Cytochromes P450, P420 & mixed-function oxidases as biomarkers of polychlorinated biphenyl (PCB) exposure in harbour seals (Phoca vitulina)

Hepatic microsomal cytochrome P450, EROD and ECOD activity were investigated as biomarkers of PCB exposure in harbour seals (Phoca vitulina). Due to the difficulty of obtaining undegraded seal liver samples, standard spectrophotometric methodology was adapted to investigate P420 (degraded P450) as a PCB biomarker with partially degraded samples. Total PCB burdens in both blubber and liver had positive correlations with P450, P420 and MFO activity levels. The use of P420 biomarkers in this study supports the inclusion of samples from by-caught marine mammals for future biomonitoring studies. P450 isozymes CYP1A (P4501A) and CYP2B (P4502B) in conjunction with MFO activity were investigated as "specific" biomarkers of PCB exposure. They were found to reliably reflect levels of [MC] and [PB]-type PCB exposure in harbour seal liver.     

Oxidation of benzo[a]pyrene by recombinant human cytochrome P450 enzymes

The oxidation of benzo[a]pyrene (B[a]P) was examined using reconstituted systems prepared with recombinant human cytochrome P450 (P450) enzymes 1A1, 1A2, 2C8, 2C10, 2E1, and 3A4 and with microsomes prepared from Saccharomyces cerevisiae expressing recombinant human P450s 2C8, 2C9, and 2C18. Products measured by HPLC included the 3- and 9-phenols, the 4,5-, 7,8-, and 9,10-dihydrodiols (detected in the presence of epoxide hydrolase), and products in the polar fraction eluting immediately after the void volume. The most active enzyme in all reactions was P450 1A1. P450 3A4 and P450 1A2 formed appreciable amounts of several of the products, including the 3-phenol. P450 2C enzymes and P450 2E1 formed relatively low amounts of all B[a]P products. Consideration of these patterns along with knowledge of levels of expression of the P450s in human tissues and previous results with microsomes leads to the conclusion that P450 1A1 should dominate the oxidation of B[a]P in tissues where it is present and inducible. In human liver the level of P450 1A1 is low and P450 3A4, P450 2C subfamily enzymes, and P450 1A2 probably all contribute. Of the human P450s considered here, P450 1A2 was the most active hepatic enzyme forming the 7,8-dihydrodiol. 7,8-Benzoflavone stimulated the oxidation of B[a]P by P450 3A4 and inhibited the oxidations catalyzed by P450 1A2. The extent of inhibition of P450 1A1 was less (than with P450 1A2), probably due to the rapid oxidation of 7,8-benzoflavone by P450 1A1. The major 7,8-benzoflavone product appears to be the 5,6-oxide.     

Induction of cytochrome P450 1A in hamster liver and lung by 6-nitrochrysene

The present study has determined the effect of 6-nitrochrysene (6-NC) on hepatic and pulmonary cytochrome P450 (P450)-dependent monooxygenases using hamsters pretreated with the nitrated polycyclic aromatic hydrocarbon (nitro-PAH) at 5 mg/kg per day for 3 days. Pretreatment with 6-NC elevated serum gamma-glutamyltranspeptidase, lactate dehydrogenase, and bilirubin levels. Liver S9 fractions prepared from controls and hamsters pretreated with 6-NC markedly increased mutagenicity of the nitro-PAH in Salmonella typhimurium tester strains TA98, TA100, and TA102. The pretreatment selectively increased 1-nitropyrene reductase activities of lung cytosol and liver and lung microsomes. Pretreatment with 6-NC resulted in increases of microsomal 7-ethoxyresorufin and methoxyresorufin O-dealkylases activities in liver and lung without affecting the monooxygenase activities in kidney. Immunoblot analysis of microsomal proteins using mouse monoclonal antibody 1-12-3 to rat P450 1A1 revealed that 6-NC induced P450 1A-immunorelated proteins in liver and lung. RNA blot analysis using mouse P450 1A1 cDNA showed that 6-NC increased liver and lung P450 1A mRNA. 6-NC had no effect on the kidney P450 protein and mRNA. The present study demonstrates that the hamster enzymes can support 6-NC metabolic activation and the nitro-PAH induces liver and lung P4501A via a pretranslational mechanism.     

Subacute cocaine treatment changes expression of mouse liver cytochrome P450 isoforms

Acute administration of single high doses of cocaine (50 or 60 mg/kg) produces liver injury in mice that have been pretreated with inducers of mixed function oxidases. Multiple low doses of cocaine (10-30 mg/kg) will produce hepatotoxicity without prior induction. To establish whether cocaine can induce its own activation, mice were given three daily injections of cocaine. Total cytochrome P450 content of the liver did not change. After 3 days the amount of cytochrome P450 2B10, as measured by pentoxy resorufin-O-dealkylase activity and immunoblotting, increased 3-fold. Cytochrome P450 2A5-catalyzed coumarin 7-hydroxylase activity and immunoreactive protein increased by about 50%. Enzyme activities and Western blotting of isoforms 1A, 2E, and 3A showed no change during this time. Chronic cocaine increased N-hydroxylation of norcocaine. Immunoinhibition studies showed that cytochrome P450 2A5 was the major isoform responsible for norcocaine N-hydroxylation. These results demonstrate that chronic cocaine can induce its own metabolism. Similar increases were also observed in mice not susceptible to liver injury from chronic cocaine.     

Suicide inactivation of cytochrome P450 by midchain and terminal acetylenes. A mechanistic study of inactivation of a plant lauric acid omega-hydroxylase

Incubation of Vicia sativa microsomes, containing cytochrome P450-dependent lauric acid omega-hydroxylase (omega-LAH), with [1-(14)C]11-dodecynoic acid (11-DDYA) generates a major metabolite characterized as 1,12-dodecandioic acid. In addition to time- and concentration-dependent inactivation of lauric acid and 11-DDYA oxidation, irreversible binding of 11-DDYA (200 pmol of 11-DDYA bound/mg of microsomal protein) at a saturating concentration of 11-DDYA was observed. SDS-polyacrylamide gel electrophoresis analysis showed that 30% of the label was associated with several protein bands of about 53 kDa. The presence of beta-mercaptoethanol in the incubate reduces 1,12-dodecandioic acid formation and leads to a polar metabolite resulting from the interaction of oxidized 11-DDYA with the nucleophile. Although the alkylation of proteins was reduced, the lauric acid omega-hydroxylase activity was not restored, suggesting an active site-directed inactivation mechanism. Similar results were obtained when reconstituted mixtures of cytochrome P450 from family CYP4A from rabbit liver were incubated with 11-DDYA. In contrast, both 11- and 10-DDYA resulted in covalent labeling of the cytochrome P450 2B4 protein and irreversible inhibition of activity. These results demonstrate that acetylenic analogues of substrate are efficient mechanism-based inhibitors and that a correlation between the position of the acetylenic bond in the inhibitor and the regiochemistry of cytochromes P450 oxygenation is essential for enzyme inactivation.     

Amiodarone N-deethylation in human liver microsomes: involvement of cytochrome P450 3A enzymes (first report)

Experiments were conducted on three different human liver samples to identify the cytochrome P450 isozyme which is involved in the biotransformation of the class III antiarrhythmic agent, amiodarone, into its major metabolite, desethylamiodarone (DEA). The classic P450 inhibitors, SKF 525A, metyrapone, and carbon monoxide provided a significant reduction in the in vitro formation of DEA by human hepatic microsomes. Amiodarone N-deethylase activities expressed by intrinsic clearance values were similar in all the livers used, although two livers were genotyped as extensive and one as a poor metabolizer for the cytochrome P450 CYP2D6 gene. DEA production was strongly inhibited (more than 80%) by the anti-P450 3A4 antibody, but not by anti-LKM1-positive serum. It seems therefore that the P450 3A subfamily is certainly implicated in human hepatic amiodarone N-deethylation.     

Inactivation of cytochrome P450 2E1 by tert-butylisothiocyanate

Several naturally occurring and synthethic isothiocyanates were evaluated for their ability to inactivate the major ethanol-inducible hepatic cytochrome P450 2E1. Of the compounds tested, tert-butylisothiocyanate (tBITC) was found to be the most selective inactivator of the 2E1 p-nitrophenol hydroxylation activity. tBITC was more specific for inactivating P450 2E1 activity than for rat P450 1A1, 1A2, 3A2, and 2B1, or the human cytochromes P450 3A4 and 2B6. The kinetics of inactivation of P450 2E1 by tBITC were characterized. P450 2E1, either in rat liver microsomes or in a purified reconstituted system containing the bacterially expressed rabbit cytochrome, was inactivated by tBITC in a mechanism-based manner. The loss of activity followed pseudo-first-order kinetics and was NADPH- and tBITC-dependent. The maximal rates for inactivation of P450 2E1 in microsomes or for the purified P450 2E1 at 30 degrees C were 0.72 and 0.27 min-1 and the apparent KI values were 11 and 7.6 microM, respectively. When cytochrome b5 was co-reconstituted with P450 2E1, the apparent KI for P450 2E1 inactivation by tBITC was similar to that seen in microsomes (14 microM). P450 2E1 T303A was also inactivated by tBITC with kinetic constants similar to that of the wild type enzyme. Co-incubations with an alternate substrate protected P450 2E1 from inactivation by tBITC. The extent of P450 2E1 inactivation by tBITC resulted in a comparable loss of the ability of the enzyme to form a reduced CO complex.     

Portal vein ligation selectively lowers hepatic cytochrome P450 levels in rats

In rats, surgical creation of a portacaval shunt leads to hepatic atrophy and lowered levels of cytochrome P450, the key component of liver enzymes involved with drug metabolism. These effects are largely attributable to diversion of portal blood away from the liver and not to decreased hepatic blood flow. The present study has established a simpler model of portal blood diversion in order to examine the role of portal blood constituents in the regulation of hepatic cytochrome P450. Portal vein ligation was performed on male Wistar rats in which portasystemic anastomoses had been produced by subcutaneous transposition of the spleen. Portal vein ligation resulted in portal hypertension, as evidenced by splenomegaly, and in hepatic atrophy. In liver of rats with portal vein ligation, microsomal cytochrome P450 levels were significantly less than in sham-operated control rats, but cytochrome b5, NADPH-cytochrome c reductase, and glucose-6-phosphatase were unaltered. The activities of four mixed function oxidases also were reduced significantly in the liver of rats with portal vein ligation, the changes being greatest for ethylmorphine N-demethylase, a prototype substrate for the phenobarbital-inducible isoenzyme of cytochrome P450. In contrast, the activity of microsomal heme oxygenase, the rate-limiting step in catabolism of heme to bilirubin, was enhanced after portal vein ligation. Experiments in pair-fed rats showed that the changes observed in liver from rats with portal vein ligation could not be attributed to caloric deprivation. Administration of phenobarbital increased liver mass, cytochrome P450 levels, and mixed function oxidase activities both in rats with portal vein ligation and in controls, indicating that the liver of the ligated rats retained considerable protein synthetic capacity. It appears that hepatic atrophy and lowering of cytochrome P450 levels that follow portal vein ligation are consequences of altered exposure of the liver to factors normally present in portal blood, and that the same alterations may also enhance heme oxygenase activity.     

Identification of human liver cytochrome P450 isoforms involved in the in vitro metabolism of cyclobenzaprine

Cyclobenzaprine (Flexeril) is a muscle relaxant, possessing a tricyclic structure. Numerous therapeutic agents containing this structure are known to be metabolized by polymorphic cytochrome P4502D6. The aim of this study was to determine if cytochrome P4502D6 and other isoforms are involved in the metabolism of cyclobenzaprine in human liver microsomes. Selective cytochrome P450 inhibitors for CYP1A1/2 (furafylline and 7,8-benzoflavone) and CYP3A4 (troleandomycin, gestodene, and ketoconazole) inhibited the formation of desmethylcyclobenzaprine, a major metabolite of cyclobenzaprine, in human liver microsomes. Antibodies directed against CYP1A1/2 and CYP3A4 inhibited the demethylation reaction whereas anti-human CYP2C9/10, CYP2C19, and CYP2E1 antibodies did not show any inhibitory effects. When a panel of microsomes prepared from human B-lymphoblastoid cells that expressed specific human cytochrome P450 isoforms were used, only microsomes containing cytochromes P4501A2, 2D6, and 3A4 catalyzed N-demethylation. In addition, demethylation catalyzed by these recombinant cytochromes P450 can be completely inhibited with selective inhibitors at concentrations as low as 1 to 20 microM. Interestingly, cyclobenzaprine N-demethylation was significantly correlated with caffeine 3-demethylation (1A2) and testosterone 6 beta-hydroxylation (3A4) but not with dextromethorphan O-demethylation (2D6) in human liver microsomes. To further determine the involvement of cytochrome P4502D6 in cyclobenzaprine metabolism, liver microsomes from a human that lacked CYP2D6 enzyme activities was included in this study. The data showed that cyclobenzaprine N-demethylation still occurred in the incubation with this microsome. These results suggested that cytochrome P4502D6 plays only a minor role in cyclobenzaprine N-demethylation whereas 3A4 and 1A2 are primarily responsible for cyclobenzaprine metabolism in human liver microsomes. Due to the minimum involvement of CYP2D6 in the vitro metabolism of cyclobenzaprine, the polymorphism of cytochrome P4502D6 in man should not be of muci concern in the clinical use of cyclobenzaprine.     

A convenient method to discriminate between cytochrome P450 enzymes and flavin-containing monooxygenases in human liver microsomes

Liver microsomes are a frequently used probe to investigate the phase I metabolism of xenobiotics in vitro. Structures containing nucleophilic hetero-atoms are possible substrates for cytochrome P450 enzymes (P450) and flavin-containing monooxygenases (FMO). Both enzymes are located in the endoplasmatic reticulum of hepatocytes and both need oxygen and NADPH as cofactors. The common method to distinguish between the two enzyme systems is to use the thermal inactivation of FMO and to inhibit P450 completely with carbon monoxide, N-octylamine or N-benzylimidazole. In the literature no indication could be found that the heat inactivation of FMO does not affect any of the human P450 enzymes or that the overall P450 inhibitors inhibit the different human P450 enzymes sufficiently and do not affect the FMO. The effect of N-benzylimidazole and heat inactivation was tested on specific activities of seven P450 enzymes in human liver microsomes, 1A2, 2A6, 2C9, 2C19, 2D6, 3A4/5, and 2E1, using methoxyresorufin O-demethylation, coumarin 7-hydroxylation, (S)-warfarin 4-hydroxylation, (S)-(+)-mephenytoin 4-hydroxylation, dextrometorphan O-demethylation, oxidation of denitronifedipine, and chlorzoxazone 6-hydroxylation respectively. The sulfoxidation of methimazole (MMI) was used as a specific probe for the determination of FMO activity. Methimazole sulfoxidation was compared with the well known assay for FMO metabolism, the formation of N,N-dimethylaniline (DMA) N-oxide, to be confirmed as an exclusively FMO mediated reaction. The participation of P450 and FMO in the sulfoxidation of four sulfur containing peptides, ametryne; terbutryne, prometryne and methiocarb was investigated using human liver microsomes. All four reactions were demonstrated to be catalysed predominantly by cytochrome P450.     

Cytochromes P450 and uridine triphosphate-glucuronosyltransferases: model autoantigens to study drug-induced, virus-induced, and autoimmune liver disease

Enzymes of phase I (cytochromes P450) and phase II (UDP [uridine diphosphate]-glucuronosyltransferases) of drug metabolism are targets of autoimmunity in the following chronic liver diseases of different etiology: 1)autoimmune hepatitis (AIH); 2) hepatitis associated with the autoimmune polyendocrine syndrome type 1 (APS-1); 3) virus-induced autoimmunity; and 4) drug-induced hepatitis. AIH is diagnosed by the following: the absence of infection with hepatitis viruses; the presence of a threshold of relevant factors, including circulating autoantibodies, hypergammaglobulinemia, female sex (female/male ratio 4:1), human leukocyte antigen (HLA) B8, DR3, or DR4; and benefit from immunosuppression. Patients with autoimmune hepatitis type 2 (AIH-2) are characterized by antibodies directed against liver and kidney microsomes, by an early onset of autoimmune hepatitis, which is a more aggressive course of the disease, and by a higher prevalence of autoimmunity directed against other organs. The major target of autoimmunity in patients with AIH-2 is cytochrome P450 2D6. Epitope mapping experiments revealed four short linear epitopes on cytochrome P450 2D6, recognized by liver/kidney microsomal autoantibodies type 1 (LKM-1) in patients with AIH-2. In addition, about 10% of the patient sera contain autoantibodies that detect a conformational epitope on UDP-glucuronosyltransferases (UGTs) of family 1. Presently, LKM-1 autoantibodies are used as diagnostic markers for AIH-2. It is unclear whether these autoantibodies have a pathogenetic role. Hepatitis is found in some patients with APS-1. Presumably this also is an autoimmune liver disease. APS-1 patients with hepatitis may develop autoantibodies directed against microsomal P450 enzymes of the liver; however, these autoantibodies do not recognize cytochrome P450 2D6, but they do recognize cytochrome P450 1A2. Autoimmunity in patients with APS-1 usually is directed against several organs simultaneously, and several organ specific autoantibodies may exist. Interestingly, APS-1 patients may produce various anti-cytochrome P450 antibodies. In addition to the hepatic anti-cytochrome P450, 1A2 autoantibodies are directed against steroidogenic cytochromes P450, namely P450 c21, P450 scc, and P450 c17. These autoantibodies correlate with adrenal and ovarian failure and often these steroidal cell autoantibodies precede the manifestation of adrenal or ovarian dysfunction. Whether anti-P450 1A2 autoantibodies have a similar predictive value is not yet known. LKM autoantibodies are further found in association with chronic hepatitis C and D. In chronic hepatitis C, the major target of LKM autoantibodies is cytochrome P450 2D6. Predominantly, conformational epitopes are recognized by LKM-1 sera of patients with chronic hepatitis C. In 13% of patients with chronic hepatitis D, LKM-3 autoantibody is detectable. The target proteins are UGTs of family 1 and in a minority of sera UGTs of family 2. The epitopes are conformational. All hepatic diseases discussed earlier have in common that autoimmunity, which is directed against enzymes of drug metabolizing multigene families. Each disease is characterized by a specific pattern of autoantibodies, with apparently little overlap. For example, LKM-1 autoantibodies, which are directed against P450 2D6, seem to overlap between AIH and chronic hepatitis C. However, a close examination of these autoantibodies shows differences between LKM-1 autoantibodies from patients with chronic hepatitis C and with AIH. In AIH, LKM autoantibodies are more homogenous, titers are higher, and major autoepitopes on cytochrome P450 2D6 are small and linear. LKM autoantibodies in viral hepatitis C are more heterogeneous and there are multiple epitopes, many of which are conformational. These differences indicate the different mechanisms that are involved in the generation of autoimmunity. (ABSTRACT TRUNCATED)     

Induction and inhibition of cytochrome P450 isoforms by imazalil, a food contaminant, in mouse small intestine and liver

1. The effects of imazalil, a food contaminant used as a fungicide, were investigated on the expression and activity of cytochrome P450 in the small intestinal mucosa and liver of mice. Imazalil was orally administered to mice daily at 1 or 10 mg/kg for 3 days. 2. Imazalil enhanced cytochrome P450-catalysed ethoxyresorufin O-deethylase and pentoxyresorufin O-depentylase (PROD) activities in both tissue microsomes at the 10 mg/kg/day dose level, indicating the induction of cytochrome P450 subfamilies CYP1A and CYP2B. In addition, immunochemical analyses also demonstrated an enhanced expression of CYP2B, CYP2C and CYP3A subfamilies in both tissues. 3. Imazalil was a potent inhibitor of cytochrome P450-dependent monooxygenase activities (PROD, aminopyrine N-demethylase and erythromycin demethylase) in in vitro assays using both small intestinal and liver microsomes. 4. From these findings, imazalil has been demonstrated to have not only a potent inhibitory activity but also a significant inducing ability of P450 isoforms in the small intestine. Prolonged ingestion of such a food contaminant may modulate the xenobiotic-metabolizing enzyme system at the site of a primary portal of xenobiotic entry to the systemic circulation.     

Specificity of self-assembly of cytochrome P450

Under certain conditions, hexamers of microsomal cytochrome P450 can self-assemble from the subunits of different isoforms. However, the possibility for free choice results in recognition between identical subunits of each form of cytochrome P450 which provides preferential association of identical monomers into corresponding hexamers. The specificity of self-assembly suggests hexameric arrangement of cytochrome P450 in native membranes as we proposed earlier. In the present study, highly purified cytochrome P450 2B4 and cytochrome P450 1A2 (CYP 2B4 and CYP 1A2), including those immobilized by covalent attachment to an insoluble carrier of one protomer of each hexamer, were employed.     

In vitro identification of the P450 enzymes responsible for the metabolism of ropinirole

The in vitro metabolism of ropinirole was investigated with the aim of identifying the cytochrome P450 enzymes responsible for its biotransformation. The pathways of metabolism after incubation of ropinirole with human liver microsomes were N-despropylation and hydroxylation. Enzyme kinetics demonstrated the involvement of at least two enzymes contributing to each pathway. A high affinity component with a K(M) of 5-87 microM and a low affinity component with a K(M) of approximately two orders of magnitude greater were evident. The high affinity component could be abolished by pre-incubation of the microsomes with furafylline. Additionally, incubation of ropinirole with microsomes derived from CYP1A2 transfected cells readily produced the N-despropyl and hydroxy metabolites. Some inhibition of ropinirole metabolism was also observed with ketoconazole, indicating a minor contribution by CYP3A. Multivariate correlation data were consistent with the involvement of the cytochrome P450 enzymes 1A2 and 3A in the metabolism of ropinirole. Thus, it could be concluded that the major P450 enzyme responsible for ropinirole metabolism at lower (clinically relevant) concentrations is CYP1A2 with a contribution from CYP3A, particularly at higher concentrations.     

Differential roles of Glu318 and Thr319 in cytochrome P450 1A2 catalysis supported by NADPH-cytochrome P450 reductase and tert-butyl hydroperoxide

The kinetic values for 7-ethoxycoumarin (7-EC) hydroxylation have been obtained in both the NADPH-cytochrome P450 reductase- and tert-butyl hydroperoxide (TBHP)-supported systems for several Glu318 and Thr319 mutants of cytochrome P450 1A2. The results with the reductase-supported system suggest that Glu318 is important for both substrate binding and catalysis, whereas Thr319 is critical for neither, although the size of the residue at position 319 influences catalytic activity. In contrast, neither Glu318 nor Thr319 appears to be important for catalytic turnover in the TBHP-supported system despite the fact that the size of the amino acid at position 319 affects the binding of TBHP and 7-EC in opposite manners. The roles of these two distal amino acids in the cytochrome P450 1A2-catalyzed oxidation of 7-EC therefore differ for the reactions supported by cytochrome P450 reductase and TBHP.     

Differential effects of flavonoids on testosterone-metabolizing cytochrome P450s

Flavonoids are widely distributed phytochemicals, whose modulation of cytochrome P450 mediated carcinogen metabolism is well established. Less well studied is their effect on P450 dependent metabolism of endogenous substrates. To address this question we evaluated a series of twelve flavonoids and hematoxylin for their effect on P450-mediated steroid hydroxylation by rat liver microsomes. Site-specific 7alpha-, 6beta- and 2alpha-hydroxylation of testosterone by P450s 2A1, 3A2 and 2C11, respectively, was measured. Highly selective patterns of inhibition or activation of these P450s were observed. 3,6-dichloro-2'-isopropyloxy-4'-methylflavone was the most potent inhibitor of P450 2C11 while cyanidin chloride most potently inhibited P450s 2A1 and 3A2. The flavonoid analogue hematoxylin was unique in that it activated 2C11 (by 2.5 fold) yet inhibited both 2A1 and 3A2 (by 60%). These results indicate that consumption of dietary flavonoids may likewise alter the metabolite profile of steroids and other physiological P450 substrates.     

Purification and characterization of constituent androstenedione 15 alpha-hydroxylase (cytochrome P450(15 alpha AD)) from mouse liver. Sex- and tissue-dependent expression

Hepatic microsomal androstenedione 15 alpha-hydroxylase (i.e.cytochrome P450(15)alpha AD was purified from female CD-1 mice. Protein purification was monitored in eluates from Fractogel, DEAE-Sephacel, and hydroxylapatite columns at heme absorbing 417 nm, by cytochrome P450 content, reactivity to monoclonal antibody against female-specific rat cytochrome P450 2C12, and androstenedione 15 alpha-hydroxylase activity. The catalytic activity for androgens of the purified cytochrome P450(15)alpha AD, exhibiting a high degree of regioselectivity and stereospecificity, was restricted to the 7 alpha- and 15 alpha-hydroxylation of androstenedione, representing, respectively, > 5% and > 93% of the total metabolites. Polyclonal antibodies against cytochrome P450(15)alpha AD exhibited a concentration-dependent and very selective inhibition of hepatic microsomal androstenedione 7 alpha- and 15 alpha-hydroxylation and a 60% inhibition of benzphetamine demethylation, the latter drug appearing to be a much more effective substrate than androgens. Cytochrome P450(15)alpha AD accounted for about 3% of the total P450 in female mouse liver microsomes. The apparent subunit molecular weight of P450(15)alpha AD was 53,000, and the protein appeared as a single band or sodium dodecyl sulfate-polyacrylamide gels. The isoform was intensely expressed in both liver and lung of CD-1 female mice and was female-predominant in the livers of five or eight strains examined; it was sex-independent in the remaining three strains. Amino-terminal sequence analysis indicates that cytochrome P450(15)alpha AD is a member of the murine cytochrome P450 2c subfamily.     

Inhibitory monoclonal antibody to human cytochrome P450 2B6

The human cytochrome P450 2B6 metabolizes, among numerous other substrates, diazepam, 7-ethoxycoumarin, testosterone, and phenanthrene. A recombinant baculovirus containing the human 2B6 cDNA was constructed and used to express 2B6 in Sf9 insect cells. The 2B6 was present at 1.8 +/- 0.4% of the total cellular protein and was purified to a specific content of 13.3 nmol/mg protein. Mice were immunized with the purified 2B6, and a total of 811 hybridomas were obtained from the fusion of NS-1 myeloma cells and spleen cells of the immunized mice. Monoclonal antibodies (MAbs) from 24 of the hybrids exhibited immunobinding to 2B6 as determined by ELISA. One of the MAbs, 49-10-20, showed a strong immunoblotting activity and was highly inhibitory to 2B6 enzyme activity. MAb 49-10-20 inhibited cDNA-expressed 2B6-catalyzed metabolism of diazepam, phenanthrene, 7-ethoxycoumarin, and testosterone by 90-91%. MAb 49-10-20 showed extremely high specificity for 2B6 and did not bind to 17 other human and rodent P450s or inhibit the metabolism of phenanthrene catalyzed by human 1A2, 2A6, 2C8, 2C9, 2D6, 2E1, 3A4, and 3A5. MAb 49-10-20 was used to determine the contribution of 2B6 to the metabolism of phenanthrene and diazepam in human liver. In ten liver samples, MAb 49-10-20 inhibited phenanthrene metabolism variably by a wide range of 8-42% and diazepam demethylation by 1-23%. The degree of inhibition by the 2B6 specific MAb 49-10-20 defines the contribution of 2B6 to phenanthrene and diazepam metabolism in each human liver. This technique using inhibitory MAb 49-10-20 determines the contribution of 2B6 to the metabolism of its substrates in a human tissue containing multiple P450s. This study is a prototype for the use of specific and highly inhibitory MAbs to determine individual P450 function.