Identification of the human cytochrome P450 enzymes involved in the two oxidative steps in the bioactivation of clopidogrel to its pharmacologically active metabolite.

The aim of the current study is to identify the human cytochrome P450 (P450) isoforms involved in the two oxidative steps in the bioactivation of clopidogrel to its pharmacologically active metabolite. In the in vitro experiments using cDNA-expressed human P450 isoforms, clopidogrel was metabolized to 2-oxo-clopidogrel, the immediate precursor of its pharmacologically active metabolite. CYP1A2, CYP2B6, and CYP2C19 catalyzed this reaction. In the same system using 2-oxo-clopidogrel as the substrate, detection of the active metabolite of clopidogrel required the addition of glutathione to the system. CYP2B6, CYP2C9, CYP2C19, and CYP3A4 contributed to the production of the active metabolite. Secondly, the contribution of each P450 involved in both oxidative steps was estimated by using enzyme kinetic parameters. The contribution of CYP1A2, CYP2B6, and CYP2C19 to the formation of 2-oxo-clopidogrel was 35.8, 19.4, and 44.9%, respectively. The contribution of CYP2B6, CYP2C9, CYP2C19, and CYP3A4 to the formation of the active metabolite was 32.9, 6.76, 20.6, and 39.8%, respectively. In the inhibition studies with antibodies and selective chemical inhibitors to P450s, the outcomes obtained by inhibition studies were consistent with the results of P450 contributions in each oxidative step. These studies showed that CYP2C19 contributed substantially to both oxidative steps required in the formation of clopidogrel active metabolite and that CYP3A4 contributed substantially to the second oxidative step. These results help explain the role of genetic polymorphism of CYP2C19 and also the effect of potent CYP3A inhibitors on the pharmacokinetics and pharmacodynamics of clopidogrel in humans and on clinical outcomes.

Effects of epitope sequence tandem repeat and proline incorporation on polyclonal antibody production against cytochrome 1A2 and 3A4.

We describe a method for producing polyclonal antibodies against peptide antigen cytochrome P450 1A2 and 3A4 using a tandem repeat of the epitope region and incorporation of proline residue between the repeated sequences. An ELISA assay revealed more efficient generation of polyclonal antibodies to tandem repeat peptide antigens than mono-epitope peptides. The incorporation of proline residues further stimulated antibody production.

Utility of polyclonal antibodies targeted toward unique tryptic peptides in the proteomic analysis of cytochrome P450 isozymes.

P450s are key enzymes responsible for biotransformation of numerous endogenous and exogenous compounds and are located in almost every tissue. This superfamily is the largest group of enzymes (>6000) that share a high degree of similarity in protein sequence. The human genome contains 57 CYP genes and 58 pseudogenes. A major gap exists in our knowledge about differences in CYP expression on a protein level. DNA and mRNA information are not sufficient because transcription and particularly translation events are not necessarily correlated with levels of expressed proteins. The data reported in this study complete the framework of an integrated proteomic method for the simultaneous qualitative and quantitative analysis of CYP isozyme composition using MALDI-TOF-MS and immunochemistry that has been developed in our laboratory over the last several years (Alterman et al., 2005a,b) and is based on the fact that each P450 isozyme possesses unique tryptic peptide(s) (UTP) that could be used for differential analysis of human CYP expression. Here we demonstrate that three different immunochemical techniques (ELISA, Western blot, and peptide affinity enrichment on magnetic beads with attached antibodies) have potential to be incorporated in an integrated proteomic method combining mass spectrometry and immunochemistry. Fundamentally, this approach is based on the measurement of the same chemical entity (isozyme-specific UTP) in the tryptic digest by two orthogonal analytical techniques, mass spectrometry and immunochemistry. The application of this approach is illustrated with two human CYP isozymes--CYP1A2 and CYP2E1.

Elucidation of the effects of the CYP1A2 deficiency polymorphism in the metabolism of 4-cyclohexyl-1-ethyl-7-methylpyrido[2,3-d]pyrimidine-2-(1h)-one (YM-64227), a phosphodiesterase type 4 inhibitor, and its metabolites in dogs.

The canine CYP1A2 1117 C>T single nucleotide polymorphism is responsible for a substantial portion of the interindividual variability seen in the pharmacokinetics of 4-cyclohexyl-1-ethyl-7-methylpyrido[2,3-d]pyrimidine-2-(1H)-one (YM-64227). The purpose of this study is to investigate the contribution of CYP1A2 to the metabolism of YM-64227 and its five metabolites (MM-1 to MM-5), as well as to determine the interindividual variability between the pharmacokinetic profiles of the compounds with respect to the CYP1A2 deficiency polymorphism. alpha-Naphthoflavone and anti-CYP1A1/2 antibody inhibited the metabolic activities at which MM-2 and MM-3 were formed from YM-64227 in C/C- and C/T-type microsomes. In T/T type, the rate of MM-2 and MM-3 formation was lower, and alpha-naphthoflavone and anti-CYP1A1/2 antibody were shown to have no effect. A positive correlation between the overall metabolism of YM-64227 and phenacetin O-deethylation, a CYP1A2 activity marker, was observed in all the genotypes. The in vitro metabolic clearances in the T/T type of MM-1, MM-3, MM-4, and MM-5 were less than 50% lower than those in the C/C type. The anti-CYP1A1/2 antibody inhibited the metabolism of MM-1, MM-3, MM-4, and MM-5 in the C/C and C/T types. These results suggest that the formation of MM-2 and MM-3 from YM-64227 is catalyzed by CYP1A2, and that CYP1A2 contributes mainly to the subsequent metabolism of the primary metabolites of YM-64227, with the exception of MM-2. It is possible that the interindividual variability of YM-64227 with respect to the CYP1A2 deficiency polymorphism is caused by a decrease in the metabolic activities of both the unchanged drug and its metabolites.

Identification of human hepatic cytochrome P450 enzymes involved in the metabolism of 8-prenylnaringenin and isoxanthohumol from hops (Humulus lupulus L.).

The female flowers of hops (Humulus lupulus L.) are used in the brewing of beer and are under investigation for use in dietary supplements for the management of menopausal symptoms in women. Hop extracts contain the weakly estrogenic compound isoxanthohumol (IX), proestrogenic xanthohumol, and the potent estrogen 8-prenylnaringenin (8PN). Because IX can be metabolized in the human liver to form 8PN, the specific cytochrome P450 (P450) enzymes responsible for this O-demethylation reaction were identified. In addition, the enzymes that convert IX and 8PN to their most abundant metabolites were identified because these metabolic pathways might also affect the estrogenicity of hop preparations. Specifically, the P450 enzymes that catalyze the oxidation of the prenyl side chains of IX and 8PN into trans- or cis-alcohols were investigated. Human liver microsomes and monoclonal antibodies that inhibit specific P450 enzymes were used in combination with liquid chromatography/mass spectrometry to identify the enzymes responsible for these transformations. CYP2C19 was found to catalyze the formation of both cis- and trans-alcohols of the prenyl side chain of 8PN with K(m) values of 14.8 +/- 3.2 and 16.6 +/- 4.6 microM, respectively. CYP2C8 converted 8PN regioselectively to the trans-alcohol of the prenyl group with a K(m) of 3.7 +/- 0.9 microM. Finally, CYP1A2 was found to catalyze the O-demethylation of IX to generate 8PN, with a K(m) value of 17.8 +/- 3.7 microM. These results suggest that the estrogenicity of hop constituents in vivo will depend in part on metabolic conversion that may show individual variation.

Relative contributions of the five major human cytochromes P450, 1A2, 2C9, 2C19, 2D6, and 3A4, to the hepatic metabolism of the proteasome inhibitor bortezomib.

VELCADE (bortezomib, PS-341), reversibly inhibits the 20S proteasome and exhibits cytotoxic and antitumor activities. Pretreatment of cancer cells with bortezomib increases the chemosensitivity of these cells, suggesting that bortezomib may be used in combination chemotherapy. The relative contributions of the five major human cytochromes P450 (P450s), 1A2, 2C9, 2C19, 2D6, and 3A4 (the focus of the present study), to the metabolism of bortezomib are an important aspect of potential drug interactions. Relative activity factor (RAF), chemical inhibition, and immunoinhibition using monoclonal antibodies were three approaches employed to determine the relative contributions of the major human P450s to the net hepatic metabolism of bortezomib. RAFs for the P450 isoform-selective substrates were determined; the ratio of the rate of metabolism of bortezomib with cDNA-expressed P450s versus rate of metabolism with human liver microsomes was normalized with respect to the RAF for each P450 isoform to determine the percentage contributions of the P450s to the net hepatic metabolism of bortezomib. CYP3A4 followed by CYP2C19 were determined to be the major contributors to the metabolism of bortezomib. Chemical inhibition and immunoinhibition confirmed that CYP3A4 and CYP2C19 were the major P450s responsible for the hepatic metabolism of bortezomib. The studies were conducted with 2 muM bortezomib, and the disappearance of bortezomib, rather than appearance of a specific metabolite, was quantified to determine the contributions of the P450s to the overall hepatic metabolism of bortezomib in humans.

Identification of cytochrome P450 1A2 as enzyme involved in the microsomal metabolism of Huperzine A.

Huperzine A is a reversible and selective cholinesterase inhibitor approved for the treatment of Alzheimer's disease. To identify which cytochrome P450 (CYP) isoenzymes are involved in the metabolism of Huperzine A, an in vitro study was performed with rat liver microsomes and immunoinhibition and chemical inhibition methods. Huperzine A metabolism was analyzed with high-performance liquid chromatography (HPLC) and expressed as Huperzine A disappearance rate. Result showed that 76.2% of Huperzine A metabolism was inhibited by CYP1A2 antibody and 17.8% by CYP3A1/2 antibody. Inhibitory effects produced by CYP2C11 and 2E1 antibodies were minor. The CYP1A2 substrate phenacetin showed an inhibitory effect of 70.3%. In conclusion, Huperzine A metabolism in rat liver microsomes is mediated primarily by CYP1A2, with a probable secondary contribution of CYP3A1/2. CYP2C11 and 2E1 are likely not involved in Huperzine A metabolism.

Autoantibodies against cytochrome P450s in sera of children treated with immunosuppressive drugs.

Treatment with the immunosuppressive drugs cyclosporin and tacrolimus, the mainstays of anti-graft rejection and autoimmune disease therapy, is limited by their hepato- and nephrotoxicity. The metabolic conversion of these compounds to more easily excretable products is catalysed mainly by hepatic cytochrome P4503A4 (CYP3A4) but also involves extrahepatic CYP3A5 and other P450 forms. We set out to study whether or not exposure to cyclosporin and FK506 in children undergoing organ transplantation leads to formation of autoantibodies against P450s. Immunoblotting analysis revealed anti-CYP reactivity in 16% of children on CyA for anti-graft rejection or treatment of nephrosis (n = 67), 31% of kidney transplant patients switched from CyA to FK506 (n = 16), and 21% of kidney and or liver transplant patients on FK506 (n = 14). In contrast, the frequency of reactive immunoblots was only 8.5% among the normal paediatric controls (n = 25) and 7% among adult kidney transplant patients on CyA or FK506 (n = 30). The CYP2C9+ sera were able to immunoprecipitate in vitro translated CYP2C9 and the immunoblot reactivity showed striking correlation to peaks in the age at onset of drug exposure. Sera were isoform selective as evidenced from Western blotting using human liver microsomes and heterologously expressed human P450s. These findings suggest that anti-cytochrome P450 autoantibodies, identified on the basis of their specific binding in immunoblots, are significantly increased among children on immunosuppressive drugs and in some cases are associated with drug toxicity and organ rejection.

Increased incidence of anti-LKM autoantibodies in a consecutive cohort of hepatitis C patients from central Greece.

OBJECTIVES: In Greece, there are insufficient data regarding the presence of non-organ and liver-related autoantibodies in hepatitis C patients. This study in a consecutive cohort of 39 such patients from central Greece investigates (1) the prevalence of non-organ and liver-related autoantibodies, and (2) the reactivity of anti-liver-kidney microsomal type 1 antibodies (in the case of positivity with at least one of the methods used) against their molecularly defined antigens. DESIGN: All serum samples were tested by standard and molecular assays for the presence of anti-nuclear antibodies, smooth muscle antibodies, anti-liver-kidney microsomal type 1 antibodies, antibodies against parietal cells, anti-CYP2A6, anti-CYP1A2 and anti-CYP2D6 autoantibodies. METHODS: Indirect immunofluorescence, competitive enzyme-linked immunosorbent assays, immunoblotting and novel radioligand assays based on immunoprecipitation of [35S]-methionine labelled recombinant CYP2A6, CYP1A2 and CYP2D6 His-taq fusion proteins produced by in vitro transcription/translation were used. RESULTS: Seven out of 39 patients (17.9%) tested positive for smooth muscle antibodies, 2/39 (5.1%) tested positive for anti-nuclear antibodies, 1/39 (2.5%) tested positive for parietal cell antibodies, and 4/39 (10.3%) were found to be anti-liver-kidney microsomal positive (with at least one of the methods used). All sera were negative for anti-CYP2A6 and anti-CYP1A2 autoantibodies. Three out of four anti-liver-kidney microsomal positive samples had the typical liver-kidney microsomal staining pattern shown by indirect immunofluorescence. However, none tested positive for anti-CYP2D6 autoantibodies using the competitive CYP2D6 enzyme-linked immunosorbent assay, the specific CYP2D6 radioligand assay, and western blot using either human microsomes or recombinant CYP2D6. The fourth patient tested negative for anti-liver-kidney autoantibodies by either indirect immunofluorescence or the competitive enzyme-linked immunosorbent assay, but was repeatedly positive for anti-CYP2D6 autoantibodies by the sensitive and specific radioligand assay. Western blot experiments using human microsomes in this patient serum revealed two bands of 50 kDa and 55 kDa that documented as anti-CYP2D6 and anti-uridine triphosphate glucuronosyltransferase autoantibodies when recombinant CYP2D6 and recombinant uridine triphosphate glucuronosyltransferase autoantigens were used for immunoblot, respectively. CONCLUSIONS: A relatively high incidence of anti-liver-kidney microsomal autoantibodies (10.3%) was found in a consecutive sample of Greek patients with hepatitis C. The expanded panel of assays, however, failed to document CYP2D6 as the target autoantigen of anti-liver-kidney microsomal autoantibodies in most patients. We report for the first time the detection of parietal cell antibodies and both anti-CYP2D6 (anti-liver-kidney microsomal type 1) and anti-uridine triphosphate glucuronosyltransferase (anti-liver-kidney microsomal type 3) autoantibodies in patients who were hepatitis C positive/hepatitis D negative. Further studies are needed to confirm our findings and to determine whether these preliminary results have a clinical importance or not.

Catalytic and immunochemical properties of hepatic cytochrome P450 1A in three avian species treated with beta-naphthoflavone or isosafrole.

Induction of cytochrome P450 1A (CYP1A) can be used as a biomarker of exposure to planar halogenated aromatic hydrocarbons (PHAHs). Our objective was to characterize the induction of CYP1A activity and protein in three avian species following in vivo treatment with beta-naphthoflavone (BNF) and/or isosafrole. Alkoxyresorufin-O-dealkylase (alk-ROD) activities of hepatic microsomes from Herring Gulls (Larus argentatus) (HGs), Double-crested Cormorants (Phalacrocorax auritus) (DCCs) and chickens (Gallus domesticus) were measured using ethoxy-, methoxy-, pentoxy- and benzyloxy-resorufin, in the presence and absence of the inhibitors ellipticine or furafylline. Immunoreactivity of microsomal proteins with antibodies to several CYP1A proteins was investigated. CYP1A protein and alk-ROD activities of HGs and DCCs, but not chickens, were induced by isosafrole. Ellipticine was a potent and non-selective inhibitor of alk-ROD activity in all three species, while furafylline inhibition of alk-ROD activities varied among species and treatments. In all three species, BNF induced a protein immunoreactive with monoclonal antibody to CYP1A1 from the marine fish Stenotomus chrysops (scup), but a CYP1A2-like protein was not detected in avian microsomes probed with polyclonal antibodies to mouse CYP1A2. Variations in responses among avian species indicate that CYP1A proteins and substrate specificities should be characterized for each species used in PHAH biomonitoring programs.

Vesicular transport of newly synthesized cytochromes P4501A to the outside of rat hepatocyte plasma membranes.

Anti-cytochrome P450 (CYP)1A2 autoantibodies are found in dihydralazine-induced hepatitis, and CYPs2B and 2C have been shown to follow vesicular flow to the plasma membrane (PM). However, it is unknown whether other CYPs follow this route, whether NADPH-CYP reductase is present on the hepatocyte surface, and whether autoimmune hepatitis-inducing drugs increase PM CYPs. In this study, we determined the transmembrane topology and transport of CYPs1A in rat hepatocytes. In cultured hepatocytes, colchicine and other vesicular transport inhibitors decreased PM CYPs1A assessed by flow cytometry. Colchicine administration also decreased PM CYPs1A in vivo. Pulse chase experiments with [(35)S]methionine showed that only the newly synthesized CYP molecules are transferred to the PM, whereas microsomal CYP1A2 was stably radiolabeled for several hours. In contrast, radiolabeled CYP1A2 reached the PM and disappeared from the PM with half-lives of less than 30 min. Confocal microscopy, biotinylation, and coimmunoprecipitation experiments showed that PM CYPs1A and CYP reductase are present on the cell surface, and that the reductase is closely associated with PM CYPs. Exposure of whole cells to an anti-CYP1A1/2 antibody at 4 degrees C, before five washes and PM preparation, abolished PM CYPs1A-supported monooxygenase activity, indicating that PM CYPs are mostly located on the external surface. Dihydralazine and other CYPs1A inducers increased PM CYPs1A. In conclusion, newly synthesized CYPs1A follow vesicular flow to the outside of the PM, and NADPH-CYP reductase also is located on the hepatocyte surface. Dihydralazine administration increases PM CYP1A2, its autoimmune target.

Immunoreactivity to various human cytochrome P450 proteins of sera from patients with autoimmune hepatitis, chronic hepatitis B, and chronic hepatitis C.

Numerous human Cytochrome P450 enzymes (CYPs) associated with 'phase I' drug metabolism have been identified. Among them, CYP2D6 is thought to be the major target autoantigen to anti-liver kidney microsome (LKM)-1 autoantibody, a characteristic feature of autoimmune hepatitis (AIH) type II. In this study, we were able to clone CYP2D6 cDNA from a human liver cDNA library and express the CYP2D6 recombinant protein, and also to prepare four other representative human CYP proteins (CYP1A2, 2C9, 2E1, and 3A4). These preparations were used to assay the immunoreactivity of patients with AIH type I (n=35) and type II (n=9). As comparison groups, sera from patients with chronic hepatitis B (n=15), chronic hepatitis C (n=55; 24 anti-LKM-1-positive, 31 anti-LKM-1-negative), and from normal controls (n=30) were included. The five CYP proteins did not react with sera from normal controls nor from patients with chronic hepatitis B. CYP2D6 reacted with sera from 100% (9/9) of AIH type II patients, 79% (19/24) of patients with anti-LKM-1-positive chronic hepatitis C, and 6.5% (2/31) of patients with anti-LKM-1-negative chronic hepatitis C. In contrast, CYP1A2 reacted with serum from one patient with AIH type I, CYP2E1 reacted with sera from two patients with AIH type I, one patient with anti-LKM-1-positive chronic hepatitis C, and two patients with anti-LKM-1-negative chronic hepatitis C, and CYP3A4 reacted with sera from one patient with AIH type II and one patient with anti-LKM-1-positive chronic hepatitis C. CYP2C9 did not react with any of the sera included in this study. From these results, it is suggested that CYPs other than CYP2D6 can function as immunotargets in certain disease conditions.

Inhibitory monoclonal antibodies to human cytochrome P450 1A2: analysis of phenacetin O-deethylation in human liver.

Human cytochrome P450 1A2 metabolizes a large number of common drugs and engages in carcinogen metabolism and activation. Baculovirus-expressed 1A2 was used to immunize mice producing hybridomas yielding monoclonal antibodies (MAbs). Three of 2050 clones assayed yielded the MAbs, MAb 26-7-5, MAb 951-5-1, MAb 1812-2-4, which were specific for 1A2 as assessed by enzyme-linked immunosorbent assay and immunoblots. The three MAbs inhibited 1A2-catalysed metabolism of phenacetin, 7-ethoxycoumarin, chlorzoxazone and phenanthrene by more than 85%. The MAbs were highly specific to 1A2 and did not inhibit 11 other human P450s. The phenancetin O-deethylation activity varied from 0.44-2.49 nmol/min/nmol P450 in eight human liver microsomes samples. MAb 26-7-5 inhibited 1A2-dependent phenacetin O-deethylation in these samples by 64-84% indicating the amount of 1A2 contribution to this reaction and in addition a role for other P450s in the O-deethylation. Independent analysis of recombinant human P450s showed that 1A1, 1A2, 2A6 and 2C19 exhibited phenacetin O-deethylation activity, with 1A1 and 1A2 being the most active followed by 2C19 and 2A6. Eight other P450s were inactive towards phenacetin O-deethylation. The role of different P450 in eight liver samples was analysed with specific individual inhibitory MAbs. Inhibitory antibodies to 1A2, 2C8/9/18/19, 2A6, 2D6, 2E1, and 1A1 were combinatorially added to the microsomes. The O-deethylation activity was inhibited by antibodies to 1A2 (64-84%), to 2C19 (4.6-20%) and to 2A6 (0-8.8%). The total activity inhibited by antibodies to P450 2E1, 2D6 and 1A1 was less than 4.5%, indicating a minor role for these P450s in phenancetin metabolism in human liver microsomes. Thus, 1A2, 2C 9 and 2A6 are the dominant P450s for phenacetin O-deethylation. These studies demonstrate the use of inhibitory MAbs to P450s for a simple and precise assessment of the quantitative role of each P450 in the metabolism of substrates, including drugs, carcinogens, mutagens, environmental chemicals and endobiotics.

Two cytochromes P450 are major hepatocellular autoantigens in autoimmune polyglandular syndrome type 1.

BACKGROUND & AIMS: Liver disease has been described in 10%-15% of patients with autoimmune polyglandular syndrome type 1 (APS-1). After the discovery of cytochrome P450 1A2 (CYP1A2) as a hepatocellular autoantigen in liver-kidney microsomal autoantibody (LKM)-positive patients with APS-1, the investigation of antiliver antibodies was extended to 11 Sardinian patients with APS-1. METHODS: Indirect immunofluorescence and Western blotting analysis were performed to study the antiliver antibodies. RESULTS: Immunofluorescence revealed LKM antibodies in 3 patients with APS-1, 1 of whom died of fulminant hepatitis. Western blotting showed a liver microsomal protein band of approximately 51 kilodaltons in the LKM-positive sera of these 3 patients. Western blotting performed with recombinant cytochrome P450 enzymes allowed the identification of CYP2A6 as a specific target antigen. CONCLUSIONS: LKM antibodies in APS-1 sera are specifically directed against CYP1A2 or CYP2A6, but their diagnostic and prognostic significance for liver disease remain to be determined.

Cytochrome P450IA2 and aromatic L-amino acid decarboxylase are hepatic autoantigens in autoimmune polyendocrine syndrome type I.

Autoimmune chronic active hepatitis (AI-CAH) is a feared component of autoimmune polyendocrine syndrome type I (APS I). In this study, immunoreactivity was assessed in sera from eight APS I patients, of whom three had AI-CAH, in an attempt to identify hepatic autoantigens. We performed indirect immunofluorescence staining of human and rat liver sections, Western blots on subcellular fractions of human and rat liver, immunoprecipitations of labelled aromatic L-amino acid decarboxylase (AADC) and cytochrome P450IA2 (CYP IA2) expressed by an in vitro transcription and translation system and studies of enzymatic activity. Autoantibodies against AADC were present in sera from all eight APS I patients, while immunoreactivity against CYP IA2 was only found in sera from the three APS I patients with AI-CAH. Enzymatic activity of CYP IA2 was inhibited by sera from APS I patients with AI-CAH but not by control sera. Our results show that CYP IA2 and AADC constitute hepatic autoantigens in patients with APS I and that immunoreactivity against CYP IA2 is associated with the presence of AI-CAH.

Specific inhibition of human CYP1A2 using a targeted antibody.

The structural similarity of related forms of P450 makes selective immunoinhibition of individual forms notoriously difficult to achieve. We report the use of a targeted antibody to overcome this problem. An antibody was raised against the synthetic peptide, Ser-Lys-Lys-Gly-Pro-Arg-Ala-Ser-Gly-Asn-Leu-Ile, corresponding to residues 291-302 of human CYP1A2. This sequence of human CYP1A2 is located in a similar position to a proinhibitory region previously identified in rat CYP1A1 and CYP1A2. The antibody bound strongly and specifically to CYP1A2 in human hepatic microsomal fraction. Binding was unaffected by denaturation of the protein. The specificity of the antibody was demonstrated by immunoblotting of human hepatic microsomal fraction where a single immunoreactive band was identified at Mr 54,000. The intensity of this band correlated strongly with high-affinity phenacetin O-deethylase activity of the microsomal fractions. In addition, the antibody bound to a single protein at Mr 54,000 in the microsomal fraction of lymphoblastoid cells expressing human CYP1A2, but not to any other recombinant P450 enzyme. CYP1A2-dependent activity (high-affinity phenacetin O-deethylase) of human hepatic microsomal fraction was inhibited >90% by whole antiserum or purified immunoglobulin. This decrease in activity represents complete inhibition of CYP1A2 activity, residual phenacetin O-deethylase activity being due to low-affinity enzymes. In contrast, the antibody, which does not bind to rat CYP1A2, had no effect on CYP1A2-dependent activity (high-affinity phenacetin O-deethylase) of rat hepatic microsomal fraction. The antiserum also had no effect on human hepatic microsomal debrisoquine 4-hydroxylase (CYP2D6) or coumarin 7-hydroxylase (CYP2A6) activities, indicating that inhibition was specific to human CYP1A2. These results demonstrate the importance of the region comprising residues 291-302 of human CYP1A2 in the catalytic activity of this enzyme.

Epitope mapping of human CYP1A2 in dihydralazine-induced autoimmune hepatitis.

Dihydralazine-induced hepatitis is characterized by the presence of anti-liver microsomal (anti-LM) autoantibodies in the sera of patients. Cytochrome P450 1A2 (CYP1A2), involved in the metabolism of dihydralazine, was shown to be a target for autoantibodies. In order to investigate further the relationship between drug metabolism and the pathogenesis of this drug-induced autoimmune disease, and since the specificity of anti-LM autoantibodies towards CYP1A2 has been determined, the antigenic site was further localized. By constructing fragments derived from CYP1A2 cDNA and probing the corresponding proteins with several anti-LM sera, we were able to define a region (amino acid 335-471) which was immunoreactive with 100% of sera. An internal deletion in this region led to the loss of recognition by anti-LM autoantibodies, confirming that the epitope was conformational. Epitope mapping studies had previously been performed for CYP2D6, CYP17, CYP21A2, and recently for CYP3A1 and CYP2C9. Those data were compared with results obtained in the present study for CYP1A2.

Oxidation of xenobiotics by recombinant human cytochrome P450 1B1.

Human cytochrome P450 (P450) 1B1 (CYP1B1) has recently been shown to be an important enzyme in the activation of diverse procarcinogens such as arylarenes, nitroarenes, and arylamines to reactive metabolites that cause DNA damage in the cells. However, it is not known whether this P450 enzyme also plays roles in the oxidation of certain drugs or model substrates commonly used in P450 assays. We examined the substrate oxidation activities of recombinant human CYP1B1 in yeast microsomes and compared these activities with those catalyzed by reconstituted systems containing recombinant CYP1A1 and CYP1A2 which were isolated from membranes of Escherichia coli in which respective cDNAs have been expressed. Catalytic activities towards some of the model substrates of other human P450 enzymes including CYP2A6, 2C9, 2C19, 2D6, 2E1, and 3A4 were also determined and compared. CYP1B1 catalyzed benzo[a]pyrene 3-hydroxylation at rates lower than those of CYP1A1 but higher than those of CYP1A2. The activity towards 7-ethoxyresorufin O-deethylation catalyzed by CYP1B1 was about one-tenth of that of CYP1A1, but the Km values were lower for CYP1B1 than those for CYP1A1 and CYP1A2. CYP1B1 was also able to catalyze the oxidation of theophylline and caffeine, two prototypic substrates for CYP1A2. CYP1B1 did not oxidize other typical P450 substrates such as coumarin, tolbutamide, S-mephenytoin, chlorzoxazone, nifedipine, and testosterone, while low rates of oxidation of bufuralol and 7-ethoxycoumarin were found for CYP1B1. These results indicate that CYP1B1 has catalytic activities overlapping CYP1A1 and CYP1A2 with respect to the oxidation of drugs and model P450 substrates, although the relative catalytic roles in these three P450 enzymes differ depending upon the substrates examined. A distinct marker activity of CYP1B1 has not been identified.