Differences in the modulation of P450IA1 and epoxide hydratase expression by benz[a]anthracene and 2,3,7,8-tetrachlorodibenzo-p-dioxin in mouse embryo versus mouse hepatoma-derived cell lines

Polycyclic aromatic hydrocarbon (PAH)-induced C3H/10-T1/2/CL8 mouse embryo fibroblasts (10T1/2) and mouse hepatoma-derived Hepa 1c1c7 cells (Hepa-1), exhibit comparable total cytochrome P450 levels and total PAH-metabolizing activities but very different distributions of PAH metabolites. Based on anti-P450IA1-IgG inhibition data, P450IA1 contributes essentially all PAH metabolism in Hepa-1 microsomes but is not involved in PAH metabolism by 10T1/2 cells. In addition, the microsomal epoxide hydratase (EHm) in Hepa-1 cells is far less effective in dihydrodiol (diol) formation compared to that in 10T1/2 microsomes [Pottenger, L.H. and Jefcoate, C.R. Carcinogenesis, 11, 321-327 (1990)]. In the present study, the levels of expression of P450IA1 and EHm proteins and the corresponding mRNAs, both prior to and following exposure to benz[a]anthracene (BA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), have been correlated with microsomal PAH metabolism by each cell type. In 10T1/2 cells, P450IA1 protein (56 kd) and mRNA (2.6 kb) were detectable at extremely low levels in only two of five cell preparations and then only after maximum induction by TCDD and BA. Thus although 10T1/2 cells contain functional Ah receptors, their capacity to induce P450IA1 is highly suppressed, representing at most 2% of the total P450. TCDD (10 nM) was 4-fold more effective than BA (10 microM) in inducing P450IA1 mRNA, while the levels of immunodetectable protein were comparable. An even greater discrepancy between P450IA1 mRNA and protein levels was seen in BA-induced Hepa-1 cells, where a 4-fold increase in mRNA was paralleled by a 20-fold increase in protein. This difference is probably due to the greater effect of BA depletion on mRNA compared to protein levels. In 10T1/2 cells, BA and TCDD were equally effective at increasing expression of an unidentified 1.9 kb mRNA sequence that blotted very weakly with the P450IA1 cDNA probe. The expression of this mRNA was independent from that of P450IA1. A similar band was visible in Hepa-1 cells less than 1% of the P450IA1 mRNA. EHm mRNA was almost 3-fold higher in 10T1/2 compared to Hepa-1 cells and was unaffected by cell treatments. In Hepa-1 cells, BA and TCDD elevated EHm protein and hydrating activity to levels comparable to those expressed in 10T1/2 cells. It is, therefore, suggested that the relative ineffectiveness of Hepa-1, compared to 10T1/2 EHm, to hydrate low levels of PAH-epoxides is due to differences between the two proteins or their disposition in the microsomal membrane.     

Mouse endometrium stromal cells express a polycyclic aromatic hydrocarbon-inducible cytochrome P450 that closely resembles the novel P450 in mouse embryo fibroblasts (P450EF)

Stromal cells from mouse endometrium, E041 cells, at passages21, 23 and 26 were metabolically active towards 7,12-dimethylbenz[a]anthracene(DMBA). The total DMBA-metabolizing activity of E041 cells waspreferentially increased by benz[a]anthracene (BA) relativeto 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment (7-foldby BA and 4-fold by TCDD), but the relative proportions of DMBAmetabolites formed remained unchanged. Profiles of DMBA metabolitesgenerated from E041 cell microsomes were very different fromthat of mouse P4501A1 but exhibited similarities to that ofP450EF, the polycyclic aromatic hydrocarbon (PAH)-induciblecytochrome P450 in the mouse embryo fibroblast cell line C3H10T1/2(10T1/2). Notably, both induced and uninduced E041 cell microsomeswere very effective in the formation of the proximate carcinogenDMBA-3,4-dihydrodiol (15–20% of total) and DMBA-10,11-dihydrodiol(13–18% of total) but ineffective in forming the 7-hydroxymethylderivative of DMBA (<1% of total). Antibodies to P450EF completelyinhibited the DMBA-metabolizing activities of both induced anduninduced E041 cell microsomes, with an effectiveness similarto that in microsomes prepared from identically treated 10T1/2cells. Anti-P4501A1 had no inhibitory effect on DMBA metabolismby either induced or uninduced E041 cell microsomes. Total DMBA-metabolizingactivities in BA- and TCDD-induced E041 cells were consistently2-fold lower compared to those in similarly treated 10T1/2 cells.In addition, both induced and uninduced E041 cell microsomesformed a lower proportion of DMBA dihydrodiols relative to phenolsin comparison to identically treated 10T1/2 cell microsomes(0.5 versus 1). Addition of exogenous epoxide hydrolase to E041cell microsomes resulted in a product distribution indistinguishablefrom that in 10T1/2 cells. Immunoblots demonstrated 5-fold lowerlevels of epoxide hydrolase in E041 cell microsomes comparedto 10T1/2 cell microsomes. Anti-P450EF immunoblotted a 55 kdprotein in E041 cell microsomes that was induced 14-fold byBA and 6-fold by TCDD, thus paralleling the increases in therespective DMBA metabolism. It is therefore concluded that followingPAH exposure endometrial stromal cells express the novel PAH-induciblemouse embryo fibroblast P450 and fail to express P4501A1.     

Use of monoclonal antibodies to identify cytochrome P450 isozymes in rat liver microsomes that hydroxylate N-nitrosomethylamylamine at each of six positions

Inhibition of enzyme activity by monoclonal antibodies (MAbs) was used to indicate which cytochrome P450 isozymes in Sprague-Dawley rat liver microsomes catalyse hydroxylation of the oesophageal carcinogen N-nitrosomethyl-n-amylamine (NMAA) to give 2- to 5-hydroxy-NMAA (HO-NMAA), formaldehyde and pentaldehyde. Liver microsomes (0.3-0.6 mg protein) were incubated (15 min, 23 degrees C) with 0.4 mg MAb and, after adding NMAA to 6 mM, incubated for 20 min at 37 degrees C. Mixtures were analysed for HO-NMAAs by gas chromatography-thermal energy analysis and for aldehydes by high-performance liquid chromatography of their 2,4-dinitrophenylhydrazones. The percentage inhibition by each MAb indicates the percentage metabolism by the corresponding P450 isozyme(s). These results indicate that the MAb to P450 IIB1 cross-reacts with P450 IIE1 and that the MAb to male-specific constitutive IIC11 cross-reacts with female-specific IIC12. Taking this into account, the main results were as follows. With uninduced male microsomes, 4-hydroxylation was catalysed mainly by IIC11 and demethylation by IIC11 and IIE1. With uninduced female microsomes, P450s reacting with the MAb to IIC11 (probably mainly IIC12) were responsible for most of the 4-hydroxylation and demethylation. With 3-methylcholanthrene-induced male microsomes, most 3-hydroxylation and some depentylation were due to IA1 or IA2. With phenobarbital-induced microsomes, all six reactions, but especially 4-hydroxylation and depentylation, were largely due to IIB1. With Aroclor-induced microsomes, all six reactions were catalysed by IIB1 and IA1 or IA2. The role of P450 IIC11 in 4-(omega-1)-hydroxylation was striking.     

Co-expression of human CYP1A1 and a human analog of cytochrome P450-EF in response to 2,3,7,8-tetrachloro-dibenzo-pdioxin in the human mammary carcinoma-derived MCF-7 cells

Cultured human mammary carcinoma (MCF-7) cells exhibited constitutivecytochrome P450-dependent metabolism of 7,12-dimethylbenz[a]anthracene(DMBA) (45–75 pmol/mg microsomal protein). Exposure ofthe cells to 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD), whichis known to induce CYP1A1, not only resulted in a 30-fold increasein the total microsomal metabolism of DMBA but produced substantialdifferences in the distribution of DMBA metabolites formed.This suggested that different cytochrome P450 (P450) forms predominatedin untreated and induced cells. Comparative studies with TCDD-inducedhuman hepatoblastoma (HepG2) and skin cell carcinoma (SCC-13)cells and also recombinantly expressed human CYP1A1, confirmedthat the DMBA metabolite profile in TCDD-induced MCF-7 cellswas that of human CYP1A1. This distribution, however, differedsubstantially from the regioselectivity of rat CYP1A1 and mouseCypla-1. Rabbit antibodies to rat CYP1A1 completely inhibitedthe DMBA-metabolizing activity of TCDD-induced MCF-7 cells buthad no inhibitory effect on constitutive DMBA metabolism whichwas, however, completely inhibited by chicken antibodies tothe novel P450 in mouse embryo fibroblasts (P450-EF). Anti-P450-EFinhibited only 10% of the DMBA-metabolizing activity in theTCDD-induced MCF-7 cell microsomes. Microsomes from untreatedMCF-7 cells expressed a 52 kDa protein that was immunodetectableby rabbit anti-P450-EF and failed to express immunodetectablelevels of human CYP1A1. DMBA metabolism, therefore, s from P450-EFin uninduced microsomes to CYP1A1 in TCDD-induced microsomes.The mobility of the P450-EF-like protein in MCF-7 cells washigher than that of P450-EF from C3H/10T1/2CL8 (10T1/2) cells(55 kDa). The 52 kDa protein from MCF-7 cells was induced     

Positional specificity for methyl-n-amylnitrosamine hydroxylation by cytochrome P-450 isozymes determined with monoclonal antibodies

Inhibitory monoclonal antibodies (MAbs) were used to determine the contribution of epitope-specific cytochrome P-450 isozymes in rat liver microsomes to hydroxylation of the esophageal carcinogen methyl-n-amylnitrosamine. These P-450-catalyzed reactions form 2-, 3-, 4-, and 5-hydroxymethyl-n-amylnitrosamine, formaldehyde (demethylation), and pentaldehyde (depentylation). With uninduced microsomes from male rats, MAb 1-68-11 inhibited 4-hydroxylation by 73% and demethylation by 46%. This indicated the major contribution of constitutive male-specific P-450 IIC11 to the metabolism. Inhibition studies with MAbs 2-66-3 and 1-91-3 indicated that P-450 IIB1 contributed 19% and IIE1 35% to demethylation. With uninduced microsomes from females, MAb 1-68-11 produced similar inhibitions to those in male rats, indicating that female-specific P-450 IIC12 (which is closely related to IIC11) also catalyzed 4-hydroxylation and demethylation. With microsomes from 3-methylcholanthrene-induced male rats, P-450 IA1 and/or IA2 were responsible for 60% of 3-hydroxylation and 40% of depentylation. With microsomes from phenobarbital-treated rats, P-450 IIB1 and IIB2 catalyzed all 6 reactions but especially 4-hydroxylation and depentylation, which were 50-75% inhibited by MAb 2-66-3. Microsomes from Aroclor-induced males behaved as if they were induced by both 3-methylcholanthrene and phenobarbital. After treatment with isoniazid (a P-450 IIE1 inducer), inhibition by MAb 1-91-3 indicated a 45% contribution of P-450 IIE1 to demethylation, and both P-450 IIE1 and IIB1 (or IIB2) appear to have been induced. A major finding with uninduced microsomes was the high specificity of MAb 1-68-11 for inhibiting 4-hydroxylation, indicating that P-450 IIC11 and IIC12 catalyzed most of this omega-1-hydroxylation. In microsomes from induced rats, the MAb inhibitions showed the role of the induced P-450 IA1 (or IA2), IIB1 (or IIB2), and IIE1 in methyl-n-amylnitrosamine hydroxylation at different positions, as well as the presence of P-450 IIC11. This study illustrates the usefulness of inhibitory MAbs for defining the contribution of individual P-450s to position-specific metabolism.     

Differential rates of metabolic activation and detoxication of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine by different cytochrome P450 enzymes

Rat liver microsomes metabolized the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to the genotoxic metabolite 2-hydroxamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (2-hydroxamino-PhIP) and to the detoxified product 2-amino-4'-hydroxy-1-methyl-6-phenylimidazo[4,5-b]pyridine (4'-hydroxy-PhIP). A 25-fold higher rate of metabolism was measured in microsomes from polychlorinated-biphenyl-treated rats (94 nmol/mg proteins/30 min) in comparison with those from untreated rats. Other effective inducers of PhIP metabolism were beta-naphthoflavone and isosafrole (ISF), whereas phenobarbital was ineffective. About twice as much 2-hydroxamino-PhIP as 4'-hydroxy-PhIP was formed in microsomes irrespective of the inducer the rats had been treated with. The metabolism was dependent on NADPH and was abolished by the cytochrome P450 inhibitor alpha-naphthoflavone. In a reconstituted enzyme system purified rat cytochrome P450 IA2 (P450ISF-G) had the highest N-hydroxylation rate (30 nmol/nmol P450/30 min) closely followed by the rat cytochrome P450 IA1 (P450BNF-B). Less activity was seen with rat P450 IIC11 (P450UT-A) and rabbit P450 IA2 (P450 LM4). Rat P450 IIE1 (P450j), P450 IIB1 (P450PB-B) and rabbit P450 IIB4 (P450 LM-2) and P450 IIE1 (P450 LM3a) were essentially inactive. Rat P450 IA1 (P450BNF-B) produced five times more 4'-hydroxy-PhIP (32 +/- 2 nmol/nmol P450/30 min) than did P450 IA2 (P450ISF-G). Hence, the measured ratio of activation to detoxication for rat P450 IA2 (P450ISF-G) enzyme was 7-fold higher than that of the other active P450 enzymes.     

Metabolism of 2-acetylaminofluorene and benzo(a)pyrene and activation of food-derived heterocyclic amine mutagens by human cytochromes P-450

The human P-450 CYP1A1 gene and a P450IA2 complementary DNA have been expressed in Cos-1 cells and the expressed proteins were assayed for their capacity to metabolize the carcinogens 2-acetylaminofluorene (AAF), benzo(a)pyrene, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was determined. The expressed human P450IA1 and P450IA2 proteins, when run on a 7.5% sodium dodecyl sulfate-polyacrylamide gel, migrated with different mobilities, with the former displaying the lower molecular weight. In human liver microsomes from 18 subjects, only a protein band corresponding to P450IA2 was detectable. Cos-1 cell-expressed P450IA1 and P450IA2 were capable of N-hydroxylating AAF and these activities were inhibited by alpha-naphthoflavone. In human liver microsomes, a correlation of r = 0.76 (P less than 0.05; n = 18) was obtained between AAF N-hydroxylase activity and P450IA2 content. AAF N-hydroxylase activity of human liver microsomes was also strongly inhibited by alpha-naphthoflavone. Except in the case of PhIP, where both proteins exhibited similar activities, P450IA2 was at least an order of magnitude more efficient than P450IA1 in activating IQ, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline to mutagens as measured in the Ames test. Statistically significant correlations were obtained between IQ activation and P450IA2 content (r = 0.75, r2 = 0.56) and PhIP activation and P450IA2 content (r = 0.71, r2 = 0.5) in human liver microsomes. The activation of both IQ and PhIP by expressed proteins and human liver microsomes was strongly inhibited by alpha-naphthoflavone. The above data suggest a major role for P450IA2 in activation (N-hydroxylation) of aromatic amides and amines in human liver. When benzo(a)pyrene hydroxylase activity was determined, only Cos-1 cell-expressed P450IA1 exhibited appreciable activity. While alpha-naphthoflavone inhibited Cos-1 cell-expressed P450IA1 benzo(a)pyrene hydroxylase activity, it caused a marked stimulation of this activity in human liver microsomes, which lack P450IA1 protein. The lack of a role for P450IA proteins in benzo(a)pyrene metabolism is further supported by the poor correlation (r = 0.43, P greater than 0.05) between this activity and P450IA2 content of human liver microsomes. However, when P450IIIA3 content of the above human liver microsomes was determined by using the Western blot technique and correlated with benzo(a)pyrene metabolism, an r value of 0.70 (P less than 0.5) was obtained. These data suggest that human P450IIIA proteins are involved in benzo(a)pyrene metabolism.     

Induction of mammary cytochromes P-450: an essential first step in the metabolism of 7,12-dimethylbenz[a]anthracene by rat mammary epithelial cells

Rat mammary epithelial cells (RMEC) in culture have been shownto activate polycydlic aromatic hydrocarbon (PAH) carcinogens.This study investigates the role of mammary cyto chrome P-450monooxygenases in these metabolic processes. Monooxygenatlonof 7,12-dimethythenz[a]anthracene (DMBA) by RMEC in cultureexhibited a 6-h lag period before reaching a constant rate.The mechanism for this time-dependent expression of DMBA monooxygenaseactivity was investigated in lysed cells, where both conjugationand in situ induction of P-450 are prevented. Although metabolismof DMBA by untreated RMEC lysates was undetectable (<1 pmol/mgcell protein/h), prior exposure of cultured cells to benz[a]anthracene(BA) induced DMBA metabolism, ({small tilde}100 pmol/mg cellprotein/h). BA pretreatment also eliminated the lag period formetabolism of DMBA by cultured RMEC but did not prevent additionalinduction of DMBA monoxygenase activity by the substrate. Thedistribution of monooxygenated DMBA metabolites formed by BA-inducedcell lysates was clearly different from that obtained with purifiedP-450c, the predominant PAH-inducible isozyme in rat liver.For example, the carcinogen precursor DMBA 3,4-dihydrodiol,which is not formed by P-450c, was a dearly detectable productin RMEC. The low epoxide hydratase activity of BA-induced lysate({small tilde}400-fold lower compared to that in the liver)limited formation of all DMBA dihydrodlols. The formation ofDMBA 3,4-dihydrodiol increased by 5-fold following additionof exogenous purified epoxide hydratase. The DMBA monooxygenaseactivity of BA-induced RMEC lysates was completely inhibitedby -naphthoflavone but was only partially inhibited (50%) bya polyclonal antibody raised against cytochrome P-450c. AntiP-450c completely inhibited formation of some of the metabolites,partially inhibited formation of others and notably stimulatedformation of DMBA 3,4-dihydrodlol by 60%. A polyclonal antibodythat recognized both rat hepatic P-450a and a group of P-450isozymes related to P-450h, and which totally inhibited DMBA3,4-dihydrodiol formation by rat liver microsomes, did not inhibitformation of any DMBA metabolite in RMEC, including DMBA 3,4-dihydrodiol.Western blot analyses of RMEC homogenates demonstrated thatBA pretreatment induces P-450c, but not P-450a or any of theP-450h-related isozymes. We conclude that metabolism of DMBAby RMEC depends on induction of P-450c and at least one additionalform of cytochrome P-450 which is immunochemically distinctfrom rat hepatic P-450a and P-450h related isozymes, but issensitive to a-naphthoflavone.     

N,N',N'-triethylenethiophosphoramide (thio-TEPA) oxygenation by constitutive hepatic P450 enzymes and modulation of drug metabolism and clearance in vivo by P450-inducing agents.

The cancer chemotherapeutic drug N,N',N'-triethylenephosphoramide (thio-TEPA) is oxidatively desulfurated to yield the active metabolite N,N',N'-triethylenephosphoramide (TEPA) in a reaction catalyzed by the phenobarbital-inducible rat liver P450 enzyme IIB1. In the current study, the role of constitutively expressed P450 enzymes in thio-TEPA metabolism was studied using purified P450s, isolated liver microsomes, and intact rats. Metabolism of thio-TEPA (100 microM) to TEPA by uninduced adult female and male rat liver microsomes proceeded at initial rates of 0.10 and 0.28 nmol TEPA formed/min/mg microsomal protein, respectively. Although these rates are low compared to those catalyzed by phenobarbital-induced liver microsomes (3.5 nmol TEPA/min/mg), they are sufficient to contribute to the systemic metabolism of this drug. Thio-TEPA metabolism catalyzed by uninduced female liver microsomes was approximately 70% inhibitable by antibodies selectively reactive with P450 IIC6. For the uninduced male liver microsomes, which exhibit a severalfold higher rate of thio-TEPA metabolism, enzyme activity was only 15-20% inhibitable by these antibodies but was 80-85% inhibited by an anti-P450 IIC6 monoclonal antibody cross-reactive with P450 IIC11, which is expressed only in the males. Consistent with these observations, purified P450s IIC11 and IIC6 both oxidized thio-TEPA in reconstituted systems (turnover, 1.1 and 0.3 min-1 P450-1, respectively, at 100 microM substrate), while several other constitutive hepatic P450s exhibited significantly lower or undetectable activities (turnover, less than or equal to 0.15 min-1 P450-1). Metabolism of thio-TEPA by purified P450 IIC11 was associated with a time-dependent inactivation of the cytochrome analogous to that previously shown to accompany thio-TEPA metabolism catalyzed by P450 IIB1. Depletion of hepatic P450 IIC11 by cisplatin treatment of adult male rats led to a 70% reduction of TEPA formation catalyzed by the isolated liver microsomes, suggesting that cisplatin may influence thio-TEPA pharmacokinetics when these two drugs are given in combination. The extent to which hepatic P450s contribute to thio-TEPA metabolism and clearance in vivo was assessed by monitoring thio-TEPA and TEPA pharmacokinetics in rats that exhibit widely differing rates of microsomal thio-TEPA metabolism, i.e., uninduced female and male rats, and male rats treated with the P450 IIB1 inducers clofibrate and phenobarbital. In accord with the microsomal activities, conversion of thio-TEPA to TEPA was less extensive and thio-TEPA elimination slower in female than in male rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

Metabolic deactivation of furylfuramide by cytothrome P450 in human and liver microsomes

Metabolic deactivation of furylfuramide by human and rat livermicrosomal cytochrome P450 enzymes has been investigated ina system measuring induction of umu gene expression responsein Salmonella typhimurium TA1535/pSK1002. Both human and ratliver microsomes catalyzed the metabolism of furylfuramide toinactive form(s) that are incapable of inducing umu gene expressionin the tester strain. The reaction required an NADPH-generatingsystem and molecular oxygen and was inhibited by carbon monoxide,suggesting that a cytochrome P450-linked monooxygenase systemis prerequisite for the deactivation reaction. With liver microsomesfrom variously pretreated rats, 3-methylcholanthrene was foundto be a powerful inducer for the furylfuramide-metabolizingactivity, and antibodies raised against rat P450IA1(BNF-B, c)and P450IA2(ISF-G, d) inhibited the microsomal activity. Humanliver microsomal furylfuramide-metabolizing activity was alsoinhibited significantly by anti-P450IA2 lgG but weakly by anti-P450IA2IgG. In liver microsomes prepared from seven different humansamples, the activities of deactivation of furylfuramide werefound to correlate with the amounts of immunoreactive proteinrelated to rat P450IA2 and with the monooxygenase activitiesof metabolic activation of 2-amino-3,4-dimethyl-imidazo [4,5-]quinoline(MeIQ) and of ethoxyresorufin O-deethylation. These resultssuggest that P450IA1 and P450IA2 in rats, and P45OPA (IA2, thephenacetin O-deethylase and ortholog of rat P450IA2) in humansare the major enzymes involved in the deactivation of furylfuramidein liver microsomes. The metabolic studies involving HPLC analysisof products followed by spectrophotometric examination havealso suggested that furylfuramide can be degraded very rapidlythrough the aerobic metabolism by liver microsomes.     

A monoclonal antibody to rat liver cytochrome P450 IIC11 strongly and regiospecifically inhibits constitutive benzo[a]pyrene metabolism and DNA binding

The monoclonal antibody MAb 1-68-11, prepared to constitutive cytochrome P450 IIC11 (2c/RLM5) from male Sprague-Dawley rat liver, was used to study the contribution of the class of cytochrome P450s epitopically related to P450 IIC11 to the regiospecific metabolism of benzo[a]pyrene (BP) and its binding to DNA. The effect of MAb 1-68-11 was determined on the conversion of BP to BP-9,10-dihydrodiol, BP-7,8-dihydrodiol, BP-4,5-dihydrodiol, BP phenols, and BP quinones, and on the P450-dependent DNA binding catalyzed by P450 in microsomes from uninduced male and female Wistar and Sprague-Dawley rat livers, as well as 3-methylcholanthrene- and phenobarbital (PB)-induced male Wistar rat livers. In liver microsomes from untreated male rats, MAb 1-68-11 inhibited BP-9,10-dihydrodiol formation by 80%; in liver microsomes from untreated female rats, the inhibition was 100%. BP-7,8-dihydrodiol formation was inhibited from 38 to 77% in microsomes from males and 50% in those from females. In microsomes from PB-induced rats, inhibition of the 9,10-dihydrodiol and the 7,8-dihydrodiol was 90% and 73%, respectively, whereas BP-4,5-dihydrodiol formation was enhanced 80%. In microsomes from 3-methylcholanthrene-treated rats, no inhibition of MAb 1-68-11 was observed on either the metabolism of BP or its binding to DNA. In contrast, the binding of BP to DNA was completely inhibited by MAb 1-68-11 in microsomes from uninduced male Wistar rats and 70% in PB-induced microsomes. 32P-postlabeling analysis showed that formation of the major stable adduct, BP diol epoxide bound at C-10 to the 2-amino of deoxyguanosine, was strongly inhibited in uninduced and PB-induced microsomes. Formation of the major labile BP-DNA adduct 7-(benzo[a]pyren-6-yl) guanine (BP-N7Gua) was inhibited about 60% in microsomes from untreated male Wistar rats. These results show that MAb 1-68-11 regiospecifically inhibits cytochrome P450 IIC11 and epitopically related P450s that metabolize BP at the 7,8 and 9,10 positions. MAb 1-68-11 also inhibits enzyme-catalyzed binding of BP to DNA in the specific formation of BP-N7Gua and adducts detected by the 32P-postlabeling technique.     

Effects of synthetic and naturally occurring flavonoids on benzo[a]pyrene metabolism by hepatic microsomes prepared from rats treated with cytochrome P-450 inducers

Activity-directed fractionation of Trifolium pratense resulted in isolation of the isoflavone biochanin A, a potent inhibitor of metabolic activation of the carcinogen benzo[a]pyrene (B[a]P) in cells in culture. To determine the structural features required for maximal inhibition of cytochrome P-450 mediated metabolism of B[a]P, the inhibitory potencies of 23 flavonoids on metabolism of B[a]P to water-soluble derivatives were examined in liver S-9 homogenate from rats induced with Aroclor 1254. Flavones were much more efficient inhibitors than their corresponding isoflavone or flavanone analogs. Most flavonols were as effective inhibitors as their flavone analogs with the exception of kaempferide. Flavones with two hydroxyl or two methoxyl groups at positions 5 and 7 were the most active. Although all eight flavonoids tested effectively inhibited B[a]P metabolism by beta-naphthoflavone-induced microsomes, none were very effective inhibitors of B[a]P metabolism by phenobarbitol-induced microsomes, and only three were effective inhibitors of B[a]P metabolism by microsomes from non-induced rats. These results indicate that flavones or flavonols that contain free 5- and 7-hydroxyls are potent inhibitors of P-450 induced by beta-naphthoflavone (P-450IA1 and/or P-450IA2) and may potentially be useful as chemopreventive agents against hydrocarbon-induced carcinogenesis.     

Stereoselective metabolism of (-)-benzo[a]pyrene-7,8-diol by human lung microsomes and peripheral blood lymphocytes: effect of smoking.

Benzo[a]pyrene (B[a]P)-tetrols formed after stereoselective cytochrome P450-dependent metabolism from (-)-trans-7,8-dihydroxy-7,8- dihydrobenzo[a]pyrene [(-)-B[a]P-7, 8-diol] by lung microsomes (n = 19) and peripheral blood lymphocytes (n = 13) from lung cancer patients were measured, and the effect of smoking explored. B[a]P-tetrols were quantified by an HPLC/fluorescence assay with a detection limit of approximately 300 attomol, after incubation with peripheral blood lymphocytes or microsomes from lung cancer patients who were current cigarette smokers, ex-smokers and non-smokers. In lymphocytes from these subjects, high, medium and low metabolic activities respectively for (-)-B[a]P-7,8-diol to tetrol conversion were found, but there was no statistically significant difference between smokers, ex-smokers and non-smokers. When the B[a]P-tetrol formation by human lung microsomes was measured, recent smokers had 4- to 7-fold higher (P = 0.04) metabolic activity than ex-smokers and non-smokers. The mean lung microsomal arylhydrocarbon hydroxylase (AHH) activity was three times higher in smokers than in non-smokers and was undetectable in ex-smokers. AHH activity was correlated with tetrol formation in the same lung microsomal samples (r = 0.62, P less than 0.01 in smokers; and r = 0.67, P less than 0.01 in all subjects). When subjects were grouped according to smoking habits, however, no correlation was detected between mean tetrol formation by lung microsomes and that of lymphocytes. Thus, lymphocytes cannot serve as a surrogate for lung microsomes concerning the pulmonary metabolism of (-)-B[a]P-7,8-diol. The much higher B[a]P-tetrol formation observed in lung microsomes from smokers is in accord with a reported higher pulmonary AHH activity, cytochrome P450IA level, and CYP1A1 gene expression in recent tobacco smokers.     

Metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-diol by human cytochrome P450 1B1

Benzo[a]pyrene (B[a]P), a ubiquitous environmental, tobacco and dietary carcinogen, has been implicated in human cancer etiology. The role of human cytochrome P450 1B1 in the metabolism of B[a]P is poorly understood. Using microsomal preparations of human P450 1A1, 1A2 and 1B1 expressed in baculovirus-infected insect cells, as well as human and rat P450 1B1 expressed in yeast, we have determined the metabolism of B[a]P, with and without the addition of exogenous epoxide hydrolase, and B[a]P-7,8-dihydrodiol (7,8-diol), each substrate at a concentration of 10 microM. HPLC analysis detected eight major metabolites of B[a]P and four metabolites of the 7,8-diol. The results of these studies indicate that cytochrome P450 1B1 carries out metabolism of B[a]P along the pathway to the postulated ultimate carcinogen, the diol epoxide 2, at rates much higher than P450 1A2 but less than P450 1A1. The rates of formation of the 7,8-diol metabolite in incubations with epoxide hydrolase are 0.17 and 0.38 nmol/min/nmol P450 for human P450 1B1 and 1A1, respectively, and undetectable for 1A2. The rates of total tetrol metabolite formation from the 7,8-diol, which are indicative of diol epoxide formation, are 0.60, 0.43 and 2.58 nmol/min/nmol P450 for 1B1, 1A2 and 1A1 respectively. In agreement with other reports of rat P450 1B1 activity, our data show this rat enzyme to be very active for B[a]P and 7,8-diol, with rates higher than human P450 1B1. In addition to the established role of P450 1A1 in B[a]P metabolism, P450 1B1 may significantly contribute to B[a]P and 7,8-diol metabolism and carcinogenesis in rodent tumor models and in humans.      

Selective interactions of cytochromes P-450 with the hydroxymethyl derivatives of 7, 12-dimethylbenz[a]anthracene

Competition between a hydroxylated metabolite and the parentpolycyclic aromatic hydrocarbon (PAH) for metabolism at cytochromesP-450 may result in the generation of hydroxylated dihydrodiolepoxides. The effectiveness of the competition between 7-hydroxymethyI-12-methylbenz[a]-anthracene(7HOMMBA) or 12-hydroxymethyl-1–7-methyl-benz[a]anthracene(12HOMMBA) and 7, 12-dimethylbenz[a]-anthracene (DMBA) is highlydependent on the form(s) of cytochrome P-450 in the microsomes.The inhibitory effects of exogenously added 7HOMMBA or 12HOMMBAon DMBA metabolism were 30- to 50-fold greater in 3-methyl-cholanthrene(MC-induced rat liver microsomes (K_i = 0.4 µM) comparedto either uninduced or phenobarbital (PB-induced liver microsomes(K_i = 14 and 11 µM, respectively). Similarly, productinhibition of total DMBA metabolism by metabolites generatedin situ was significant only in MC-induced liver microsomes(K'_i = 2.5 µM). Metabolism of 7HOMMBA in these microsomeswas strongly restricted by an unusual substrate inhibition derivedfrom the inhibitory binding of a second molecule of 7HOMMBA.This same phenomenon was observed with reconstituted cytochromeP-450c but not with PB-induced or uninduced microsomes. Complexformation by binding of DMBA, 7HOMMBA, and 12HOMMBA to purifiedP-450c reconstituted in phospholipid micelles was determinedby optical spectroscopy and fluorescence quenching. Bindingaffinities of both the 7HOMMBA and 12HOMMBA (K_d = 95 and 110nM, respectively), were 2.5-fold higher compared to that ofDMBA (K_d = 265 nM). These results provide a first demonstrationthat hydroxylation of a PAH can lead to preferential metabolismthrough an increased affinity for cytochrome P-450.     

Glutathione depletion suppresses conjugation of benzo[a]pyrene metabolites and ({+/-})-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene metabolites with glutathione but does not affect their binding to DNA in C3H/10T1/2 mouse fibroblasts

The study was aimed at determining the role of glutathione (GSH)conjugation in the binding of reactive benzo[a]pyrene (BaP)species to DNA of C3H/10T1/2 cells. In order to suppress GSHconjugation cells were depleted of GSH by treatment with buthioninesulfoximine for 18 h and 1-chloro-2,4-dinitrobenzene for 1 hprior to incubation with radiolabelled substrates. Under theseconditions GSH levels decreased to <1% of the control value.C3H/10T1/2 cells produced GSH conjugates with 7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydro-benzo[a]pyrene(BaPDE) comprising 6% of the total metabolites formed from BaPor (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene(BaP-7,8-diol). In GSH-depteted cells formation of GSH conjugateswith metabolic products of BaP or BaP-7,8-diol was suppressedto 1% of total metabolites during an 8-h incubation period.Metabolic activation of BaP and BaP-7,8-diol by C3H/10T1/2 cellsresulted in the formation of DNA adducts which largely consistedof BaPDE:deoxyguanosine. Depletion of GSH altered neither thedegree of DNA binding nor the pattern of DNA adducts to anysignificant extent. When C3H/10T1/2 cells were co-incubatedwith microsomes from liver of 3-methylcholanthrene-treated ratsfor 1 h in order to activate BaP or BaP-7,8-diol extracellularly,the same pattern of GSH conjugates and DNA adducts was generatedas by intracellular metabolism of the polycyclic hydrocarbons.No GSH conjugates were detected following co-incubation of microsomeswith GSH-depleted C3H/10T1/2 cells. The formation of DNA adductsagain remained unaffected by the suppression of conjugation.C3H/10T1/2 cells are apparently capable of conjugating BaPDEwith GSH but are not capable of trapping by GSH conjugationthose BaPDE moieties which bind to DNA. The results are compatiblewith the notion that BaPDE is partially contained in a cellularcompartment—presumably the lipid environment of membranes—whereit is inaccessible to GSH transferases of C3H/10T1/2 cells.     

Relative contribution of various forms of cytochrome P450 to the metabolism of benzo[a]pyrene by human liver microsomes

A variety of cytochromes P450 have been implicated in the hepaticmetabolism of benzo[a]pyrene (BP), including forms that areconstitutively expressed and those that are highly inducible.In the present study the metabolism of BP to organic solvent-solublederivatives by eight forms of cytochrome P450 isolated fromrat liver and by a series of 11 human liver microsomal sampleswas investigated. The relative contribution of specific P450forms to the human hepatic metabolism was evaluated. A 4-foldvariation in formation of total organic solvent-soluble BP metaboliteswas observed, as well as differences in the regio- and stereo-selectivityof this metabolism between the three individuals studied. Thelevels of expression of cytochromes P450 from five gene sub-families,as determined by Western blot analysis, did not show any correlationwith the rate of BP metabolism to organic solvent-soluble derivativesin these livers. No reduction in metabolism was observed inthree livers in which either the debrisoqulne P450 (P450IID1)was not expressed or bufuralol 1-hydroxylase activity was low.Of six different antibodies to forms of rat liver P450 tested,only those to P450s MC_la (P450IA2), MC_lb (P450IA1) and UT (P450IIA1)consistently inhibited BP metabolism. This inhibition was generallylimited and rarely exceeded 30%. An antibody to cytochrome P450PB (P450 did, however, inhibit the formation of metabolitesat the 4,5- and 9,10-positions of BP by microsomal fractionsof livers from one individual who had been receiving the drugphenytoin. These data indicate that several forms of P450 inhuman liver are involved in the metabolism of BP and that bothconstitutively expressed as well as inducible forms are importantin its disposition in man.     

Role of P450IIE1 in the metabolism of 3-hydroxypyridine, a constituent of tobacco smoke: redox cycling and DNA strand scission by the metabolite 2,5-dihydroxypyridine

The metabolism of 3-hydroxypyridine, a significant constituent of tobacco smoke, to 2,5-dihydroxypyridine has been characterized in hepatic microsomes and in the reconstituted enzyme system using purified forms of P450. The redox cycling activity of the metabolite and its ability to damage DNA in vitro have been examined. Pyridine-induced microsomes, which contain elevated levels of P450IIE1 (Kim et al., J. Pharmacol. Exp. Ther., 246: 1175-1182, 1988), catalyzed an 8-fold increase in the production of 2,5-dihydroxypyridine, relative to control, which showed biphasic kinetics. Pyridine-induced rabbit hepatic microsomes exhibited a Vmax of 5.9 nmol 2,5-dihydroxypyridine/min/mg protein and a Km value of 110 microM. In contrast, phenobarbital- and isosafrole-induced microsomes had Vmax values of 2.5 and 1.2 nmol/min/mg protein and Km values of 590 and 134 microM, respectively. Pyridine-induced rat hepatic microsomes also exhibited elevated catalytic activity toward the hydroxylation of 3-hydroxypyridine, with an 8-fold increase in Vmax (2.74 nmol/min/mg protein) relative to uninduced rat hepatic microsomes (Vmax = 0.34 nmol/min/mg protein). In the reconstituted system, cytochrome P450IIE1 displayed the greatest activity in the production of 2,5-dihydroxypyridine of the major forms of rabbit P450 examined. P450IIE1 was 34-fold more active than P450IIB1 and 12-fold more active than P450IA2 in the production of 2,5-dihydroxypyridine. The redox cycling activity of 2,5-dihydroxypyridine has been characterized. The rate of NADPH oxidation in the presence of 0.5 mM 2,5-dihydroxypyridine was stimulated approximately 4-fold (69.2 nmol NADPH oxidized/min/mg protein), relative to control (16 nmol/min/mg protein). 2,5-Dihydroxypyridine at 0.5 and 1.0 mM produced a 12- and 17-fold increase, respectively, in the rate of superoxide anion production compared to control, as monitored by the SOD-inhibitable reduction of acetylated cytochrome c. 3-Hydroxypyridine alone failed to increase the rate of superoxide production. Inclusion of reduced glutathione in the incubation resulted in a pronounced decrease in the 2,5-dihydroxypyridine-stimulated rate of cofactor oxidation and superoxide production. The ability of 2,5-dihydroxypyridine to damage DNA was assessed by monitoring phi X-174 DNA strand scission. The band intensity of the supercoiled form of DNA, when incubated with 1 mM 2,5-dihydroxypyridine, decreased substantially, with a concomitant increase in intensity of the band associated with the open circular form of DNA. The change in phi X-174 DNA topology produced by 2,5-dihydroxypyridine was accelerated in a dose-dependent manner, with an estimated EC50 of approximately 60 microM.(ABSTRACT TRUNCATED AT 400 WORDS)     

Metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) by liver microsomes and isolated rabbit cytochrome P450 isozymes

The cytochrome P450-dependent metabolism of the heterocyclic amine mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) has been determined. We investigated the in vitro metabolism of PhIP by polycyclic hydrocarbon-induced mouse and rabbit liver microsomes, and by purified rabbit liver P450 isozymes. Following a 60 min incubation, 3-methylcholanthrene-induced mouse microsomes converted 36% of the PhIP to two major metabolites, N-hydroxy-PhIP and 4'-hydroxy-PhIP, with 43% total metabolism. Rabbit P450 form 6 and form 4 produced the same two major metabolites (20 and 5% total metabolism respectively). Additional metabolites were produced in low yields and amounts varied depending on the isozyme used (1-5%). Metabolites were not detected in incubations of PhIP with P450 forms 2 and 3C. N-Hydroxy-PhIP was found to be directly mutagenic to Salmonella TA98, while the 4'-hydroxy-PhIP was not mutagenic either with or without additional metabolic activation. These data suggest that the cytochrome P450IA isozymes are involved in the metabolism of PhIP by rabbit liver and that formation of N-hydroxy-PhIP is involved in the mutagenicity of PhIP.