Evidence for distinct binding sites in the cumene hydroperoxide-dependent metabolism of benzo[a]pyrene catalyzed by cytochrome P-450

A few constitutive cytochrome P-450 isozymes in male rat liver microsomes catalyzed the metabolism of benzo[a]pyrene (BP) in cumene hydroperoxide (CHP)-dependent reactions, which produced predominantly 3-hydroxyBP and BP quinones. This process varied with the concentration of CHP. At 0.05 mM CHP, 3-hydroxyBP was the major metabolite. An increase in CHP concentration reduced 3-hydroxyBP formation but increased the level of BP quinones. This change in metabolic profile was reversed by preincubation with pyrene. Pyrene selectively inhibited quinone formation and enhanced 3-hydroxyBP formation. Naphthalene, phenanthrene and benz[a]anthracene nonspecifically inhibited total metabolism. BP binding to microsomal protein correlated with quinone formation, suggesting a common precursor reactive intermediate. BP metabolism by female rat liver microsomes also depended on CHP concentration but was much less effective than that in the male. With females, quinones were the major metabolites at all CHP concentrations, and their formation was again modulated by pyrene. These data indicate that two distinct binding sites are responsible for the formation of 3-hydroxyBP and BP quinones.     

Inhibition of cytochrome P-450c-mediated benzo[a]pyrene hydroxylase and ethoxyresorufin O-deethylase by dihydrosafrole

1. Inhibitory activity of dihydrosafrole towards benzo[a]pyrene (BP) hydroxylase activity in hepatic microsomes from beta-naphthoflavone (BNF)-induced rats, and in reconstituted systems containing cytochrome P-450c, increased dramatically on preincubation of the inhibitor with NADPH; no inhibition occurred without preincubation. The level of BP hydroxylase inhibition was associated with the progressive formation of the 456 nm dihydrosafrole metabolite-cytochrome P-450c spectral complex during preincubation. 2. Inhibition of BP hydroxylase by dihydrosafrole in control microsomes, and inhibition of ethoxyresorufin O-deethylase (EROD) in microsomes (control or BNF-induced) and in reconstituted systems with cytochrome P-450c, did not require preincubation and apparently was not dependent on prior formation of the dihydrosafrole metabolite-cytochrome P-450 complex. 3. Kinetic studies established that, following preincubation with NADPH, dihydrosafrole was a noncompetitive inhibitor of both BP hydroxylase and EROD activities. In the absence of preincubation, dihydrosafrole was an effective competitive inhibitor of EROD in BNF-induced microsomes and in reconstituted systems with cytochrome P-450c. 4. Both ethoxyresorufin and benzo[a]pyrene inhibited the development of the type I optical difference spectrum of dihydrosafrole in reconstituted systems containing cytochrome P-450c. Inhibition by ethoxyresorufin was competitive while that caused by benzo[a]pyrene was noncompetitive in nature. 5. The type II ligand phenylimidazole was an effective noncompetitive inhibitor of EROD activity but failed to exert any inhibitory effect on cytochrome P-450c-mediated BP hydroxylase activity. Phenylimidazole inhibited formation of the dihydrosafrole type I optical difference spectrum non-competitively. 6. The results indicate that ethoxyresorufin and benzo[a]pyrene may occupy different binding sites on cytochrome P-450c and that dihydrosafrole binds primarily to the site utilized by ethoxyresorufin.     

Inhibition of cytochrome P-450c-mediated benzo[a]pyrene hydroxylase and ethoxyresorufin O-deethylase by dihydrosafrole

1. Inhibitory activity of dihydrosafrole towards benzo[a]pyrene (BP) hydroxylase activity in hepatic microsomes from β-naphthoflavone (BNF)-induced rats, and in reconstituted systems containing cytochrome P-450c, increased dramatically on preincubation of the inhibitor with NADPH; no inhibition occurred without preincubation. The level of BP hydroxylase inhibition was associated with the progressive formation of the 456 nm dihydrosafrole metabolite-cytochrome P-450c spectral complex during preincubation.

2. Inhibition of BP hydroxylase by dihydrosafrole in control microsomes, and inhibition of ethoxyresorufin O-deethylase (EROD) in microsomes (control or BNF-induced) and in reconstituted systems with cytochrome P-450c, did not require preincubation and apparently was not dependent on prior formation of the dihydrosafrole metabolite-cytochrome P-450 complex.

3. Kinetic studies established that, following preincubation with NADPH, dihydrosafrole was a noncompetitive inhibitor of both BP hydroxylase and EROD activities. In the absence of preincubation, dihydrosafrole was an effective competitive inhibitor of EROD in BNF-induced microsomes and in reconstituted systems with cytochrome P-450c.

4. Both ethoxyresorufin and benzo[α]pyrene inhibited the development of the type 1 optical difference spectrum of dihydrosafrole in reconstituted systems containing cytochrome P-450c. Inhibition by ethoxyresorufin was competitive while that caused by benzo[α]pyrene was noncompetitive in nature.

5. The type II ligand phenylimidazole was an effective noncompetitive inhibitor of EROD activity but failed to exert any inhibitory effect on cytochrome P-450c-mediated BP hydroxylase activity. Phenylimidazole inhibited formation of the dihydrosafrole type 1 optical difference spectrum non-competitively.

6. The results indicate that ethoxyresorufin and benzo[α]pyrene may occupy different binding sites on cytochrome P-450c and that dihydrosafrole binds primarily to the site utilized by ethoxyresorufin.     

Benzo[a]pyrene metabolism by purified cytochrome P-450 from 3-methylcholanthrene-treated rats

The metabolism of benzo[a]pyrene in reconstituted pulmonary mono-oxygenase systems has been studied. Metabolites formed by pulmonary cytochrome P450MC, a major form of pulmonary cytochrome P-450 isolated from 3-methylcholanthrene-treated rats, were analysed by h.p.l.c. The profiles of benzo[a]pyrene metabolites formed by the reconstituted P-450MC systems were different from that obtained with rat-lung microsomes, indicating the presence of several unknown metabolites in the reconstituted systems containing NADPH-cytochrome P-450 reductase and epoxide hydrolase. 3-Hydroxybenzo[a]pyrene was a major product formed by pulmonary cytochrome P-450MC, in the absence or presence of epoxide hydrolase. The addition of purified epoxide hydrolase to the reconstituted systems increased the formation of dihydrodihydroxy-benzo[a]pyrenes, particularly 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene. The 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene was the major dihydrodiol formed by pulmonary cytochrome P-450MC. By the addition of epoxide hydrolase the total amount of phenols decreased in parallel with an increased production of dihydrodiol, but the amount of quinones was not changed. Similar results concerning the related production of phenols and dihydrodiols, in the absence and presence of epoxide hydrolase, were obtained in reconstituted systems of hepatic cytochrome P-450MC, the major form of hepatic cytochrome P-450 from 3-methylcholanthrene-treated rats.     

Metabolism of benzo[a]pyrene to trans-7,8-dihydroxy-7, 8-dihydrobenzo[a]pyrene by recombinant human cytochrome P450 1B1 and purified liver epoxide hydrolase.

Recombinant human enzymes expressed in membranes obtained from Escherichia coli transformed with cytochrome P450 (P450) and NADPH-P450 reductase cDNAs were used to identify the human P450 enzymes that are most active in catalyzing the oxidative transformation of benzo[a]pyrene in vitro. Activation of benzo[a]pyrene to genotoxic products that cause induction of umu gene expression in Salmonella typhimurium NM2009 by P450 1A1 and P450 1B1 enzymes was found to be enhanced by inclusion of purified epoxide hydrolase (isolated from rat or human livers) with the reaction mixture. High-performance liquid chromatographic analysis showed that P450 1B1 catalyzed benzo[a]pyrene to trans-7, 8-dihydroxy-7,8-dihydrobenzo[a]pyrene at level of approximately 3 nmol min(-)(1) nmol of P450(-)(1) only when epoxide hydrolase was present and P450 1A1 (with the hydrolase) was able to catalyze benzo[a]pyrene at one-tenth of the activity catalyzed by P450 1B1. Kinetic analysis showed that ratio of V(max) to K(m) for the formation of trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene in this assay system was 3.2-fold higher in CYP1B1 than in CYP1A1. Other human P450s (including P450s 1A2, 2E1, and 3A4) were found to have very low or undetectable activities toward the formation of trans-7, 8-dihydroxy-7,8-dihydrobenzo[a]pyrene. A reconstituted system containing purified P450 1B1, rabbit liver NADPH-P450 reductase, and human liver epoxide hydrolase was found to catalyze benzo[a]pyrene to trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene at a rate of 0.86 nmol min(-)(1) nmol of P450(-)(1); the activities were found to be largely dependent on the presence of sodium cholate in the system. These results suggest that P450 1B1 is a principal enzyme in catalyzing the oxidation of benzo[a]pyrene to trans-7,8-dihydroxy-7, 8-dihydrobenzo[a]pyrene and that the catalytic functions of P450 1B1 may determine the susceptibilities of individuals to benzo[a]pyrene carcinogenesis.     

Anticarcinogenic effects of cadmium in B6C3F1 mouse liver and lung

The B6C3F1 mouse liver has been widely used for the evaluation of carcinogenic or tumor promoting efficacy of various organic compounds, although little is known about the actions of metallic carcinogens in this system. Thus, the ability of cadmium to initiate or promote tumors in B6C3F1 mouse liver was studied. In promotion studies, diethylnitrosamine (DEN; 90 mg/kg, ip) was given as an initiator to 5-week-old mice followed 2 weeks later by 500 or 1000 ppm of cadmium in drinking water for 50 weeks. DEN caused an elevation of liver tumor incidence (13 tumor bearing mice/45 total) over control (1/48) which was prevented by cadmium (DEN + 500 ppm cadmium, 3/42; DEN + 1000 cadmium, 0/47). Cadmium alone did not further reduce the very low spontaneous liver and lung tumor incidence at approximately 1 year of age. DEN-induced lung tumor incidence (15/45) was also reduced by cadmium (DEN + 500 ppm cadmium, 11/42; DEN + 1000 ppm cadmium, 1/47) to control levels (0/48). In initiation studies, cadmium (20 or 22.5 mumol/kg, sc) was given to 5-week-old mice (n = 30-60) 2 weeks before an established promoting regimen of sodium barbital (BB) in drinking water at 500 ppm level was begun. Barbital in drinking water was given continuously for up to 92 weeks. Such cadmium doses caused acute, focal hepatic necrosis. Mice treated with BB and killed at 97 weeks of age showed an elevation of liver tumor multiplicity (7.44 tumors/liver) over control (2.24) that was prevented by cadmium in a dose-related manner (20 mumol/kg cadmium + BB, 3.93; 22.5 mumol/kg cadmium + BB, 1.87). Cadmium alone given by injection also reduced spontaneous liver tumor multiplicity. These results indicate that cadmium inhibits tumor formation in the B6C3F1 mouse liver initiation/promotion system regardless of route of exposure or sequence of administration. The possibility exists that cadmium has a specific toxicity toward previously initiated cells within liver and lung.     

Specificity of four forms of cytochrome P-450 in the metabolic activation of several aromatic amines and benzo[a]pyrene

The involvement of four forms of cytochrome P-450 in the activation of the promutagens, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-6-methyl-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-aminofluorene and 4-aminobiphenyl has been investigated using a Salmonella test system. A high-spin form, P-448 II-a, catalysed the activation of IQ and Glu-P-1 28 and 12 times faster, respectively, than a low-spin form, P-448 II-d, whereas benzo[a]pyrene was metabolized to the phenols 60 times faster by P-448 II-d than P-448 II-a. Both P-448 II-a and P-448 II-d were highly active in the activation of Trp-P-2 and 2-aminofluorene. Treatment of CDF1 mice with polychlorinated biphenyls (PCB) increased the microsomal-activating ability for the promutagens in various degrees. More than a ten-fold increase was observed with Trp-P-2, while the increase was only two-fold with IQ. No sex-related difference was observed for the hepatic microsomal activating ability of male and female CDF1 mice for Trp-P-2, Glu-P-1 or IQ. These results indicate that more than two forms of cytochrome P-450, which are inducible by treatment with PCB or 3-methylcholanthrene, mediate the metabolic activation of heteroaromatic amines in rats and mice.     

Correlations between cytochrome P-450 and oxidative metabolism of benzo[a]pyrene and 7-ethoxycoumarin in human liver in vitro and antipyrine elimination in vivo

Cytochrome P-450 content was correlated to aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-de-ethylase activities in human liver biopsy samples in vitro. Antipyrine half-life and clearance were measured in the same patients in vivo. The results were compared with data obtained in rat liver in vitro. The correlation of cytochrome P-450 content with aryl hydrocarbon hydroxylase activity in human liver biopsy samples was relatively good (r = 0.75, p < 0.001), but that with 7-ethoxycoumarin O-de-ethylase activity was much poorer (r = 0.42, p < 0.001). The good correlation between cytochrome P-450 content and aryl hydrocarbon hydroxylase activity in biopsy samples from patients with widely varying hepatic disease processes, exposure to inducers, history of cigarette smoking, etc., is in contrast with the data obtained in the rat, where the corresponding correlation in a population treated with different inducers and inhibitors was rather poor (r = 0.37, p < 0.01). This poor correlation was caused mainly by the nonequal effects of polycyclic aromatic hydrocarbons on cytochrome P-450 and aryl hydrocarbon hydroxylase. Cigarette smoking was not found to induce human liver drug or carcinogen metabolism. Aryl hydrocarbon hydroxylase activity in vitro and antipyrine half-life or clearance in vivo were correlated (r = 0.36, p < 0.01 and r = 0.35, p < 0.01, respectively), indicating a weak, but statistically significant association between these parameters. This study suggests that correlations between in vitro and in vivo measurements of drug metabolism are not strong enough for the tests to be used as predictive tests in experimental or clinical research.     

Oxidation of troglitazone to a quinone-type metabolite catalyzed by cytochrome P-450 2C8 and P-450 3A4 in human liver microsomes.

Troglitazone, a new oral antidiabetic drug, is reported to be mostly metabolized to its conjugates and not to be oxidized by cytochrome P-450 (P-450) enzymes. Of fourteen cDNA-expressed human P-450 enzymes examined, CYP1A1, CYP2C8, CYP2C19, and CYP3A4 were active in catalyzing formation of a quinone-type metabolite at a concentration of 10 microM troglitazone, whereas CYP3A4 had the highest catalytic activity at 100 microM substrate. In human liver microsomes, rates of the quinone-type metabolite formation (at 100 microM) were correlated well with rates of testosterone 6beta-hydroxylation (r = 0.98), but those at 10 microM troglitazone were not correlated with any of several marker activities of P-450 enzymes. Quercetin efficiently inhibited quinone-type metabolite formation (at 10 microM troglitazone) in human samples that contained relatively high levels of CYP2C, whereas ketoconazole affected these activities in liver microsomes in which CYP3A4 levels were relatively high. Anti-CYP2C antibodies strongly inhibited quinone-type metabolite formation (at 10 microM troglitazone) in CYP2C-rich human liver microsomes (by approximately 85%); the intensity of this effect depended on the human samples and their P-450 status. The results suggest that in human liver both CYP2C8 and CYP3A4 have major roles in quinone-type metabolite formation and that the hepatic contents of these two P-450 forms determine which P-450 enzymes play major roles in individual humans. CYP3A4 may be expected to play a role in formation of quinone-type metabolite from troglitazone even at a low concentration in humans.     

Distribution and induction of cytochrome P-450 and two cytochrome P-450-dependent monooxygenase activities in rat liver parenchymal cell subpopulations separated by centrifugal elutriation

Liver parenchymal cells from the periportal and centrilobular zones differ in their morphological, biochemical and functional characteristics. In an effort to obtain fractions enriched in either periportal or centrilobular cells, isolated rat liver parenchymal cells were separated into five subpopulations by centrifugal elutriation. The mean diameters of the cells present in fractions I–V were 19.6, 21.1, 21.8, 22.7 and 23.5 m, respectively. The content of cytochrome P-450 as well as benzphetamine N-demethylase and 7-ethoxyresorufin O-deethylase activities were higher in the larger parenchymal cells than in the smaller ones. After administration of phenobarbital the content of cytochrome P-450 was approximately two-fold greater in the cells present in fractions 3–5, when compared to the same subpopulations isolated from untreated rats; the activity of benzphetamine N-demethylase was enhanced to a similar extent in all five fractions. 3-Methylcholanthrene treatment resulted in a significant increase of cytochrome P-450 content and 7-ethoxyresorufin O-deethylase activity in all five fractions: both parameters were slightly higher in fractions 4 and 5 than in fractions 1 and 2. In conclusion, the elutriated liver parenchymal cells seem to preserve the biochemical heterogeneity observed in the intact liver; the potential enrichment of periportal and centrilobular cells in the different fractions by centrifugal elutriation is discussed.     

Metabolism of territrem B and C in liver microsomes from 14-wk-old Wistar rats is catalyzed by cytochrome P-450 3A

The metabolism of territrem B (TRB) and territrem C (TRC) in liver microsomes of 14-wk-old male and female Wistar rats was investigated. Metabolism of TRB to 4beta-hydroxylmethyl-4beta-demethylterritrem B (MB2), O-demethylation of the methoxy group of the aromatic moiety of TRB to form MB4 (same structure as TRC), and metabolism of TRC to 4beta-hydroxylmethyl-4beta-demethylterritrem C (MC) were observed in both genders. However, the amounts of MB2, MB4, and MC formed in females were much lower than in males. To investigate which cytochrome P-450 (CYP450) isoforms were involved in each step, four CYP450 isotype-specific inhibitors (furafylline, orphenadrine, cimetidine, and troleandomycin) and antibodies against CYP1A1, CYP2B1, CYP2C11, or CYP3A2 were used. Formation of MB2, MB4, and MC was markedly inhibited by cimetidine and troleandomycin, but less by furafylline and orphenadrine. Anti-CYP3A2 antibody completely inhibited MB, MB, and MC formation, while antibodies against CYP1A1, CYP2B1, or CYP2C11 produced no marked effect. Of the seven tested supersomes from baculovirus-transformed insect cells expressing rat CYP450 isoforms (1Al, 1A2, 2B1, 2C11, 2C12, 3A1, and 3A2), only those expressing CYP3A1 and CYP3A2 metabolized TRB and TRC. The amounts of MB2, MB4, and MC formed by male and female rat liver microsome preparations were related to the testosterone 6beta-hydroxylase activity and CYP3A1/2 protein content of the preparation. Immunoblotting showed that CYP3A1 was expressed in both genders, but at different levels, while CYP3A2 was only expressed in males. These results suggest that the formation of MB2, MB4, and MC in liver microsomes from 14-wk-old rats of either gender is mediated by CYP3A1 and CYP3A2.     

Metabolism of phenanthridine to phenanthridone by rat lung and liver microsomes after induction with benzo[a]pyrene and aroclor

The comparative metabolism of phenanthridine (3,4-benzoquinoline) by rat lung and liver microsomes has been investigated. The array of metabolites produced by induced lung and liver are qualitatively similar. Phenanthridone has been identified as a phenanthridine metabolite produced by induced rat liver and lung. Phenanthridone is directly mutagenic in Salmonella tester strain TA-98 while phenanthridine is not. Although phenanthridone is more mutagenic than phenanthridine after incubation with rat liver 9000g supernatant fraction, it is less cytotoxic to Chinese hamster ovary cells in vitro.     

Deoxyribonucleic acid binding of 3-hydroxy- and 9-hydroxybenzo[a]pyrene following further metabolism by mouse liver microsomal cytochrome P1-450.

In the presence of NADPH and microsomes from 3-methylcholanthrene-treated C57BL/6N mice, [³H]3-OH-benzo[a]pyrene is metabolized to reactive intermediates which covalently bind to deproteinized salmon sperm DNA in vitro. Enzymatically digested DNA, containing bound [³H]3-OH-benzolajpyrene derivatives, generates an elution profile from Sephadex LH20 chromatography which resembles similar chromatograms with [³H]benzo[a]pyrene. All peaks resulting from [³H]benzo[a]pyrene activation appear to be prominently represented in [³H]3-OH-benzo[a]pyrene activation, except that several peaks which emerge near the end of the eluting gradient of methanol and water are much reduced. Notably, a peak designated E, and attributed to benzo[a]pyrene-7, 8-diol-9, 10-oxide binding in [³H]benzo[a]pyrene incubations, is also prominently represented in incubations with [³H]3-OH-benzo[a]pyrene. Radioactivity in all of these peaks is inhibited effectively if one-seventh the concentration of 1-OH-benzo[a]pyrene is included in the incubation with [³H]3-OH-benzo[a]pyrene. Microsomes from 3-methylcholanthrene-treated DBA/ 2N mice cause insignificant binding. UDP-glucuronic acid markedly reduces all peaks except E, and 1, 2-epoxy-3, 3, 3-trichloropropane reduces all peaks except C and E. 9-Hydroxybenzo[a]pyrene is further metabolized to DNA binding species by microsomes from either 3-methylcholanthrene-treated DBA/2N or C57BL/6N mice. UDP-glucuronic acid prevents about 50 per cent of the binding with microsomes from DBA/2N mice but not with microsomes from C57BL/6N. In contrast, UDP-glucuronic acid does prevent binding in some of these same peaks when [³H]benzotaipyrene is the starting substrate with microsomes from C57BL/6N mice. UDP-glucuronic acid does not prevent binding in peak E in incubations with [³H]benzo[a]pyrene or [³H]3-hydroxybenzo[a]pyrene.     

Benzo[a]pyrene-hydroxylase catalyzed by purified isozymes of cytochrome P-450 from beta-naphthoflavone-fed rainbow trout

We have purified five isozymes of liver microsomal (LM) P-450 from beta-naphthoflavone-fed rainbow trout. Four forms (LM3, LM1, LM4a and LMx) were resolved on DEAE-Sepharose. Chromatography on hydroxylapatite further resolved LMx into two components, LM2 and LM4b. This latter form, obtained in highest yield (5%), had an apparent minimum molecular weight (Mr), as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), of 58,000, a specific content of 11.9 nmoles/mg, a lambda max in the carbon monoxide-ligated, reduced difference spectrum of 447.0 nm, and was active towards benzo[a]pyrene in a reconstituted system. A second form, LM4a, obtained in a final yield of 2%, had a specific content of 10.3 and was indistinguishable from Lm4b by Mr, lambda max, or activity towards benzo[a]pyrene. Form LM2 (2% yield) had a specific content of 10.8, a Mr of 54,000, a lambda max of 449.5 nm, and was not effective in reconstitution of benzo[a]pyrene-hydroxylase. In addition, two other forms with lower specific contents were obtained, LM1 and LM3. Neither LM1 nor LM3 was active towards benzo[a]pyrene. The properties of LM2, LM4a and LM4b were further examined with the aid of antibodies prepared from rabbits. Antibodies to LM4a and LM4b each cross-reacted with the other antigen and formed lines of identity on Ouchterlony plates, and both IgGs exhibited some cross-reaction to P-448 from rat. Neither antibody cross-reacted with trout LM2, and LM2-IgG did not cross-react with any other purified P-450. Benzo[a]pyrene-hydroxylase, catalyzed by either LM4a or LM4b, was inhibited by LM4b-IgG but not by LM4a-IgG, suggesting that these antibodies recognize different antigenic sites. Further comparison of LM4a and LM4b by amino acid composition, peptide mapping, kinetic properties, sensitivity to alpha-naphthoflavone, and regioselectivity towards benzo[a]pyrene-dihydrodiol formation indicates that these forms are highly similar in structure and function.     

Metabolic activation of benzo[c]phenanthrene by cytochrome P450 enzymes in human liver and lung.

The environmentally occurring polycyclic aromatic hydrocarbon (PAH) benzo[c]phenanthrene (B[c]PH) is a weak carcinogen in rodents. In contrast, the dihydrodiol-epoxides of B[c]PH are among the most carcinogenic PAH metabolites tested so far. In rodents, B[c]PH is predominantly metabolized to B[c]PH-5,6-dihydrodiol (B[c]PH-5,6-DH) and only to a minor extent to B[c]PH-3,4-DH, the proximate precursor of the highly potent ultimate carcinogen, B[c]PH-3,4-DH-1,2-epoxide. This might explain why in rodents B[c]PH is a weak carcinogen. However, little is known about human metabolism of B[c]PH. Using microsomal preparations from human liver and lung, we investigated the metabolic activation of B[c]PH. In contrast to the findings in experimental animals, human liver microsomes predominantly generated B[c]PH-3,4-DH and only to a minor extent B[c]PH-5,6-DH. Only one lung tissue sample was found to be metabolically active, producing B[c]PH-5,6-DH together with small amounts of B[c]PH-3,4-DH. Catalytic activities known to be associated with specific cytochrome P450 (P450) enzyme activities were determined and correlated with the spectrum of B[c]PH metabolites. The results indicate that B[c]PH-DH formation in human liver is mainly mediated by P450 1A2. Studies with P450 enzyme selective inhibitors confirmed these findings. Further support was obtained using preparations of the respective human recombinant P450 enzymes expressed in Escherichia coli and yeast. In addition to P450 1A2, P450 1B1 effectively mediated B[c]PH-metabolism. The umu-assay for induction of SOS repair response in Salmonella typhimurium TA 1535 pSK 1002 containing a umuC-lacZ reporter gene was used to study metabolic generation of genotoxic metabolites from B[c]PH-DHs in human microsomal preparations. B[c]PH-3,4-DH was activated by human liver microsomes to a potent genotoxic agent. Taken together, the results clearly demonstrate that human liver microsomes can effectively catalyze the biotransformation of B[c]PH into highly genotoxic metabolites. The results provide evidence that B[c]PH should be considered a potentially potent carcinogen in humans, and that rodent models may underestimate the risk.     

Purification and characterization of a mouse liver cytochrome P-450 induced by cannabidiol

A cytochrome P-450 isozyme (Mr = 51,600) was purified to apparent homogeneity from hepatic microsomes of mice pretreated with cannabidiol (CBD), a major constituent of marijuana. The isozyme exhibited high pentoxyresorufin O-dealkylase, hexobarbital hydroxylase, and 16 alpha- and 16 beta-testosterone hydroxylase activities and formed a Fe+2-metyrapone complex, properties characteristic of the major hepatic cytochrome P-450s previously purified from phenobarbital (PB)-pretreated animals. In addition, the CBD-induced cytochrome P-450 was immunoreactive with an antibody raised against the major rat hepatic PB-inducible cytochrome P-450 and exhibited an NH2-terminal amino acid sequence greater than 90% homologous with that of the PB-inducible rat liver isozyme. Because of the many similarities between the CBD-induced isozyme and certain other isozymes previously purified from PB-pretreated animals, a cytochrome P-450 isozyme was purified from PB-pretreated mice by a chromatographic procedure similar to that employed for purification of the CBD-induced isozyme. The PB-inducible isozyme was indistinguishable from the CBD-inducible cytochrome P-450 on the bases of apparent molecular weight, absorption spectra, NH2-terminal amino acid sequence, peptide mapping, immunoreactivity, and catalytic activity. Although the CBD- and PB-inducible P-450 isozymes appear to be qualitatively very similar, PB appears to be a quantitatively better inducer of the isozyme. Thus, CBD exposure results in the induction of an isozyme that is refractory to CBD-mediated inactivation, thereby apparently altering the cytochrome P-450 isozymal composition of mouse hepatic microsomes.     

Oxidation of cyclopenta[cd]pyrene by human and mouse liver microsomes and selected cytochrome P450 enzymes.

The metabolism of the environmental pollutant and suspected human carcinogen, cyclopenta[cd]pyrene (CPP), was investigated. Human liver microsomes from three individuals were examined, as well as CD-1 mouse liver microsomes. Five new metabolites recently identified in our lab, 4-hydroxy-3,4-dihydroCPP, 3,4-dihydroCPP-cis-3,4-diol, 4-oxo-3,4-dihydroCPP, 3,4,9,10-tetrahydroCPP-trans-3,4-trans-9,10-tetrol, and trans-3,4-dihydroCPP-3, 4,x-triols, as well as the known major metabolite, 3,4-dihydroCPP-trans-3,4-diol, were all observed from the incubations of human liver microsomes and CPP. Even though all three human samples were capable of producing all the metabolites identified from the mouse liver microsomal incubations of CPP, the quantity of each metabolite varied among the microsomal samples. In an attempt to explain the variation among human liver samples, the microsomes derived from genetically engineered cells containing specific cytochrome P450 isozyme cDNAs were employed. It was found that the 3,4-cyclopenta double bond can be oxidized by the cytochrome P450 enzymes 1A1, 1A2, and 3A4. The 9,10 K-region double bond was not efficiently oxidized by cytochrome P450 1A1, but by P450 1A2 either from CPP or from the t-3,4-dihydrodiol. The lack of catalytic activity of 3A4 toward the t-3,4-dihydrodiol, despite its high activity toward CPP oxidation to tetrahydrotetrols, suggests the possibility of two dihydrodiol epoxides, 3,4-dihydrodiol 9,10-epoxide and 9,10-dihydrodiol 3,4-epoxide, of CPP.     

Formation of benzo[a]pyrene metabolite-nucleoside adducts in isolated perfused rat and mouse liver and in mouse lung slices.

The formation of benzo[a]pyrene metabolite-nucleoside adducts in perfused rat and mouse liver and in mouse lung slices was studied by Sephadex LH20 chromatography. In liver from β-naphthoflavone-pretreated rats, four different deoxyribonucleoside complexes were observed; these are tentatively attributed to DNA modification by the 7,8-diol-9, 10-epoxide(s), secondary metabolites of benzo[a]pyrene quinones, the 4,5-oxide, and secondary metabolites of benzo[a]pyrene phenols. The diol-epoxide-deoxyribonucleoside adduct was also detected in perfused liver and in lung slices from 3-methylcholanthrene-treated genetically responsive C57BL/6N mice, whereas no adducts were detectable in such samples from 3-methylcholan-threne-treated genetically nonresponsive DBA/2N mice. In perfused liver of phenobarbital-pretreated rats, the 4,5-oxide-deoxyribonucleoside adduct was present. These results suggest that some of the benzo[a]pyrene metabolite-nucleoside complexes generated by microsomes and deproteinized DNA in vitro also occur in the intact rodent liver and lung tissues.Furthermore, complexes with the diol-epoxide(s) were observed with RNA from perfused liver of β-naphthoflavone-treated, but not from untreated or phenobarbital-treated rats. Complexes between ribonucleoside(s) and the diol-epoxide(s) were also found in perfusedliver or lung slices from genetically responsive but not from genetically nonresponsive mice.     

Hydroxylation of benzo[a]pyrene and binding of (−)trans 5-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene metabolites to deoxyribonucleic acid catalyzed by purified forms of rabbit liver microsomal cytochrome P-450: Effect of 7,8-benzoflavone,butylated hydroxytoluene and ascorbic acid

The catalytic activities of hepatic microsornes from untreated, phenobarbital-treated and 3-methylcholanthrene-treated adult rabbits with respect to benzo[a]pyrene hydroxylation and the activation of (?)(rflw-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene[(?)trans-7,8-diol] to DNA-binding metabolites were determined in the absence and presence of mixed-function oxidase inhibitors and compared to the corresponding activities of the individual enzyme systems. Treatment of rabbits with phnobarbital led to induction of P-450LM₂ and a concomitant 3-fold enhancement in microsomal benzo[a]pyrene hydroxylase activity, whereas the conversion of (?)trans-7,8-diol to DNA-binding products was unaffected. Homogeneous phenobarbital-inducible P-450LM₂ exhibited the highest activity and specificity toward benzo[a]pyrene and the lowest activity toward (?)trans-7,8-diol. Conversely, P-450LM₄ was the major form of cytochrome P-450 induced in rabbit liver by 3-methylcholanthrene or β-naphthoflavone, and this was associated in microsomes with an increase in the metabolism of (?)trans-7, 8-diol but not of benzo[a]pyrene. Homogeneous P-450LM₄ preferentially Catalyzed the oxygénation of (?)trans-7,8-diol, but was largely ineffective with benzo[a]pyrene. Partially purified P-450LM₇ lacked substrate specificity, for it metabolized both benzo[a]pyrene and (?)trans-7, S-diol at comparable rates. Additionally, 7,8-benzoflavone strongly inhibited benzo[a]pyrene hydroxylation by P-450LM₄ and phenobarbital-induced microsomes, as well as (?)trans-7,8-diol metabolism by P-450LM₄ and 3-methyl-cholanthrene-induced microsomes; in contrast, the activity of control microsomes with either substrate, and the activities of P-450LM₄ and LM₂ with benzo[a]pyrene and (?)trans-7 ,8-diol, respectively, were only partially or slightly decreased by 7,8-benzoflavone. Unlike 7,8-benzoflavone, butylated hydroxytoluene inhibited benzo[a]pyrene hydroxylation only. Thus, different forms of rabbit liver microsomal cytochrome P-450 were involved in the metabolism of benzo[a]pyrene and its 7,8-dihydrodiol. The results also demonstrate that the changes in substrate specificity and inhibitor sensitivity seen in phenobarbital- and 3-methylcholanthrene-induced microsomes relative to control rabbit liver microsomes can be accounted for by the catalytic properties of a specific form of cytochrome P-450 that prevails in these preparations, P-450LM₂ and LM₄, respectively.