The human hepatic cytochromes P450 involved in drug metabolism.

Critical Reviews in Toxicology, 22( 1): 1-2 1 (1992)

The Human Hepatic Cytochromes P450 Involved in Drug Metabolism Steven A. Wrighton* and Jeffrey C. Stevens

Critical Reviews in Toxicology Downloaded from informahealthcare.com by University of North Carolina on 05/12/13 For personal use only.

Department of Drug Metabolism and Disposition, Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Mail Drop 0825, Indianapolis, IN 46285

*

To whom all correspondence should be addressed.

ABSTRACT: The cytochromes P450 are a superfamily of hemoproteins that catalyze the metabolism of a large number of xenobiotics and endobiotics. The type and amount (i.e., the animal’s phenotype) of the P450s expressed by the animal, primarily in the liver, thus determine the metabolic response of the animal to a chemical challenge. A majority of the characterized P450s involved in hepatic drug metabolism have been identified in experimental animals. However, recently at least 12 human drug-metabolizing P450s have been characterized at the molecular and/or enzyme level. The characterization of these P450s has made it possible to “phenotype” microsomal samples with respect to their relative levels of the various P450s and their metabolic capabilities. The purpose of this review is to compare and contrast the human P450s involved in drug metabolism with their related forms in the rat and other experimental species.

KEY WORDS: human, liver, microsomal, cytochromes P450, drug metabolism, mixed function oxidases, expression of the P450s, regulation of the P450s, inhibition of the P450s.

1. INTRODUCTION The cytochromes P450 are a superfamily of hemoproteins that are the terminal oxidases of the mixed function oxidase system. At the last official count,’ the P450 superfamily was comprised of 154 genes found in 23 eukaryotes (both plants and animals) and 6 prokaryotes. The rat and human genomes were found to contain, respectively, 39 and 28 P450 genes at last count.’ A recommended nomenclature system has been devised based on the evolutionary relationships of these oxidases. In this nomenclature system, the deduced amino acid sequences from the genes are compared and first divided into families, which are comprised of those P450s that share at least 40%identity (currently, 27 gene families are documented). The mammalian families are divided further into subfamilies, which are comprised of those forms that are at least 55% related by their

deduced amino acid sequences. Only 3 P450 gene families (i.e., CYPl, CYP2, and CYP3) of the 27 families identified are currently thought to be responsible for the majority of hepatic drug metabolism. Representatives of each of these three families have been identified in the human liver and are the subject of this review. The hepatic microsomal P450s catalyze the metabolism of an amazingly large number of lipophilic endogenous and exogenous compounds. The P450s actually catalyze only a limited number of reactions including carbon hydroxylation, heteroatom oxygenation, dealkylation, and epoxidation, as recently reviewed by Guengerich.* In addition, the P450s can catalyze reductive reactions, and with certain compounds, metabolites can be formed that inactivate the P45Os.* The metabolites formed from the majority of substrates by the P450s are more hydrophilic than the parent compound and are thus more readily

1040-8~&4/92/$. 50 0 1992 by CRC Press, Inc.

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excreted from the body. In some cases, however, electrophilic metabolites are formed, which can react with cellular nucleophiles resulting in toxic or carcinogenic i n ~ u l t . ~ . ~ In the past, the P450s were often referred to as having broad and overlapping substrate specificities. However, it has become apparent that this broad substrate specificity can be accounted for by the facts that (1) the liver contains multiple forms of P450, and (2) at concentrations of the substrates found under physiologic conditions, enzyme kinetics often favors a single form of P450 being the primary catalyst of the metabolism. Thus, the phenotype of an individual with respect to the forms and amounts of the individual P450s expressed in the liver can determine the rate and pathway of the metabolic clearance of a compound. The majority of studies on the catalytic activities and regulation of expression of the P450s has been performed using various experimental systems. These systems include the common experimental animals,s plants,6 and microbial systems.' In addition, the past several years has seen an explosion of information about the human hepatic P450s. The accumulation of this information on the human hepatic P450s was greatly facilitated by the application of the knowledge gained and experimental techniques developed (such as metabolic assays, specific antibodies, and cDNA probes) through the use of experimental systems. However, the studies with the human hepatic P450s have reinforced the notion that drug -metabolism and the regulation of the expression of the drug-metabolizing enzymes is often quite different in man compared to that found in experimental animals. That is, it has become apparent that significant differences exist between man and experimental species with respect to the catalytic activities and regulation of the expression of the hepatic drug-metabolizing P450s. In general terms, three basic differences were identified. First, different P450s in the various species may perform, with high specificity, the same metabolic function. For example, mephenytoin is metabolized in the rat by a 3A subfamily member,9 whereas in man mephenytoin is metabolized by a member of the 2C subfamily. l o Furthermore, even highly structurally related P450s in different species (i.e., structural homologs) may have different substrate ' v 8

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specificities (see Section 1I.A. 1). Second, the regulation of expression of related forms of P450 can vary among the various species, including man. The classic case of this is the sexual bimorphism observed for the metabolism of many compounds by rats that is not observed with the other species, including man.s In addition, strain differences were observed in rats and mice with respect to their hepatic drug-metabolizing capabilities. In man, the poor metabolizer phenotype for the 4'-hydroxylation of S-mephenytoin is observed in 20% of Oriental populations and only 5% of Caucasian populations (see Section 1II.C. 1). Furthermore, humans exhibit a genetic polymorphism in the metabolism of debrisoquine, which is inherited as an autosomal recessive trait. However, in the only strain of rats to demonstrate a polymorphism in the metabolism of debrisoquine, the defect is inherited in a sexlinked fashion (see Section III.D.l). The third general difference is that, through gene duplication, species-specific P450s have evolved. 1 1 , 1 2 Thus, these differences among experimental species and man often make it difficult to extrapolate to man the P450-mediated metabolism studies performed in those other species. l 3 Therefore, only through a complete understanding of these differences can the best extrapolation to man be performed. This review addresses what is known about the catalytic functions and regulation of expression of the human hepatic P450s involved in drug metabolism. When possible, direct comparisons are made between what has been observed in the experimental systems and man.

II. THE HUMAN CYPl FAMILY

A. The CYPlA Subfamily 1. P450 1Al

The CYPl family is described as the most straightforward P450 gene family to be studied thus far in that only two genes to date have been identified in both humans and rodents. '.I4 Human 1Al cDNA was fmt isolated by screening a cDNA library generated from TCDD-treated MCF-7 human breast carcinoma cells with cDNAs encod-


ing mouse 1A 1 and 1A2.l 5 The predicted amino acid sequence of human 1Al shares 68% identity with human 1A2.I6 In rodents, the orthologs of human 1Al are rat P450c (78% similarity)" and mouse PI-450 (80% similarity). l 5 Analyses of interspecies differences in P450 primary sequence showed a strong evolutionary conservation of mammalian 1A genes. This is in sharp contrast to the divergence seen in the members of the CYP2 family, which has been postulated to be an evolutionary response to dietary and general environmental factors in higher mammals. "*I4 In experimental animals, 1Al is constitutively expressed at low levels in the liver and in extrahepatic tissues such as the lung.5 However, following treatment with polycyclic aromatic hydrocarbons (PAH), such as 3-methylcholanthrene, 1Al levels are greatly induced. In a historical perspective, initial evidence for the multiplicity of P450s came from the early induction studies of Conney and co-workers over 30 years ago, which demonstrated that the P450 induced by 3-methylcholanthrene had different spectral and enzymatic properties than the P450(s) in the untreated rat.18 The mechanism of this induction process and the general regulation of expression of rat 1A is the subject of several reviews. I 9 v 2 O Extrapolation to man of information on P450 1A 1-dependent metabolism of xenobiotics in experimental animals may have little relevance since the current information about these forms indicates that large differences exist between man and the various species. Immunoblot analyses on microsomes prepared from 14 human liver specimens found that only 1 contained a protein that reacted with a rat 1Al antibody.21 Additional immunoblot analyses by the authors' laboratory detected 1Al in only 1 of more than 50 human liver samples.174It is currently thought that human 1Al is predominantly an extrahepatic P450 and there is good evidence that it is inducible in extrahepatic tissues by cigarette smoke and PAH.22,z3In fact, the inducibility of human 1Al and its relationship to lung cancer is currently being investigated. Two independent laboratories recently identified genetic differences in individuals with a high risk of developing lung cancer.24-25 Specifically, they found that, of the three restriction fragment length polymorphisms

(RFLP) of the CYPlAl gene observed in DNA isolated from normal and lung cancer patients, individuals that are homozygous for a specific rare allele are at significantly greater risk for developing lung cancer. Whether these individuals can take any preventative measures to decrease this genetic risk is unknown. However, these technical advances are important steps for future diagnostic work on the link between P450 forms and cancer.

2.P450 1A2 When the original cDNA clone for human 1Al was used in genomic Southern blot analyses to probe for related genes, Jaiswal et al.I5 suggested that the human genome contained only the CYPlAl gene. However, a related form, termed human IA2,' is now characterized in terms of catalytic activity, gene sequence, and regulation of expression. In fact, 1A2, which was originally purified by Disterlath et a1.26as the phenacetin 0-deethylase, was found to be universally expressed in adult human liver.21 The 3-demethylation of caffeine was shown to be a 1A2 activity marker, based on data using purified lA2 and correlation of caffeine demethylase activity with microsomal 1A2 levels. 27 Several studies using 1A2-specific metabolism of in vivo probe drugs or microsomal metabolism of phenacetin or caffeine and correlation to immunodetectable levels of human 1A2 showed that this enzyme was present in the human liver at widely varying leve l ~ . This ~ ~high - ~ level ~ of interindividual variation (Sesardic et al.30reported a > 180- and 60fold difference in phenacetin 0-deethylation rates and immunodetectable protein, respectively) led to speculation that 1A2 levels are regulated by genetic and/or environmental factors. Higher 1A2 levels and associated enzyme activities have been positively correlated to increased cigarette s m ~ k i n g . ~In ~ - vivo ~ ' studies using caffeine metabolism as a marker for 1A2 found that other factors influencing 1A2 levels include enzyme induction by physical exercise and the ingestion of charbroiled meats or cruciferous ~ e g e t a b l e sIn. ~addition, ~ drug interactions are suspected to occur via 1A2. For example, omeprazole, a proton pump inhibitor, was found

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by Diaz et al.33 to induce the levels of human lA2 both in vivo and in human hepatocytes in primary monolayer culture. However, separate studies did not find an increase in the in vivo metabolism of caffeine after omeprazole admini ~ t r a t i o n Although .~~ higher 1A2 levels may increase the bioactivation of carcinogens, there are also several studies linking 1A2 induction by dietary factors to a lower incidence of breast cancer.35*36The proposed mechanism for the decrease in breast cancer incidence is that increased 1A2-catalyzed estrogen 2-hydroxylation decreases the levels of endogenous estrogens, which have been associated with metastatic breast cancer. However, unlike 1A1, 1A2 does not appear to be expressed in extrahepatic tissues. Larger, more comprehensive studies are needed in the area of chemoprevention by the alteration of endogenous metabolic pathways by dietary changes. Finally, reduced levels of 1A2 were seen in livers from patients with cirrhosis and other liver diseases;28and of the five P450s examined, only the levels of 1A2 were found to vary significantly in the different lobes of a normal human liver.37 The orthologs of human 1A2 in experimental animals play a major role in the bioactivation of a wide variety of carcinogens and mutagen^.^'^ Specifically, the bioactivation of 4-aminobiphenyl via N-oxidation in human liver microsomes was correlated strongly with immunodetectable 1A2 levels and inhibited by antibodies raised to this P450.27From a cancer epidemiological perspective, an estimate of an individual's susceptibility to arylamine-induced cancers could be made using caffeine 3-demethylation as an in vivo probe of 1A2 activity. Another tool for studying the metabolic potential of 1A2 in vivo and in vitro is the methylxanthine derivative furafylline, shown by Sesardic et al.38to be a potent and selective inhibitor of human 1A2 in vitro.

111. THE HUMAN CYPP FAMILY

A. The CYP2A Subfamily 1. P4502A6

The P450 2A subfamily has been extensively studied in the rat and the mouse. In the rat, the

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2A subfamily is composed of three members.'.'' The most extensively characterized is 2A1, which was originally referred to as P450a or the testosterone 7 a - h y d r o ~ y l a s eThe . ~ ~ second rat form is 2A2, and although highly related to 2Al by amino acid sequence, it does not demonstrate the same degree of specificity with respect to testosterone hydroxylation.40Furthermore, 2A 1 and 2A2 are not expressed similarly in the untreated rat and, unlike 2A1, 2A2 is not induced by PAH.5.39.40The final rat 2A subfamily member, 2A3, appears to be expressed exclusively in the lung, where the levels of the mRNA encoding 2A3 are induced after 3-methylcholanthrene treatment. 41 In the mouse, the 2A subfamily is composed of 2A4 and 2A5, which are 98% similar and more closely related to rat lung 2A3 than the other rat 2A P450s as determined from their deduced amino acid sequence^.^^,^^ Originally isolated as the mouse liver testosterone 15a-hydroxylase, the expression of 2A4 was found to be developmentally regulated.44The specificity of 2A5 for testosterone hydroxylation is more relaxed than that of 2A4. However, 2A5 was shown to be the It is incoumarin 7-hydroxylase in the teresting to note that the rat 2A P450s do not actively catalyze the 7-hydroxylation of coumarin. In addition, 2A5 is induced in mice treated with phenobarbital or pyrazole.46 Thus, major differences between the rat and mouse 2A subfamily exist not only in substrate specificity, but also in the regulation of their expression. The human 2A subfamily has not been as extensively investigated. Microsomes prepared from human livers do not hydroxylate testosterone at either the 7a- or 1 5 a - p o ~ i t i o nHowever, .~~ Raunio et al.48 demonstrated that human liver microsomes contain a protein immunochemically related to mouse 2A5 and capable of hydroxylating coumarin. Two cDNAs isolated from a human liver cDNA library were assigned to the 2A subfamily based on their deduced amino acid sequences . 4 9 When cloned into an expression system, only one of these cDNAs, 2A6, produced a functional enzyme that was a coumarin 7-hyd r o x y l a ~ e When . ~ ~ compared by deduced amino acid sequence to other 2A subfamily members, 2A6 was 85% similar to the rat lung 2A3 and Yun et 75% of the mutant alleles.92The application of similar chimeric enzyme technology has yielded a wealth of information on structure-function relationships of other P450s.93-94Additional constructions of inter- or intraspecies 2D hybrids should help to identify what peptides or amino acids are responsible for catalytic activity. In the past few years, several research groups have focused on the development of diagnostic tests for genotyping individuals for this genetic polymorphism. The first such test developed was an RFLP analysis, which takes advantage of altered restriction sites recognized by Xba I endonuclease in the mutated 2D6 genes to produce a characteristic “fingerprint” for the PM phentype.^^ However, this method positively predicted the PM phenotype in only approximately one fourth of the subjects t e ~ t e d .The ~ ~ devel.~~ opment of a genotyping test that utilizes PCRbased DNA amplification improved the accuracy of genotyping by adding three major advantages: (1) it is >95% accurate in predicting an individual’s genotype when combined with RFLP analysis; (2) it can identify heterozygous carriers of mutant alleles and may therefore explain why

these individuals are often distinct from EM or PM phenotypes; and (3) samples for the PCRbased tests can be collected noninvasively using body hair or urine ~ e d i m e n t . ~An ~ . ~additional ’ benefit of genotyping is that genotyping is not affected by the environmental and medical factors that influence the results of phenotyping through the use of the in vivo metabolism of marker 2D6 substrates. From a clinical perspective, the development of accurate 2D6 genotyping could significantly benefit patients that require a drug metabolized by 2D6 that also has a narrow therapeutic index.98.99A standard dose of debrisoquine may produce a dangerous hypotensive response in PM patients; however, other therapeutic indices such as analgesia and depression, which are effected by drugs metabolized by 2D6, are more difficult to measure. Therefore, the lack of drug efficacy or low toxicity may be attributed to an individual’s perception rather than differences in metabolism. For example, interindividual differences to the analgesic effect of codeine have been reported for years, but separating the pharmacological and metabolism responses was difficult. loo.loLEvidence that the demethylation of codeine to morphine is catalyzed by 2D6 in vivo and in v i m has been published and it strongly suggests that alterations in the pharmacology of codeine can be linked directly to its polymorphic metabolism. 102-105 The interaction of psychoac9


tive drugs, such as haloperidol, imipramine, and nortryptyline, with 2D6 indicates that maximum drug efficacy requires both patient behavior and drug level monitoring. Several studies have used large control (EM phenotype) and PM populations as the data base to investigate whether a link exists between 2D6 levels in man and several diseases of complicated etiology, such as cancer, Parkinsonism,Io6 and lupus erythematosus. lo' At least three independent studies report a positive correlation between lung cancer and the EM phenotype.'08-"0 Proposed hypotheses for these results are that the 2D6 gene may be linked to an oncogene or tumor suppressor gene, or that 2D6 is responsible for the bioactivation of an as yet unidentified procarcinogen. However, other studies using the debrisoquine phenotyping of patients"* or 2D6 genotyping by RFLP analysis"3 found no correlation between 2D6 expression and lung cancer. In contrast, the link between Parhnson's disease and debrisoquine metabolism may be just the opposite to that proposed for lung cancer. Io6 The PM phenotype was more prevalent among patients with Parkinson's disease, suggesting that defective metabolism may allow an accumulation of neurotoxins and the subsequent neural degeneration. Finally, a rare form of autoimmune chronic active hepatitis was identified in which the antigen in human liver microsomes is 2D6."4,"5 This disease occurs mainly among young girls and does not appear to be linked with exposure to a particular virus or drug, or with either 2D6 phenotype. Further predictive and mechanistic studies on the role of P450s in the genetic susceptibility to these disease states are needed. Several useful biochemical tools are now available to assist both basic and clinical investigations of 2D6-dependent metabolism. In addition to debrisoquine phenotyping, the relative contribution of 2D6 to the in vivo metabolism of a xenobiotic can be assessed by the coadministration of the specific 2D6 inhibitor quinidine. The K, of quinidine for the inhibition of bufuralol metabolism is -150 M."However, despite this high affinity of quinidine for 2D6, quinidine is not metabolized by this P450.88Leemann and coworkers illustrated the utility of this compound by essentially converting EM subjects to the PM

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phenotype with a single dose of quinidine. ' I 6 This technique allows an accurate assessment of the contribution of 2D6 to the in vivo metabolism of an additional compound. Metabolism that is dependent on 2D6 can also be studied using a number of in v i m approaches. With a fresh human liver sample, metabolism can be studied using isolated perfused lobes, hepatocyte culture, precision cut liver slices, or subcellular fractions (i.e., microsomes). Because of the inconsistent availability of liver samples from healthy individuals, the preferred technique is quick freezing of small liver cubes for storage and later use in microsome preparation. In addition, metabolically competent cells expressing mainly 2D6 have been developed through cDNA expression systems and are now commercially available. The use of these relatively simple and inexpensive means of assessing whether the metabolism of a new chemical entity is under genetic control are currently being vigorously pursued by the pharmaceutical industry. Although such studies are currently considered only supplementary data to regulatory agencies, they should ultimately speed the development process and yield compounds less likely to cause adverse effects in patients.y8

E. The CYP2E Subfamily 1. P450 2E7 The 2E subfamily is toxicologically a very important metabolic system. In the rat, the 2E subfamily contains only one member, 2E1 . I The regulation of expression and catalytic activities of rat 2E1 have been extensively studied.S*"7This P450 is of unusual toxicologic significance since 2E1 is induced by many commonly encountered small organic molecules. Furthermore, 2E1 often catalyzes the conversion of numerous low molecular weight agents to metabolites that are more chemically reactive than the parent compound. In fact, 2E1 was first isolated as the low K, Nnitrosodimethylamine N-demethylase, which was induced after treatment with pyrazole, acetone, isoniazid, and ethanol."8."9 As reviewed by Yang et al. , ' I 7 the number of compounds that induce the activity of rat 2E1 is very extensive and includes pyrazole, isoniazid, ethanol, acetone, ke-


tones, and isopropanol. Rat 2E1 is also induced by physiologic states that result in the accumulation of acetone or ketones such as fasting, diabetes, and obesity. It has been demonstrated that rat 2E1 is responsible for the metabolic activation of a large number of common toxins including halogenated small organics like carbon tetrachloride and enflurane, alcohols, N-nitrosodimethylamine, aniline, benzene, acetone, and acetaminophen. 'I7 In contrast to the majority of the comparisons between the rat P450s and related human forms, the information obtained concerning human 2E 1 correlates well with the rat data. Human 2E1 was first purified by immunoaffinity chromatography using an antibody against rat 2E1 bound to a solid support. 120 Structural studies with this inactive human 2E1 preparation and immunoinhibition and immunoquantification studies demonstrated that rat and human 2E1 were highly similar.'2oAdditional studies using active preparations of human 2E 1 confirmed these observations. Specifically, human 2E 1 metabolized N-nitrosodimethylamine,52 aniline,52*'21 ethano1,121.122 carbon tetrachloride, 1 2 2 and acetaminophen. 1 2 3 Furthermore, Guengerich et al. 124 demonstrated that human 2E1 appears to be responsible for the metabolic activation of a large number of low molecular weight suspected carcinogens including chloroform, vinyl chloride and bromide, ethylene dibromide and dichloride, vinyl and ethyl carbamate, benzene, and styrene. Chlorzoxazone, a centrally acting muscle relaxant, has been shown to be specifically hydroxylated in v i m by human 2El. 125 Thus, the metabolic conversion of chlorzoxazone may be a useful noninvasive probe of the in vivo metabolic potential of human 2E1. Finally, d i ~ u l f i r a m 'and ~ ~ methoxypsora1en'26has been shown to be specific inhibitors of human 2E1. The regulation of expression of rat 2E1 is more complicated than that of the other rat Specifically, the induction of rat 2E1 P450s. was shown to involve both transcriptional and post-transcriptional events. 1 7 , 1 2 8 Although limited in number, studies on the expression of human 2E1 indicate that the levels of human 2E1 are elevated in patients receiving ethanol and isoniazid, known inducers of rat 2E1 .120'129The human 2E subfamily, like the rat subfamily, con'173127

tains only one gene.'.'28 This is unlike the rabbit 2E subfamily, which is comprised of an ortholog of 2E1 and a second form, 2E2.' Rabbit 2E1 appears to be very similar to human and rat 2E1 with respect to its substrate specificity and regulation of expression. However, the expression of rabbit 2E2 is not coordinately controlled with the expression of 2E1 and their developmental expression is also very different, suggesting that rabbit 2E1 and 2E2 do not serve the same function.'30-'3' Furthermore, this dichotomy appears to be unique to the rabbit. Thus, the rat appears to be an excellent model for human 2E1 expression and function, however, the rabbit may not be an appropriate model.

IV. THE HUMAN CYP3 FAMILY

A. The CYP3A Subfamily The final subfamily of P450s involved in hepatic drug metabolism is the 3A subfamily. The various members of this subfamily have been widely studied since they were shown to be responsible for the metabolism of a wide array of clinically and toxicologically important agents, and to be inducible by steroids, macrolide antibiotics, imidazole antifungals, and phenobarbita1.14*132 Currently, two genes, 3Al and 3A2,' have been identified in the rat.133*134 However, it is important to note that various purification procedures and immunochemical characterizations indicate that there may be three to four proteins in the rat CYP3 family.'35-'37 Using oligonucleotide probes that specifically recognize 3A 1 or 3A2, it was found that rat 3A1 is induced by treatment with glucocorticoids and is not expressed in untreated adult animals. 134 Furthermore, these studies demonstrate that 3A2 is not induced by glucocorticoids and is expressed in only adult male rats.'34 Finally, both 3A1 and 3A2 are induced by phenobarbital.134 These forms of rat P450 are specifically responsible for erythromycin N-demethylation, steroid 6P-hydroxylation, and the formation of a stable metabolicintermediate complex with triacetyloleandomycin.138,139 Little is known about the regulation of expression or substrate specificity of the other rat 3A subfamily members.136

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1. P450 3A3/4 The P450 3A subfamily in man appears to be composed of at least four genes, CYP 3A3, 3A4, 3A5, and 3A7.I The regulation of the expression of these genes does not seem to parallel that described for rat 3A1 or 3A2. It appears that the first human 3A P450 to be isolated from the liver of an adult was isolated by Wang et a1." and was identified as human liver P450,. However, P450, was only partially characterized with respect to substrate specificity and structure.140 Watkins et al. using immunoblots developed with an antibody to rat 3A1 to monitor their purification, and Guengerich et al. ,I4' using nifedipine oxidase activity to monitor their purification, isolated structurally and functionally similar 3A P450s termed, respectively, HLp and P450NF. In fact, a monoclonal antibody prepared against P450, recognizes both HLp and P450NF. 143 Using the same human liver cDNA library, Molowa et a1.Ia and Beaune et isolated two slightly different 3A cDNAs now termed 3A3 and 3A4, respectively.' The deduced amino acid sequences for these two 3A P450s are in fact 97% similar.L46These two clones appear to be separate genes and not allelic variants despite their high degree of sequence identity. As indicated earlier with the antibody to P450,, 3A3 and 3A4 are so highly related that studies using immunochemical methods to determine their levels of expression and enzymatic activities cannot differentiate between them. Thus, when the experiments described cannot differentiate between 3A3 and 3A4, the term 3A3/4 will be used to described these P450s. However, by using oligonucleotide probes specific for either 3A3 or 3A4, it was shown that the mRNA encoding for 3A4 appears to be expressed to a greater degree in the human liver than that for 3A3. 14' Furthermore, through the use of 3A314-specific catalytic activities and immunochemical methods, it was determined that 3A3/4 is one of the major forms of P450 normally expressed in the human liver. In addition, the levels of 3A3/4 appear to be elevated in patients administered glucocorticoids,14' macrolide antibiotic^,'^^ or phenobarbital .47.141 Studies using immunoinhibition experiments, individual human 3A forms obtained from purification procedures or cDNA expres-

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sion systems, and/or the correlation of metabolic activity and 3A3/4 level in microsomal samples demonstrated that human 3A forms are responsible for the majority of the metabolism of a large number of structurally diverse exogenous and endogenous compounds. The endogenous compounds metabolized by the human 3A subfamily are various steroids including the 6P-hydroxylation of t e s t o s t e r ~ n e ,p~r~o g e s t e r ~ n e , ' ~ ~ and andr~stenedione;'~~ the 2- and 4hydroxylations of e ~ t r a d i o l ; and ' ~ ~ the 16a-hydroxylation of dehydroepiandrosterone 3-sulfate.'49 The xenobiotics metabolized by the human 3A subfamily include erythromycin , I 4 ' nifedipine and at least 20 other 1,4-dihydropyr~~ idines, 142, IS0 cyclosporine, Is' l i d ~ c a i n e , 'aflaquinidine,Is4 17a-ethynylestradi01,~~~ midazolam and triazolam,1s6 the plant alkaloid ~enecionine,'~'l o v a ~ t a t i n , 'ben~phetamine,'~~ ~~ and aldrin.'42 In addition, several P450 3A-specific inhibitors have been identified. Specifically, these P450s form a stable metabolic-intermediate complex with triacetyloleandomycin, which results in the inhibition of these P45Os,l4I and the acetylenic steroids g e s t ~ d e n eand ' ~ ~17a-ethynylestradioll" are mechanism-based inactivators of the 3A subfamily. In the rat, 3A2 was expressed exclusively in the adult male but not female. In contrast, no human 3A subfamily member has been shown to be expressed exclusively in the male. However, Watkins et demonstrated that 3A activity is two times higher in female than in male patients by using a I4CO, breath test for erythromycin N-demethylation, which is specifically catalyzed by 3A P450s. Cortisol 6PhydroxylationI6' and lidocaine dealkylation162 have also been used to phenotype the in vivo metabolic capabilities of patients and may reflect the in vivo activities of the 3A subfamily.

2. P450 3A5

The next member of the human 3A subfamily is 3A5.I This P450 has been found to be polymorphically expressed in that it was detected in only about 25 to 30% of the adult liver specimens e ~ a m i n e d . ~The ~ . 'levels ~ ~ of 3A5 in those samples in which it was detected range from 6 to 100% of the total 3A P450 p r e ~ e n t . ~ ~ The .'~~-'~~


expression of 3A5 does not appear to be influenced by the gender or the drug history of the liver d o n ~ r . ~However, ~ . ' ~ ~ 3A5 was detected in a significantly greater number of livers from adolescents as compared to adults and was also detected in fetal liver. 163 The metabolic capabilities of 3A5 have been shown to be very limited by comparison to 3A3/4. In fact, 3A5 does not appear to metabolize erythromycin, quinidine, or 17a-ethynyestradiol at significant rates. However, testosterone, nifedipine, cortisol, and dehydroepiandrosterone 3-sulfate were metabolized by 3A5, albeit at rates slower than 3A4.479'63It is interesting to note that, in human liver microsomes containing what appears to be 3A5, the rate of midazolam 1-hydroxylation was significantly greater than the rate observed with microsomes containing only 3A3/4. 156 Therefore, the relative expression of the various 3A subfamily members in the human liver (i.e., the contribution of 3A5 to total 3A expression) would govern the rate and extent of the metabolism of the large number of compounds indicated earlier as substrates of 3A3/4.

treated fetal rats do not contain 3A-related P450. 17' However, a 3A form can be induced in the fetal liver by treating the pregnant dams with glucoc o r t i ~ o i d s . ~In~ man, ' there is no evidence that administration of exogenous steroids are required for the expression of fetal 3A7. Thus, the rat would appear to be a poor model for human fetal drug metabolism. Several other P450s, including lA2, 2C8, 2C9 and 2E1, were not detected in human fetal liver.'68 However, three yet to be fully characterized P450s have been recently purified from human fetal liver. 172 In conclusion, it is apparent from the above discussion that the regulation of expression of the members of the rat and human 3A subfamilies is not similar. In addition, not all substrates metabolized by the rat 3A subfamily (i.e., mephenytoin9 and d i g i t o ~ i n ' ~are ~ ) metabolized by the structurally related human subfamily. Therefore, when extrapolating the results of studies on the regulation of the expression or the metabolic capabilities of the 3A subfamily in experimental animals to man, extreme caution should be used.

3. P450 3A7

V. CONCLUDING REMARKS

The human fetus is capable of metabolizing a large number of compounds; this is in sharp contrast to what has been reported for the fetuses of the experimental species.'66'167 The major form of P450 in the human fetus was shown to be a member of the 3A subfamily, specifically 3A7,I and this single P450 comprises 30 to 50% of the total fetal P450. 168*'69 Despite being expressed at high levels in the fetus, 3A7 has not been detected in adult l i ~ e r . ~On~ the . ' ~other ~ hand, 3A3/4 does not appear to be expressed in the fetus, however, 3A5 and mRNA encoding for 3A5 were detected in fetal liver.47,163,165 Although little is known about the substrate specificity of 3A7 relative to the other members of the human 3A subfamily, it was shown that microsomes from fetal livers catalyze the 16a-hydroxylation of dehydroepiandrosterone 3-sulfate at a rate greater than that of microsomes from adult liver. 149.170 This suggests that 3A7 may be more efficient at catalyzing the 16a-hydroxylation of dehydroepiandrosterone 3sulfate than 3A3, 3A4, or 3A5. Livers from un-

Over the past several years, a great deal of information regarding the regulation of the expression and the catalytic activities of the human hepatic cytochromes P450 involved in drug metabolism has become available. Currently, 12 human drug-metabolizing P450s are characterized to varying degrees. In a majority of cases, the characterization is of both the P450 and its gene. However, in some cases, recombinant DNA techniques identified genes encoding for human P450s that are not yet characterized at the enzyme level. This is the current situation for 2B6 and 2C18. The danger in this is that data generated in experiments using, for example, 2B6 obtained from cDNA-directed expression systems to determine the role of the P450 in the metabolism of a compound is difficult to interpret. That is, since it is not currently known to what level, if at all, that 2B6 is found in microsomal samples much less in vivo, the observation that 2B6 catalyzes the metabolic conversion of a compound at the greatest rate of all the expressed P450s

13


may be meaningless. It is only through a complete understanding of how and when a P450 is expressed can its true role in the metabolic clearance of a compound be determined. Despite the explosion of information on the human P450s, much is yet to be discovered. The vast data base on the P450s found in the experimental systems suggests that more human P450s exist and need to be characterized. Those P450s that are identified need to be characterized further with respect to their functions, structures, and regulation of expression. Because of the inconsistent supply of human livers, model systems of human drug metabolism need to be developed and refined. It is clear from what is already known that no one or combination of animal models reflects the metabolic capabilities of man. Thus, in the future, in vitro systems such as human hepatocytes, liver slices, subcellular fractions, isolated P450s, and transformed cell lines may be utilized to help predict the human metabolism of a new chemical entity. Various combinations of these in vitro systems along with the use of specific inhibitors and antibodies have been successful in accurately determining the enzyme responsible for the metabolic clearance of many drugs. By having a complete understanding of the factors (such as inducers, inhibitors, effect of disease state, etc.) that alter the activity of the enzyme responsible for the metabolism of a compound, it soon may be possible to predict drug interactions and eventually the metabolic clearance of the compound.

ACKNOWLEDGMENTS The authors wish to thank Debbie Brandkamp for her excellent secretarial assistance, Dr. Paul Watkins for his review of this manuscript, and Mark VandenBranden for his superb technical assistance.

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Role of cytochromes P450 in drug metabolism and hepatotoxicity.

The role of individual human cytochromes P450 in drug metabolism and clinical response.

Metabolism of palmatine by human hepatocytes and recombinant cytochromes P450.

4-hydroxylations of estradiol in human liver microsomes.

Effects of thiamine deficiency on hepatic cytochromes P450 and drug-metabolizing enzyme activities.

Expression of two cytochromes P450 involved in carcinogen activation in a human colon cell line.

Modulation of rat hepatic cytochromes P450 by chronic methapyrilene treatment.

Interspecies features of hepatic cytochromes P450 IA1 and P450 IA2 in rodents.

Dietary effects on cytochromes P450, xenobiotic metabolism, and toxicity.

Small intestinal cytochromes P450.

Metabolism of 7,12-dimethylbenz[a]anthracene in hepatic microsomal membranes from rats treated with isoenzyme-selective inducers of cytochromes P450.

Effects of testosterone and estrogen on hepatic levels of cytochromes P450 2C7 and P450 2C11 in the rat.

Combination of on-line CE assay with MS detection for the study of drug metabolism by cytochromes P450.

Hepatic cytochromes P450: structural degrons and barcodes, posttranslational modifications and cellular adapters in the ERAD-endgame.

Immunoisolation of human microsomal cytochromes P450 using autoantibodies.

Identification of human cytochrome P450 isozymes involved in the metabolism of naftopidil enantiomers in vitro.

Monepantel induces hepatic cytochromes p450 in sheep in vitro and in vivo.

Membrane topology of the mammalian P450 cytochromes.

Dose-response of oridonin on hepatic cytochromes P450 mRNA expression and activities in mice.

Effects of simvastatin, a lipoprotein-lowering drug, on the hepatic enzymes involved in drug metabolism in the Wistar rat.

1-Aminobenzotriazole-induced destruction of hepatic and renal cytochromes P450 in male Sprague-Dawley rats.

microRNA-34a is associated with expression of key hepatic transcription factors and cytochromes P450.

Peroxygenase reactions catalyzed by cytochromes P450.

Bacterial cytochromes P450: isolation and identification.