 2A genes have been extensively characterized in several mammalian species including mouse, rat, hamster, rabbit and human. It is interesting to note that marked interspecies diå erences exist in CY P2A gene expression which exempli® es the diæ culties in the use of experimental animals as predictive models of human drug metabolism (Fernandez-Salguero and Gonzalez, 1995). These diå erences are re¯ ected in both the substrate speci® city and turnover number of these enzymes. For example, rat CYP 2A3 and mouse Cyp2a-4 are expressed and inducible in rat and mouse lung tissue respectively, but no CYP 2A proteins have been identi® ed in human lung (Fernandez-Salguero and Gonzalez 1995). In addition, coumarin, a know n substrate for human CYP 2A6 and murine Cyp2a-5, is not rapidly metabolized in the rat but in man is hydroxylated by CY P2A6 to generate an intermediate which can then be conjugated with glucuronic acid (Miles et al. 1990).

Cytochrome P450 CY P2A6 A partial CYP 2A6 cDNA was the ® rst human P450 cDNA to be isolated (Phillips et al. 1985). CYP2A6 protein was subsequently puri® ed from human liver and shown to catalyse coumarin oxidation (Maurice et al. 1991, Yun et al. 1991). The role of CY P2A P450s in coumarin 7-hydroxylation was ® rst suggested by the inhibition of coumarin metabolism in human liver microsomes by an antibody raised to murine Cyp2a-5 (Raunio et al. 1988). Subsequently, a full-length CYP 2A6 cDNA clone was isolated, using a probe from the C-terminal 211 amino acid residues of the rat CYP 2B1 gene to screen a human liver cDNA library. This was shown to encode a protein with coumarin hydroxylase activity (Miles et al. 1990a). A second CY P2A6 cDNA clone, CYP 2A6v1, which diå ers from CYP 2A6 by a single amino acid was isolated from a human liver cDNA library using a rat CYP 2A1 cDNA probe (Yamano et al. 1990a). Studies using human liver microsomes to analyse coumarin 7-hydroxylase activity have demonstrated marked inter-individual diå erences in CYP2A6 enzyme activity (Kapitulnik et al. 1977, Pelkonen et al. 1985). In some cases the catalytic activity towards coumarin was very low or undetectable and was re¯ ected in a very low level of CYP2A6 mRNA (Miles et al. 1990a, Yamano et al. 1990a). This


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variation has been partly attributed to the variant CYP 2A6v1 allele, which has a single amino acid diå erence (Leu " ’ ! ! His), resulting in the production of an unstable, catalytically inactive enzyme (Yamano et al. 1990a). CYP2A6v1 represents approxim ately 10± 20 % of CY P2A6 alleles in Caucasian and Asian subjects, but is either absent or present at a very low frequency in African-Americans (FernandezSalguero et al. 1995). From phenotyping studies, it was found that a minority of individuals who were unable to metabolize coumarin were homozygous for the CY P2A6v1 allele, while heterozygous individuals had levels of metabolism that ranged between 50 and 60 % of the maximum value in the population studied (Fernandez-Salguero et al. 1995). However, a minority of individuals, homozygous for the CYP 2A6v1 allele, had similar catalytic activities towards coumarin as those exhibited by heterozygous subjects. Interestingly, no additional sequence diå erences were found between homozygous individuals completely lacking coumarin metabolism and those having 40± 50 % of the maximum coumarin hydroxylase activity (Fernandez-Salguero et al. 1995), suggesting that another P450 gene or an as yet unidenti® ed CYP2A6 allele can metabolise coumarin under these circumstances. M ore recently, a further CY P2A6 allelic variant (CYP 2A6v2) has been identi® ed as part of a 350 kb CYP2-family cluster on chromosome 19 (Fernandez-Salguero et al. 1995). CYP2A6v2 shares a high degree of homology with CYP 2A7, especially in exons 3, 6, and 8. It has been proposed that, due to the high degree of homology of genes within the CYP 2A gene family, the similarity between these two tandemly arranged genes is due to previous gene-conversion events between CYP 2A6 and CY P2A7 (Fernandez-Salguero et al. 1995). The CY P2A6v2 allele is particularly common in Japanese, representing 28 % of CY P2A6 alleles, compared with 2± 6 % in other ethnic groups (Fernandez± Salguero et al. 1995). To date, the phenotypic consequences of inheriting the CYP2A6v2 allele have not been determined. If, however, CYP2A6v2 is inactive towards coumarin, as is the case for CYP2A7, approxim ately 10 % of the Japanese population can be predicted to be poor metabolizers of coumarin and other compounds which are CYP 2A6 substrates. In addition to their role in detoxi® cation, the cytochrome P450s also activate a wide range of com pounds to cytotoxic and mutagenic intermediates. For example, CY P2A6 metabolises and activates a number of promutagens such as Nnitrosodiethylamine (NDEA) (Crespi et al. 1990, Camus et al. 1993), 4(m ethylnitrosamino)-1-3(-pyridyl)-1-butone (NNK) (Crespi et al. 1991) and a¯ atoxin B " (Aoyama et al. 1990). Therefore, allelic variation at the CYP 2A6 gene locus could be of importance in the assessm ent of cancer risk.

Cytochrome P450 CY P2A7 The CYP 2A7 cDNA encodes a 49 kDa protein which has 96 % nucleotide sequence identity and 94 % amino acid sequence identity to CYP2A6 (Miles et al. 1990a, Yamano et al. 1990a). mRN A analysis indicates that, in certain individuals, CY P2A7 is expressed at higher levels than CYP 2A6 (Ding et al. 1995). However, CY P2A7 is catalytically inactive toward coumarin (Yamano et al. 1990a, Ding et al. 1995) and, to date, no CYP 2A7 substrates have been identi® ed. The CY P2A7 gene can be alternatively spliced to produce a transcript (CY P2A7AS) lacking exon 2, but containing three amino acids derived from exon 1 (Yamano et al. 1990a, Ding et al. 1995). CYP 2A7AS produces a truncated 44 kDa protein containing the conserved

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P450 heme-binding region which could therefore still function as a monooxygenase enzyme. However, CYP 2A7AS does not contain exon 2, which may contain speci® c amino acid residues predicted to be responsible for substrate recognition (Gotoh 1992, Ding et al. 1995). In addition, it is not yet known whether the truncated CY P2A7AS protein binds haem eæ ciently, as the level of CYP 2A7A S protein obtained from the COS-cell based expression system used was too low for this to be determined (Ding et al. 1995). Interestingly, it appears that CYP 2A7A S is the major CY P2A7 mRN A in ® broblasts (Ding et al. 1995). To date, no genetic polymorphism has been identi® ed at the CY P2A7 gene locus.

Cytochrome P450 CY P2A13 CYP 2A13 is the most recently discovered CYP 2A gene and was identi® ed within a 350-kb region of chromosome 19 together with CYP 2A and CYP 2B genes (H oå man et al. 1995). CYP2A13 shares 95 % nucleotide and 93 % amino acid sequence identity with CYP2A6. Interestingly, murine Cyp2a-4 and Cyp2a-5 catalyse the 7-hydroxylation of coumarin and the 1 5 a -hydroxylation of testosterone respectively, but diå er in a total of only 11 of 494 amino acids. Using site-directed mutagenesis, Lindberg and Negishi (1989) demonstrated that the activity diå erence depended critically on the amino acid residues at positions 117, 209 and 365. Further, a single mutation converting Phe # ! * ! Leu was suæ cient to convert Cyp2a-4 into a steroid 15 a hydroxylase. It is interesting to note that both CYP2A7 and CYP 2A13 have alanine at the position corresponding to amino acid 117 in murine Cyp2a-4 and Cyp2a-5, instead of the valine present in CY P2A6. The identical amino acid substitution (Ala ! Val" " ( ) in Cyp2a-5 results in a signi® cant loss of coumarin 7-hydroxylase activity. To date, no genetic polymorphism has been identi® ed at the CYP 2A13 gene locus.

Cytochrome P450 CY P2B The human CY P2B subfamily contains two active genes, CYP 2B6 and CY P2B7, and has been localized to human chromosome 19q12-13.2, in close proximity to the human CYP 2A and CYP 2F genes (Hoå man et al. 1995). CYP 2B6 is the only member of the CYP2B family to be expressed in human liverÐ the expression of CY P2B7 is restricted to the lung (Nhamburo et al. 1989). A 3 .2 kb cDNA clone for CYP2B6, encoding a 48-kd protein, was isolated from a human liver cDNA library (Yamano et al. 1989b). Two alternatively spliced CY P2B6 mRNAs have also been describedÐ the ® rst contained part of intron 5 and lacked exon 8 (Miles et al. 1989b), while the second clone arose from an aberrant splicing mechanism at the intron 3 } exon 4 splice junction (Miles et al. 1990b). Both splice variants were found to be present in all human liver samples examined, and were always found in the presence of the full-length, correctly spliced transcript. Neither splice variant is capable of producing a functionally competent CY P2B6 protein. Hepatic CYP2B6 expression varies widely between individuals, with certain individuals having no immunodetectable CY P2B protein expression (Forrester et al. 1992). No molecular mechanism has yet been proposed to account for this variation.


1139

Constitutive levels of hepatic CYP 2B6 are relatively lowÐ CYP 2B6 comprises only 1 % of the total hepatic P450 expression (Mimura et al. 1993). As such, CY P2B6 is thought to play a relatively minor role in xenobiotic metabolism. In vitro experiments with recombinant protein have shown that CYP2B 6 will activate several substrates including 6-aminochrysene and nicotine (Mimura et al. 1993). The relative contribution of this enzym e to these activities in vivo is less clear. Certain substrates, including the anti-cancer drugs cyclophosphamide and ifosphamide, however, are speci® cally metabolized by CY P2B6 (Chang et al. 1993), and any interindividual variation in the level of hepatic CYP 2B6 expression may therefore have signi® cant clinical consequences, both in terms of therapeutic outcomes as well as side eå ects experienced. CYP 2B expression in rodents is highly induced by administration of barbiturates such as phenobarbital (Waxman and Azaroå 1992). Experiments using primary cultures of human hepatocytes and human tumours grown as xenografts in the immune-suppressed mouse have demonstrated that human CYP 2B6 expression is also induced following barbiturate administration (Smith et al. 1992, 1993). Although the induction of CYP 2B gene expression is thought to be regulated at the transcriptional level, the molecular mechanisms regulating phenobarbital induction of gene expression in mammalian cells are not yet understood. Several phenobarbital-responsive regions, including a putative ` barbie-box ’ element have, however, been identi® ed in the 5 ´ promoter sequences of CYP 2B and other mammalian barbiturate inducible genes (Shephard et al. 1994). In bacterial systems, the molecular mechanisms which regulate increased gene transcription following exposure to barbiturates have been more extensively characterized. A palindromic sequence element in the 5 ´ promoter region of the barbiturate-inducible bacterial P450 BM- $ from Bacillus megaterium (CY P102) has been shown to bind a repressor protein, Bm3R1. Exposure to phenobarbital and similar compounds is thought to release this repressor protein from its binding site, thus activating gene transcription (Shaw and Fulco 1992).

Cytochrome P450 CY P2C The CYP 2C gene family consists of 4 genesÐ CYP 2C8, CY P2C9, CYP 2C18 and CYP 2C19, which span 500 kb of genomic DNA and are located on chromosome 10q24 in the order CYP 2C18-CY P2C19-CYP2C9-CYP 2C8 from centromere to telomere (Gray et al. 1995). A fourth gene, CYP 2C10, has also been reported (U mbenhauer et al. 1987) which is identical to CY P2C9 with the exception of two single base pair substitutions in the coding region. This was initially classi® ed as a separate gene product due to a divergent 3 ´ non-coding region. There has, however, been no subsequent evidence to support the existence of an additional CYP2C gene, and it now seems likely that CY P2C10 is a cloning artefact. The four members of the CY P2C family described above are primarily expressed in human liver.

Cytochrome P450 CY P2C8 CYP 2C8 cDNAs were cloned from human liver cDNA libraries using probes from both the rat CYP2C6 cDNA (Kimura et al. 1987) and the rabbit progesterone21-hydroxylase P450 1 cDNA (Okino et al. 1987). CYP2C8 metabolizes a large number of substrates including benzo(a)pyrene, carbamazapine, 7-ethoxycoumarin,

1140

G. Smith et al. Table 3. Differences in published CYP2C9/2C10 cDNA sequences. Sites of nucleotide differences Source of CYP2C9 sequenceö

4

1

2C9 Romkes et al. (1991) ² M61857

Leu CTT

Val Pro GTT CCT

2

2C9 Yasumori et al. (1987) ² D00173

3

2C9 Meehan et al. (1988) §

4

2C9 Kimura et al. (1987) ² Y00498

5

2C9 Ged et al. (1988) ² M21940 ³

6

2C9 Romkes et al. (1991) ² M61855

Leu CTT

7

2C10 Um benhauer et al. (1987) ² M15331

Leu CTT

X

Only sequence 1 of the above sequences was identified as a true allele CYP2C9 allele in both Caucasians and Orientals (CYP2C9*1). Previously reported os Ile (4) and Ser (6) respectively. Accession number for GenBank EM BL database cDNA not sequenced over this region. This cDNA sequence was rechecked and found to be as reported. = 100 % sequence homology between all CYP 2C9 cDNAs. = homology between two CYP 2C9 cDNA sequences. = alteration seen in one cDNA only.

No.

* ² ³ §

6

144

175

239

280

Arg CGT

Cys TGC

Phe TTT

Ser Glu Tyr TCT GAA TAC

Ile Gly Gly ATT GGC GGA

Leu CTT

Val Pro Arg GTT CCG CGT

Cys TGC

Phe TTT



Leu CTT

Val Pro GTT CCT

Cys TGT

Cys TAC

Phe CTT



Leu* Val* Pro CTT GTT CCT

Arg TGT

Cys TGC

Phe TTT

Ser TCA

Tyr TAC


Arg CGT

Cys TGC

Phe TTT

Ser Glu Cys TCA GAA TGC


Val Pro GTT CCT

Arg CGT

Cys TGC

Phe TTT


Leu CTT

Val Pro GTT CCT

Arg CGT

Cys TGC

Phe TTT

Ser Glu Cys TCA GAA TGC

Ile Asp Gly ATT GAC GGA

³

63

³

281

Stop TAA

358

359

testosterone, benzphetamine, retinol and retinoic acid (Leo et al. 1989, Yun et al. 1992, Kerr et al. 1994). Puri® ed CYP2C8 protein was more active than either CY P2C9 or CY P3A4 in activating benzo(a)pyrene to mutagenic products in Salmonella typhimurium indicating that CYP2C8 may play an important role in the genotoxicity of benzo(a)pyrene in human liver (Yun et al. 1992). CYP 2C8 is also the major enzym e responsible for the 6 a -hydroxylation of the anti-cancer drug taxol to its principal human metabolite and detoxi® cation product, 6 a -hydroxytaxol (Rahman et al. 1994). Several variant CYP 2C8 cDNA sequences have been reported, but it has not yet been determined whether these represent true CY P2C8 alleles. CY P2C8 protein expression has been reported to vary appreciably in human liver (Wrighton et al. 1987), but a genetic basis for this variation has not yet been identi® ed.

Cytochrome P450 CY P2C9 In most individuals, CYP 2C9 is the major CY P2C protein expressed in liver (G oldstein and de M orais 1994). CY P2C9 cDNA clones have been isolated by several laboratories. To date, six distinct CYP 2C9 cDNA sequences have been reported (table 3), which diå er by nine single base substitutions (Kimura et al. 1987, Yasumori et al. 1987, Ged et al. 1988, M eehan et al. 1988, Rom kes et al. 1991). The CYP 2C9 gene has been cloned and contains nine exons, spanning approxim ately 55 kb of genomic DNA (de M orais et al. 1993). The 5 ´ promoter

417

475

Gly Gly GGC GGT


1141

region of CY P2C9 shows higher homology with CYP 2C8 (75 % nucleotide identity), than with other CYP 2C family members. Analysis of a 2200 bp region of the CY P2C9 promoter reveals canonical TAT A boxes situated 57 bp upstream from the ® rst codon, several potential glucocorticoid regulatory elements (GREs), as well as AP-1, HNF-1 and C } EBP liver-enriched transcription factor binding sites (de M orais et al. 1993). The upstream regions of CY P2C8, CYP 2C9 and CYP 2C19 contain a sequence related to the consensus sequence for the hepatocyte-speci® c factor 1 (HPF-1). This sequence, which is similar to the consensus sequence for hepatic nuclear factor-4 (HNF-4), has been shown to be essential for the functional expression of rabbit CY P2C genes in the human hepatocyte-derived H epG 2 cell line (Venepally et al. 1992), but is not required for expression in cell lines which are not liver-derived (de M orais et al. 1993). This HPF-1 element appears to be conserved in CYP2A, CYP 2C and CYP 2D genes, and may be important for the hepatic expression of these genes (Venepally et al. 1992). CYP 2C9 was ® rst described as the P450 responsible for the 4 ´-hydroxylation of S-mephenytoin. However, recent evidence using recombinant CYP 2C proteins has shown that CYP 2C9 and associated allelic variants had very low catalytic activity towards S-mephenytoin, which is eæ ciently metabolized by CY P2C19 (Goldstein et al. 1994). CYP2C9 does, however, metabolize of a wide variety of commonly prescribed drugs including phenytoin, tolbutamide, torsemide, tienilic acid, ibuprofen and S-warfarin (Relling et al. 1990, Page et al. 1991, Rettie et al. 1992, Goldstein and de M orais 1994, M iners et al. 1995). The metabolism of tolbutamide and phenytoin by CYP2C9 is the major pathway of elimination of these drugs (Veronese et al. 1991). Phenotyping studies have identi® ed individuals with an impaired capacity to metabolize both tolbutamide and phenytoin (Kutt et al. 1964, Scott and Poå enbarger 1979, Vasko et al. 1980, Vermeij et al. 1988, Page et al. 1991), indicating that there may be a genetic polymorphism at the CYP 2C9 gene locus. Recently, CYP 2C9 has been shown to be polym orphic in both Caucasian and Oriental populations (Wang et al. 1995, Stubbins et al. 1996). Three alleles have been identi® ed in Caucasians, CY P2C9*1, CYP 2C9*2 and CY P2C9*3 (Stubbins et al. 1996) (table 1). Only two of these, CYP 2C9*1 and CY P2C9*3, have been identi® ed in Orientals (Wang et al. 1995). CYP 2C9*1 is the most common allele with an allele frequency of 79 % in Caucasians and 98 % in Orientals. A C ! T nucleotide substitution at codon 144 in CY P2C9*1 leads to an Arg ! Cys amino acid substitution (CYP 2C9*2). CY P2C9*2 has an allele frequency of 12 .5 % in the Caucasian population but has not been found in Orientals. An A ! C substitution at codon 359 in CYP2C9*1 results in the conversion of Ile ! Leu (CYP 2C9*3), and occurs with an allele frequency of 8 .5 % in Caucasians and 2 % in Orientals (Stubbins et al. 1996). W hether the presence of these allelic variants of CY P2C9 plays a role in the wide variability observed in CY P2C9 substrate metabolism remains to be elucidated. However, heterologous expression of each allelic variant in mammalian cells suggests that both CY P2C9*2 and CYP2C9*3 have altered abilities to metabolize CY P2C9 substrates (Kaminsky et al. 1993, Veronese et al. 1993). The observed activity diå erences may be explained by the location of Arg" % % in a region predicted to be associated with coupling to P450 reductase, and the localization of Ile $ & * in a region of the protein which has been associated with substrate binding (SRS 5) (Gotoh 1992).

1142

G. Smith et al.

Cytochrome P450 CY P2C18 The CY P2C18 cDNA was cloned from a human liver cDNA library screened with a cDNA for rat liver P450IIC13 and an oligonucleotide probe for human CY P2C8 (Rom kes et al. 1991). Two allelic variants of CY P2C18 were isolated from the same cDNA library, diå ering by only a single nucleotide and resulting in a M et ! Thr substitution at codon 385. A further variant CYP2C18 cDNA, containing six silent nucleotides and a single amino acid substitution (Phe # " * ! Leu), was isolated by PCR analysis of genomic DNA using primers generated from CY P2C9 (Furuya et al. 1991). Recombinant CYP2C18 was unable to metabolise compounds known to be substrates for CYP 2C9 and CY P2C19 and, to date, speci® c CY P2C18 substrates have not been identi® ed. CYP 2C18 shares 84, 86 and 86 % nucleotide sequence and 89, 93 and 93 % amino acid sequence identity with CY P2C8, CYP 2C9 and CY P2C19 respectively (Godstein and de M orais, 1994). However, CYP 2C18 mRNA is found at an approximately 7± 8-fold lower level in human liver than either CY P2C8 or CYP2C9 (Furuya et al. 1991). The CY P2C18 gene has been isolated and spans approximately 55 kb of genomic DNA (de M orais et al. 1993). Analysis of the CYP 2C18 promotor reveals canonical TATA boxes situated 57 bp upstream from the initiating codon, multiple glucocorticoid response element (GRE) consensus sequences, and a 15 base pair sequence with high homology to a 5 ´-¯ anking sequence responsible for barbiturate-inducible expression of P450 BM- $ (CYP102) in Bacillus megaterium. In addition, the promoter region of CYP 2C18 contains consensus sequences for AP-2, Sp1 and D BP elements, which are not found in CYP2C8 or CYP2C9 (de M orais et al. 1993). DBP in a liverspeci® c transcription factor which has been shown to play a role in the developmental regulation of expression of the rat CYP 2C6 gene (Yano et al. 1992). To investigate the activity of the CYP 2C18 promoter, a comparison was made of the activity of a 2 .2 kb 5 ´-¯ anking region of the CY P2C9 gene and a 1 . 3 kb 5 ´¯ anking region of CYP 2C18 (Ibeanu and Goldstein 1995). It was found that the ability of the 2 .2 kb region of CYP 2C9 to direct expression of a luciferase reporter gene in HepG2 cells was 25 times greater than that of the 1 .3 kb region of CYP 2C18 (Ibeanu and Goldstein 1995). However, care must be taken in the interpretation of these results, as it is possible that there are additional regulatory elements situated in the longer 2 .2 kb stretch of the CYP 2C9 promotor. Recently, a single base pair change has been identi® ed in the 5 ´-¯ anking region of the CY P2C18 gene, which has an allele frequency in Orientals of 21 . 4 % . This variant was found to be linked with the low activity m1 allele of the polym orphic CY P2C19 gene (Tsuneoka et al. 1996). The phenotypic consequences of these variations in CYP2C18 sequence are, as yet, unknown.

Cytochrome P450 CY P2C19 A CYP 2C19 cDNA was originally cloned from a human liver cDNA library screened with a cDNA for rat liver P450IIC13 and an oligonucleotide probe for human CYP 2C8 (Rom kes et al. 1991). CYP 2C19 has been identi® ed as the major human S-mephenytoin 4 ´-hydroxylase (Goldstein et al. 1994), but is also responsible for the metabolism of a number of other com monly prescribed drugs including omeprazole (Andersson et al. 1992), proguanil (Ward et al. 1991), certain barbiturates (Adedoyin et al. 1994, Ku$ pfer and Branch 1985), citalopram (Sindrup


1143

et al. 1993), and diazepam (Bertilsson et al. 1989). The CYP2C19 gene shares 91, 93 and 96 % amino acid sequence homology with CYP2C8, CYP 2C18 and CYP 2C9 respectively (Goldstein and de M orais 1994). A genetic polymorphism in the metabolism of S-mephenytoin is welldocumented (Ku$ pfer and Preisig 1984, W edlund et al. 1984, W ilkinson et al. 1989), with individuals classi® ed as either poor metabolizers (PMs) or extensive metabolizers (EMs) of S-mephenytoin. The PM phenotype is inherited as an autosomal recessive trait (Inaba et al. 1986, W ard et al. 1987). There are m arked ethnic diå erences in PM frequency, ranging from 2 to 5 % in Caucasians to 18± 23 % in Japanese (Nakamura et al. 1985, W ilkinson et al. 1989). The major genetic defect leading to the PM phenotype is a single-base pair (G ! A) substitution in exon 5 of CY P2C19 (CYP 2C19 m " ) which introduces an alternative splice site and leads to aberrant splicing of the CYP2C19 mRNA from codon 215 (de M orais et al. 1994a). As a consequence, a premature stop codon is introduced 20 amino acids downstream of the splice site (de M orais et al. 1994a). CYP2C19 m " accounts for approximately 80 % of defective alleles in both the Caucasian and Japanese populations. A second gene-inactivating allele (CYP 2C19 m # ) has recently been identi® ed in Japanese (de M orais et al. 1994b), where a G ! A base substitution in exon 4 creates a premature stop codon at amino acid 212 (de M orais et al. 1994b). In combination, CYP 2C19 m " and CYP 2C19 m # account for all Japanese PMs. CY P2C19 m # has not, however, been identi® ed in Caucasians, indicating the presence of one or m ore additional geneinactivating CYP 2C19 alleles.

Cytochrome P450 CY P2D In most individuals, the human CYP 2D gene cluster contains three genesÐ CY P2D6, CYP2D7 and CYP2D8 (Kimura et al. 1989)Ð which are arranged in tandem on human chromosome 22q 13.1 (Gough et al. 1993). Both CYP 2D7 and CY P2D8 are non-functional pseudogenes. CYP 2D8 contains a number of geneinactivating mutations and does not possess appropriate transcriptional machinery, e.g. a functional TATA box. In contrast, CY P2D7 has only a single geneinactivating mutation, a single base pair insertion in the ® rst exon, which leads to a frame shift and the generation of a premature stop codon (Kimura et al. 1989). Expression of CYP2D6, the only active gene, is genetically polym orphic (Mahgoub et al. 1977), with certain individuals exhibiting a severely compromised ability to metabolize CY P2D6 substrates. This is of particular importance as many CY P2D6 substrates are commonly prescribed drugs, several of which have a low therapeutic index. Indeed, interindividual variation in hepatic P450-mediated drugmetabolizing capacity was ® rst observed in man in patients experiencing toxic side eå ects following prescription of the anti-arrythmic drug sparteine and the antihypertensive drug debrisoquine, which are now well-characterized CYP 2D 6 substrates (Tucker et al. 1977, Distlerath and Guengerich 1984). Alteration in drug pharmacokinetics as a consequence of polymorphism in CYP2D6 can often lead to toxic and even life-threatening side eå ects (Tucker 1994). Drugs that are CYP2D6 substrates fall into several distinct pharmaceutical classes, and include anti-arrythmics (e.g. propafenone, ¯ ecainamide), antidepressants (e.g. desipramine, amitryptiline) and substances of abuse such as M DM A (ecstacy) (Smith et al. 1995 and references therein). Although structurally diverse, the majority of CYP 2D6 substrates are small molecules, containing a basic

1144

G. Smith et al. Table 4.

Allele" CYP2D6*1 CYP2D6*2A

CYP2D6*3 CYP2D6*4 CYP2D6*5 CYP2D6*6 CYP2D6*7 CYP2D6*8 CYP2D6*9 CYP2D6*10

CYP2D6*11

Cytochrome 450 CYP2D6 alleles. Nucleotide diå erences from wild-type CYP2D6

Trivial name Wild-type CYP2D6L1 CYP2D6N CYP2D6L2 CYP2D6L23 CYP2D6L23 CYP2D6A CYP2D6B CYP2D6D CYP2D6T CYP2D6E CYP2D6G CYP2D6C CYP2D6J CYP2D6Ch CYP2D6M CYP2D6F

none T ;G %#’) G C %#’) G C;C T ;G "(%* #*$) %#’) G C;C T ;G "(%* #*$) %#’) G C;C T ;G G

2 12

"(%*

"(%*

C;C

#*$)

#*$)

A 2 3 6 7 dele tion 2

%#’)

normal normal normal increased increased increased

C C C C

C T ;C A ;A G; ")) "!’# "!(# C G;G C ; G 1 9 34 A ; G "!)&

Enzyme activity

"(%*

inactive inactive %#’)

C

C Y P2D6 deleted T 1 7 9 5 deleted ; G A #!’% A 3023C G C ; G 1846T ; G T "(%* #*$) A 2 7 0 3 , G 2 7 0 4 , A 2 7 0 5 deleted C 188T ; C G ;G C "(%* %#’) C 188T ; C T ;G C;G C ""#( "(%* %#’) C 188T ; C T ;G C #*$) %#’) G 971C ; C A ;A G;C G "!’#

"!(#

"!)&

inactive inactive inactive inactive

reduced reduced reduced reduced inactive

" Allele nomenclature is according to Daly et al. (1996). Individual alleles are referenced therein. # Mutations leading to an altered phenotype are highlighted in b old .

nitrogen atom which is charged at physiological pH. It has recently been demonstrated (Ellis et al. 1996) that interaction of this positively charged nitrogen with the carboxylic acid group of an aspartic acid residue at position 301 in CYP 2D 6 is one mechanism by which the substrate is anchored in the active site of the protein. To date, 18 allelic variants of CYP 2D6 have been described (table 4 ; Daly et al. 1996). Several of these, the ` loss of function ’ alleles, contain single nucleotide insertions, deletions or substitutions which lead to the generation of a premature stop codon. All the gene-inactivating mutations detected to date lie upstream of the conserved cysteine residue which provides the sixth axial ligand to the P450 haem. Individuals inheriting two copies of these loss of function alleles, so called ` poor metabolizers ’ , therefore cannot produce CYP 2D6 protein and are unable to metabolize compounds which are CYP 2D6 substrates. Certain individuals have the entire CYP 2D6 gene deleted, i.e. they are homozygous for the CYP2D 6*5 allele (table 4). The exact position of the breakpoint within the CYP 2D gene cluster has not yet been identi® ed (Gaedigk et al. 1991). In addition to the above allelic variants, certain individuals inherit multiple copies of a CYP 2D6 variant sequence, presumably as a consequence of a gene duplication event. The num ber of CYP2D6 copies can be either 2, 3 or 13, arranged in tandem (Johansson et al. 1993). Aå ected individuals, ` ultra-rapid metabolizers ’ , require greatly increased doses of compounds which are CYP2D6 substrates in order to achieve therapeutic eå ect (Bertilsson et al. 1993). Further allelic variants of CY P2D6 have been reported (Daly et al. 1996 ; table 4), the phenotypic consequences of which are less well characterised and detailed analysis of the metabolic consequences is still required. Using a combination of PCR , RFLP and SSCP analysis, all CYP 2D6 alleles can now be identi® ed, and CYP2D6 genotype unequivocally assigned. PCR-based assays are available for detection of the most common alleles (CYP 2D6*3 and


1145

CY P2D6*4 ; table 4)Ð these are extremely sensitive and rapid techniques which can be used to screen large populations, using minimally invasive procedures (Gough et al. 1990, Spurr et al. 1991, Sm ith et al. 1992). The PM phenotype segregates as an autosom al recessive M endelian trait which occurs at a frequency of approximately 7 % in the Caucasian population (Eichelbaum et al. 1979). In other populations, however, the PM frequency is considerably lower, at only 2 % in black Americans (Evans et al. 1993) and ! 1 % in Orientals (Lou et al. 1987). The marked interethnic variation in PM frequency can be partly explained by the low prevalence of the CYP 2D6*3 and CYP2D6*4 alleles, the most common gene-inactivating alleles in Caucasians, in Oriental populations. There are, however, certain alleles, e.g. CYP 2D6*10 which have a particularly high frequency in Orientals (D ahl et al. 1995). The CYP2D6*10 allele has reduced activity and, as a consequence of the high frequency of this allele in Orientals, anti-depressant drugs which are substrates for CYP2D6 are routinely prescribed at lower doses in Oriental compared with Caucasian populations (Kalow and Bertilsson 1994). It is therefore extremely im portant that the racial origin of a sample is known before attempting to correlate CY P2D6 genotype and phenotype and to predict an individuals drug metabolising capability. It is equally important to select an appropriate, raciallymatched control population before attempting to associate the CYP2D6 PM phenotype with disease susceptibility.

Cytochrome P450 CY P2E The human CYP2E subfamily contains a single gene, CYP 2E1, which has been localized to chromosome 10q 24.3-qter (Ko$ ble et al. 1993). This gene spans 11 .4 kb of genom ic DNA, and has a promoter region which contains a basal transcription element (BTE) and several hepatocyte nuclear factor (HNF1) binding sites (Ingelman-Sundberg et al. 1995). Regulation of CYP 2E1 expression in rodents is complex and occurs by several distinct mechanisms, including developmental and hormonal regulation, mRNA and protein stabilisation (Gonzalez 1990). The mechanisms which regulate the expression of human CYP 2E1 are less well understood, but are thought to have at least some features in common with the rodent genes (Perrot et al. 1989). CYP 2E1 protein expression in man has been detected in a number of tissues, including liver, lung, placenta, skin and brain (Farin and Omiecinski 1993, Botto et al. 1994). M etabolism by CY P2E1 is one route of ethanol metabolism in the body. As such, interindividual variation in CYP 2E1 expression has been associated with the pathogenesis of diseases such as alcoholism and hepatic cirrhosis (Kato et al. 1995, Lucas et al. 1995). CYP 2E1 is also responsible for the metabolism of endogenous chemicals including acetone and acetal, key metabolites in the gluconeogenic salvage pathway, activated on glucose starvation and in the diabetic state. Indeed, acetone has been proposed to be an endogenous substrate for CYP 2E1 (Koop and Casazza 1985). CYP2E1 levels are induced in the diabetic state and by starvation (Johansson et al. 1991) and are decreased by insulin treatment (Richardson et al. 1992). The presence of a functional insulin-responsive element has been demonstrated in the 5 ´ promoter region of CYP 2E1 (Pernecky et al. 1994), which may be one mechanism by which the levels of CYP 2E1 are regulated in diabetes. M any other low molecular weight compounds including nitrosamines and numerous solvents such as benzene, carbon tetrachloride and ethylene glycol are

1146

G. Smith et al.

substrates and inducers of CY P2E1 (Guengerich 1991). A number of these compounds are known or suspected carcinogens, or are converted to toxic or mutagenic forms by this enzyme. CYP 2E1 is one of the most highly conserved P450 genes, both in terms of structure and absolute sequence, indicating that it may have an important physiological function, in addition to its role in xenobiotic m etabolism. To test this hypothesis, the m urine Cyp2e 1 gene has recently been disrupted by homologous recombination in embryonic stem cells, resulting in a mouse line lacking CY P2E1 expression (Lee et al. 1996). These animals did not display any physiological abnormalities. However, on administration of the analgesic acetaminophen CY P2E1-null anim als were less sensitive to drug-induced hepatotoxicity. Acetaminophen (paracetamol) is metabolized to N-acetyl-p-benzoquinoneimine by CY P2E1, a metabolite which is considerably more toxic than the parental compound. CY P2E1 null animals cannot form the toxic metabolite and are therefore protected from the paracetamol-induced toxicity, even at high doses of the drug. Several polymorphisms have now been identi® ed in human CYP 2E1. RFLP analysis with Taq I, Rsa I and Dra I has revealed the presence of several base pair diå erences between individuals in regions which lie outwith the coding region of the gene (Persson et al. 1993, Stephens et al. 1994). The diå erences identi® ed with either Rsa I and Pst I lie in the 5 ´ ¯ anking region of CY P2E1, upstream of the transcription start site (Watanabe et al. 1990). It has been suggested that the allele associated with the Rsa I site aå ects CYP2E1 transcription by disrupting one of the HNF-1 binding sites (Hayashi et al. 1991b). The phenotypic consequences of these changes are, however, unknown. Allelic variation in the promoter region of CY P2E1 is relatively rare in the Caucasian population, occuring with a frequency of only 2± 5 % (Persson et al. 1992). A T ! C substitution in intron 6 of the CYP2E1 gene, giving an informative RFLP with Dra I, is present at an allele frequency of 10 % in the Caucasian population, but is present in up to 26 % of Orientals (Uematsu et al. 1991, Persson et al. 1993). The presence of this allele has been associated with lung cancer susceptibility in several populations (Uematsu et al. 1991b, Hirvonen et al. 1992, Persson et al. 1993). The results of these studies are inconclusive, how ever, and no metabolic rationale has been proposed to support the observed associations. A further RFLP is obtained with Rsa I as a consequence of a mutation in intron 5 of CY P2E1 (Uematsu et al. 1991). This allele is particularly common in the Oriental population, where it is present at a frequency of 43 % (Uematsu et al. 1991). Again, no phenotype has been associated with this mutation. Two further CYP2E1 alleles have recently been described (Ingelman-Sundberg 1996), where the base pair changes lie within the coding regions of the gene. The ® rst of these results in the substitution of a histidine for an arginine at codon 76 and the second, a valine to isoleucine in exon 8. The phenotypic consequences of these alleles and any association with disease susceptibility are currently under study.


The CYP3 gene cluster is located on chromosome 7q22-qter (Spurr et al. 1989) and encodes at least four highly homologous genes. cDNA clones have been isolated for each of theseÐ CY P3A3 (P450HLp or hPCN2) (Molow a et al. 1986), CYP 3A4 (N F10, NF25 and hPCN1) (Beaune et al. 1986, Gonzalez et al. 1988, Bork et al.


1147

1989), CYP3A5 (hPCN3 and P450HLp2) (Aoyam a et al. 1989, Schuetz et al. 1989) and CYP3A7 (P450HFL33) (K omori et al. 1989a, b). Two CYP 3A proteins have been puri® ed from human adult and foetal liver respectively, CYP3A4 (P450 NF ) and CY P3A7 (P450HFLa) (Guengerich et al. 1986, Kitada et al. 1987). The diå erent gene products of the CYP3 locus are involved in a wide number of reactions including the oxidation of nifedipine (Guengerich et al. 1986a), quinidine (G uengerich et al. 1986b) and midazolam (Kronbach et al. 1989), the 2- and 4hydroxylation of 17 b -oestradiol (Guengerich et al. 1986a), the 6 b -hydroxylation of testosterone, the metabolism of cyclosporin A (Kronbach et al. 1988, Aoyama et al. 1989), aldrin epoxidation and several other reactions involving dihydroxypyrimidine compounds. Enzymes from the CYP3 family are major components of the total hepatic P450 content and can be induced by glucocorticoids, phenobarbital-like compounds and macrolide antibiotics (Wrighton et al. 1985). CYP 3A genes share over 85 % sequence identity at the amino acid level and are encoded by relatively large genes, with a single gene spanning approximately 30 kb of genomic DNA (H ashimoto et al. 1993, Kolars et al. 1994). M embers of this subfamily not only constitute the major P450s in human liver but are also expressed in extrahepatic tissues. By far the most abundant CYP3A protein in human liver is CYP 3A4, whereas CY P3A5 is the major form found in the stomach (Kolars et al. 1994). By reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, both CY P3A4 and CYP 3A5 mRN As have been detected throughout the gastrointestinal tract, whereas CYP 3A7 is consistently detected only in the liver (Kolars et al. 1994). Similarly, CYP 3A4 and CYP 3A5, but not CYP 3A7 mRNAs have been detected in human placenta (Hakkola et al. 1996). Cytochrome P450 CY P3A3 The CYP 3A3 and CY P3A4 genes (table 5) are highly homologous, sharing 97 % amino acid sequence homology (Gonzalez et al. 1988). It has been suspected for some time that CYP3A3 is in fact an allele of CYP 3A4, rather than the product of a separate gene. In fact, no unique substrates have yet been identi® ed for CYP 3A3 and both CY P3A3 and CY P3A4 share a number of common substrates, including the endogenous steroids progesterone and androstenedione (Aoyama et al. 1989), as well as a number of commonly prescribed drugsÐ nifedipine, erythromycin, lidocaine, cyclosporin and tamoxifen (Kronbach et al. 1989, Schuetz et al. 1994). Cytochrome P450 CY P3A4 CYP 3A4 encodes a 52 kDa protein responsible for the metabolism of a wide variety of substrates including nifedipine, erythromycin, troleandomycin, quinidine, cyclosporin A, 17 a -ethynylestradiol, lidocaine and diltiazem (Peyronneau et al. 1993 and references therein). The m etabolism of nifedipine has been reported to exhibit wide interindividual variability, with 17 % of a Dutch population being phenotypically de® cient in the ® rst step of nifedipine metabolism (Kleinbloesem et al. 1984). The level of expression of CY P3A4 has been studied in human liver samples and has been shown to vary up to 60-fold and to be highly correlated with the total hepatic P450 levels (Forrester et al. 1992). Several distinct CYP3A4 cDNA clones have been isolated : N F25 (Beaune et al. 1986), NF10 (Bork et al. 1989) and hPCN1 (Gonzalez et al. 1988) between which there are only three amino acid diå erences within the coding region (table 5).

NF25Œ ~

hPCN1Œ **

u

u

CTC Leu CTC Leu CTC Leu CTC Leu

3

92

105

106

164

TGG CTA CGC GAG CGA Trp Leu Arg Glu Arg TGG ACA CGG AGG GCA Trp Thr Arg Arg Ala TGG ACA CGG AGG GCA Trp Thr Arg Arg Ala TGG ACA CGG AGG GCA Trp Thr Arg Arg Ala

72

193

200

203

TCA GTC CAG CTT Ser Val Gln Leu ACA ATC CAA TTT Thr Ile Gln Phe ACA ATC CAA TTT Thr Ile Gln Phe ACA ATC CAA TTT Thr Ile Gln Phe

187

225

ACA GTC Thr Val ATC Ile ACA GTC Thr Val ACA GTC Thr Val

224

Sites of nucleotide diå erences

GCT Ala TCT Ser TCT Ser TCT Ser

252

279 } 280 CAT AAG His Lys CAG Gln CAG Gln CAG Gln

Published diå erences in CYP3A3} 3A4 cDNA sequences.

TGG Trp TGG Trp TGG Trp GTG Val

392

ATA Ile ACA Thr ATA Ile ATA Ile

431

Bork (1989) ‹ J04449 Beaune (1986) ‹ M14096 Gonzalez (1988) ‹ M18907

CYP3A3

Source of sequence

u All expressed in yeast (Peyronneau et al. 1993) ; ~ , diå erent clones from the same cDNA library ; Œ , functionally equivalent alleles ; ‹ , accession number for GenBank EMBL database ; ** hPCN1 was found to be identical to another clone isolated by Spurr et al. (1989) (‹ X12387).

NF10~

u

CYP3A3

Allele

Table 5.

1148 G. Smith et al.


1149

All three CYP3A4 variants have been expressed in Saccharomyces cerevisiae (Peyronneau et al. 1993). Substrate binding aæ nities and catalytic activities were measured for the each variant with a range of substrates (Peyronneau et al. 1993). The two point mutations (Trp $ * # and Thr % $ " in NF25 ! Val$ * # and Ile% $ " in hPCN1) were found to have no eå ect on substrate binding with the substrates erythralosamine 2 ´-monobenzoate (2 ´-MBEM ), cyclosporin and dihydroergotamine, but with testosterone and erythralosamine, small but signi® cant diå erences in substrate binding were detected. In contrast, P450 NF10, which diå ers from both NF25 and hPCN1 by a single amino acid deletion im mediately adjacent to the splice acceptor site of intron 7, Ile# # % replacing Thr # # % -Val# # & , was unable to bind haem and was therefore inactive. Yeast microsomes containing P450 NF25 and hPCN1 displayed comparable catalytic activities toward all the substrates tested, although P450 hPCN1 was always found to be slightly less active than P450 NF25 (Peyronneau et al. 1993). Due to the proximity of the single amino acid deletion in P450 NF10 to the intron 7 } exon 8 boundary, this variant could be the result of alternative mRNA splicingÐ a mechanism that has already been shown to regulate the expression of several cytochrome P450 genes (Miles et al. 1989b, Ding et al. 1995). In many cases, although an aberrantly spliced mRNA can be translated into a truncated protein, its haem-binding capacity is lost and therefore the protein cannot function as a P450 monooxygenase. For example, human CYP 2B6 mRNA is subject to alternative splicing, and at least four mRNA species can be derived from this gene (Kimura et al. 1989, M iles et al. 1989b). Preliminary results from our own laboratory suggest that the NF10 cDNA sequence either does not exist in the Caucasian population, or that it is a very rare allele. It would therefore be unable to account for the large interindividual variation observed in the metabolism of nifedipine. Cytochrome P450 CY P3A5 The CYP3A5 gene encodes a 502 amino acid, 57 .1 kDa protein which shares 84 % amino acid and 89 % nucleotide sequence homology to CYP 3A4. CYP 3A5 is only expressed in 10± 20 % of human livers (Aoyam a et al. 1989). Intriguingly, it has been reported that CYP 3A5 is present in a statistically signi® cantly higher percentage of children and adolescents compared with the adult population (Wrighton et al. 1990). A CY P3A5 cDNA was isolated from a human liver cDNA library constructed from a liver in which CY P3A5 was the only CYP 3A protein expressed (Aoyama et al. 1989). Catalytic activity measurements using recombinant vaccinia virus-expressed CY P3A4 and CYP3A5 in mammalian cells have shown that both enzymes are similarly active in the oxidation of nifedipine. Both enzymes also catalyse the 6 b -hydroxylation of the steroid hormones testosterone, progesterone and androstenedione, but CYP 3A4 exhibits several-fold higher activity than CYP 3A5. In addition, it was noted that several minor steroid oxidation products were formed (e.g. 15 b -hydroxytestosterone) by CY P3A4 but not by CY P3A5, indicating that CYP 3A5 is a more highly regiospeci® c monooxygenase catalyst with steroid substrates (Aoyama et al. 1989). Diå erences were also noted in the metabolism of cyclosporine, with two hydroxylated metabolites and one demethylated metabolite formed by CY P3A4, but only one metabolite formed by CY P3A5. Interestingly, in a recent report Haehner et al. (1996) determined the relative levels of CYP 3A3, CY P3A4, CYP 3A5 and CYP 3A7 mRNA expression in human

1150

G. Smith et al.

kidney by RT-PCR. All kidneys examined expressed CYP 3A5. CYP 3A4 mRNA was detected in 40 % of the kidney samples, and 70 % of those that contained detectable CYP3A4 mRNA also expressed detectable levels of the corresponding protein. Therefore, in contrast to hepatic tissue, where CYP 3A4 is ubiquitously expressed, in renal tissue CY P3A5 protein appears to be the major member of the CY P3A subfamily to be expressed. Also contained within the CYP 3 gene cluster is a 1627 bp CYP 3A5 variant, CY P3A5P, which was isolated by screening a human liver cDNA library with a fragment of the CYP 3A5 cDNA (Scheutz and Guzelian, 1995). Com pared with the parental cDNA, CYP 3A5P contains several large insertions, deletions and in-frame termination codons and is thought to be a pseudogene derived from CYP3A5.

Cytochrome P450 CY P3A7 A partial length CY P3A7 cDNA was isolated from a human fetal liver cDNA library using antiserum to P-450 HFLa (Komori et al. 1989a). This cDNA was then used to probe the same library resulting in the isolation of a cDNA clone containing the entire coding region of CYP3A7 (K omori et al. 1989b). The full-length clone contains an open reading frame of 1509 nucleotides, encoding a 503 amino acid protein. There have been con¯ icting reports as to the tissue speci® city of CYP 3A7 gene expression. CYP3A7 was originally identi® ed as a 51 . 5 kDa foetal-speci® c protein related to the human nifedipine oxidase gene (Komori et al. 1989a, b, Schuetz et al. 1994), but has now been identi® ed in adult human liver, placenta and endometrial tissues, with the levels of mRNA and protein increased during pregnancy and at diå erent points during the m enstrual cycle (Schuetz et al. 1993). CY P3A7 catalyses dehydroepiandrosterone 3-sulphate hydroxylation, a physiologically important reaction for the formation of estriol in pregnancy (Kitada et al. 1987). The mechanism of regulation of CYP3A7 is to date unknown, and no genetic polymorphism has been identi® ed at this gene locus.


Cytochrome P450 CY P4A The CYP 4A gene family contains two members, CYP4A9 and CYP 4A11, both of which are localized on chromosome 1 (Bell et al. 1993). The cDNA clone for CY P4A11 was isolated from a kidney cDNA library as CYP 4A11 expression is higher in the kidney than in the liver (Imaoka et al. 1993, Palmer et al. 1993). Although there is marked inter-individual variation in hepatic CYP4A11 expression, no genetic basis for this variation has been identi® ed. A variant CY P4A11 sequence has, how ever, been reported, in which a single nucleotide insertion disrupts the open reading frame and results in an extension of the coding sequence by 72 amino acids (Imaoka et al. 1993). Although this clone was isolated from the same cDNA library as the principal CYP4A11 cDNA, it is not yet clear whether this variant has any phenotype. This again could arise from an alternative splicing mechanism or be due to genetic polymorphism at the CYP 4A locus. CYP 4A proteins catalyse the x -hydroxylation of m edium and long chain fatty acids including lauric acid, palmitic acid and arachadonic acid (Palmer et al. 1993, Kawashima et al. 1994). Like CYP2E 1, CYP 4A expression is induced in rats in the diabetic state (Freeman et al. 1992). This induction is also seen following exposure


1151

of rodents to a variety of structurally diverse compounds including clo® bric acid and nafenopin, collectively know n as peroxisome proliferators. A cytosolic receptor protein, the peroxisome proliferator activated receptor (PPAR), mediates induction of CYP 4A proteins in rodents following exposure to these compounds (Issemann and Green 1990). W hether a similar mechanism of gene regulation is present in man is presently under debate. PPAR is a member of the nuclear receptor superfamily which binds as a heterodimer with the retinoic acid receptor, RX R, to a speci® c recognition sequence, the PPAR responsive element (PPRE), in the 5 ´ promoter region of responsive genes. These include CYP 4A genes and several genes which encode proteins involved in lipid metabolism including peroxisomal fatty acid b oxidation enzymes and fatty acid binding proteins.

Cytochrome P450 CY P4B CYP 4B1 was cloned from a human lung cDNA library which was probed with a cDNA for the orthologous rat gene, CYP 4A1 (Nhamburo et al. 1989). In contrast with the majority of human P450s, CYP4B1 is not expressed in the liver, but has been detected at low levels in the colon using sensitive in situ hybridization techniques (McKinnon et al. 1994) and in placenta by RT-PCR (Hakkola et al. 1996). CYP 4B1 is the m ajor P450 expressed in human lung (Czerwinski et al. 1994) and may therefore be responsible for mediating lung-speci® c toxicities arising from the metabolic activation of xenobiotics. Although CYP4B1 substrate speci® city is relatively well de® ned in rodents where it has been shown to activate aromatic amines to mutagens, there are marked interspecies diå erences in the ability of CY P4B1 proteins to metabolise pulmonary toxins such as 4-ipomeanol (Czerwinski et al. 1991). The substrate speci® city and catalytic activity of the hum an CY P4B1 protein remain to be fully characterized.

O ther h um an P450s

P450s in the higher families, in general, do not play a signi® cant role in drug and xenobiotic metabolism. These enzymes, the majority of which are expressed in the liver, catalyse stereo- and regiospeci® c reactions of steroid metabolism and other important endogenous biochemical processes, including vitamin metabolism (table 6). Detailed analysis of the expression and regulation of these genes is outwith the scope of this review articleÐ these aspects of P450 structure and function have previously been expertly reviewed (Martucci and Fishman 1993, Okuda 1994, Parker and Schimmer 1995). CYP 5 (thromboxane synthase) encodes a secondary enzyme in the arachadonic acid cascade, catalysing the formation of thromboxane A from prostaglandins (Ohashi et al. 1992). Unlike the majority of P450 proteins, CYP 5 does not perform monooxygenase reactions, but is classi® ed as a P450 gene based on sequence homology to other family members. CYP 5 has a similar intron } exon structure to members of the CY P3A subfamily, although it has only 35 % identity to CYP 3A4 (Miyata et al. 1994). Like the CYP3A gene cluster, CYP5 is found on chromosome 7, where it has been localized to 7q34-35 (Chase et al. 1993). CYP 7, cholesterol 7 a -hydroxylase, catalyses the ® rst step in bile acid synthesis and the hydroxylation of cholesterol at the 7 a position (Lee et al. 1994). The CY P11A1 gene encodes P450scc, the cholesterol side chain cleavage enzym e, which

1152

G. Smith et al. Table 6.

Functions of non-xenobiotic metabolising P450s.

P450 family CYP1± CYP3 CYP4 CYP5 CYP7 CYP11 CYP17 CYP19 CYP21 CYP24 CYP27

Function xenobiotic} steroid metabolism fatty acid hydroxylation, foreign compound metabolism thromboxane synthesis cholesterol 7a -hydroxylase cholesterol side chain cleavage steroid 11b hydroxylase steroid 17a hydroxylase steroid aromatase steroid 21-hydroxylase vitamin D metabolism cholesterol 27-hydroxylase

is the ® rst, rate-lim iting step in steroid biosynthesis. De® ciencies in the expression of CYP 11A1 have been linked with the pathogenesis of congenital adrenal hyperplasia (CAH ), a rare inherited disorder which manifests as a consequence of insuæ cient cortisol biosynthesis. Altered expression of CYP 17 and CYP 21 have also been linked to CAH (White and New 1988). M utations in CYP 11A1 have also been associated with the pathogenesis of autoimmune polyendocrine system type I and Addison’ s disease (Winqvist et al. 1993, Uibo et al. 1994). Antibodies to CYP 11A1 are routinely found in blood samples collected from patients with these autoimmune diseases. Interindividual variation in the expression of mitochondrial P450 genes can therefore have profound clinical consequencesÐ mutations in several mitochondrial P450 genes have now been associated with de® ned clinical phenotypes. Although the frequency of these inherited mutations is relatively low, the molecular basis of a number of rare inherited syndromes has been attributed to altered expression of speci® c P450 genes which plays vital roles in regulating key biochemical processes in the cell.

C linical im plications of gen etic polym orphism in hu m an P450 gen es

Human P450 genes are thought to have evolved as a consequence of ` molecular drive ’ , i.e. the need for an organism to defend itself from the toxic eå ects of environm ental chemicals (Wolf 1990). As such, the P450 superfamily contains many distinct genes, with diå ering substrate speci® cities, tissue-speci® c patterns of expression and mechanisms of regulation. This catalytic diversity allows P450 proteins to metabolize a wide range of structurally diverse substrates. As a consequence, many compounds which are commonly prescribed drugs or are pollutants generated by our industrial environment are m etabolized by one or more P450-catalysed reaction. The majority of foreign compound metabolism is performed by one or more of the six most abundant P450 isozymes (Shimada et al. 1994, ® gure 3). Based on the relative levels of hepatic expression, CYP 3A4 is thought to be the m ost abundant isozyme (30 %), followed by CY P2C proteins (20 % ), CYP1A2 (13 % ), CY P2E1 (7 % ), CYP2A6 (4 % ) and CY P2D6 (2 % ). Together, these isozymes account for 70 % of the total hepatic P450 content. Interindividual variation in P450 expression can therefore have profound clinical consequences. The level of many human P450 proteins is subject to m arked


1153

Figure 3. Relative levels of P450 isozyme expression in human liver. The relative level of expression of individual P450 isozymes is illustrated. The expression levels shown are based on average values obtained from a panel of Caucasian human liver samples (Shimada et al. 1994). By far the most abundant P450 isozyme is CYP3A4. The level of expression of the rarer P450 enzymes are combined.

interindividual variation and, for some of these, a molecular genetic basis for this variation has been determined. The phenotypic consequences of interindividual variation in P450 expression can be signi® cant and there are many examples of idiosyncratic pharmacological responses to prescribed medication which have been attributed to atypical metabolism by a polymorphically expressed P450 (Tucker 1994). Interindividual variation in P450 expression can also in¯ uence disease susceptibility. For example, inheritance of CYP 1A1 alleles encoding proteins which are more inducible on exposure to polycyclic aromatic hydrocarbon substrates than the wild-type enzyme has been reported, in aå ected individuals, to lead to increased toxicity and carcinogenicity from the PAH components of cigarette smoke (K iyohara et al. 1996). CYP 2D6, which has a relatively high mutant allele frequency within the Caucasian population, has been shown to catalyse several compounds which are either agonists or antagonists of neurotransmission. It is therefore particularly interesting that inheritance of the CYP 2D 6 PM genotype (CYP2D6*3 and CY P2D6*4 alleles) has been linked to the development of Parkinson’ s disease (Smith et al. 1992, Kurth and Kurth 1993), where individuals with the PM genotype have been shown to be more than twice as likely to develop PD than a control population. M PTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is a potent neurotoxin found in synthetic meperidine narcotics, exposure to which can result in the development of Parkinson-like symptoms. It has recently been demonstrated that M PTP is both a substrate for and competitive inhibitor of CYP 2D6 (Coleman et al. 1996, Gilham et al. 1997). Attempts have also been made to link CYP 2D6 PM frequency with a number of other diseases, including Alzheimer’ s disease (Benitez et al. 1993), rheumatoid arthritis (Beyeler et al. 1994) and several types of cancer

1154

G. Smith et al.

(Smith et al. 1995 and references therein). The results of many of these studies remain equivocal, how ever, often as a consequence of insuæ cient sample sizes, inappropriate selection of control populations and the use of phenotypic rather than genotypic assessment of PM status. Although the phenotypic consequences of polymorphism in P450 genes can be considered in isolation, the eå ects of inheriting mutant forms of more than one drug-m etabolizing enzyme have the potential to be far more profound. Polymorphisms in phase II enzymes, including the glutathione S-transferases and Nacetyl transferases, are well characterized. For example, the GSTM 1 and GSTT1 genes are deleted in up to 50 % of the Caucasian population (Board et al. 1981, Pemble et al. 1994), with aå ected individuals unable to detoxify reactive intermediates by conjugation with glutathione, GSTM 1 catalyses the detoxi® cation of reactive epoxides, generated from the CYP1A1-mediated metabolism of polycyclic aromatic hydrocarbons. Individuals inheriting both the high-activity allelic form of CY P1A1 and who are also nulled for GSTM 1 are therefore at a higher risk of druginduced toxicity than individuals inheriting only one of the susceptibility alleles. The eå ects of mutations within the human drug-metabolizing enzymes are particularly signi® cant when they are considered in association with mutations in key regulators of cell growth, such as the tumour suppressor protein p53 and the oncogene H-ras (Ryberg et al. 1994). Individuals who inherit susceptibility alleles of CY P1A1 and GSTM 1 were also found to have a high frequency of p53 mutations, either through direct germ line transmission or as a consequence of carcinogen exposure. The presence of these mutations has been linked to lung cancer incidence, and has been found to correlate with cigarette dose (Kawajiri et al. 1996). In conclusion, major advances have been made in recent years in our understanding of both the molecular mechanisms and phenotypic consequences of interindividual variation in the levels of expression of human P450 proteins. Rapid and minimally invasive procedures have been developed using which an individuals drug-m etabolizing capacity may be accurately determined. Not only will this assist in tailoring doses of prescription medication according to individual metabolizing capacity, but also predictions may be made about individual disease susceptibilities and advice given to susceptible individuals on avoiding exposure to speci® c initiating chemicals.

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