Dependence of benzo[a]pyrene metabolic profile on the concentration of cumene hydroperoxide with uninduced and induced rat liver microsomes

The effect of cumene hydroperoxide (CHP) in microsomal metabolism of benzo [a]pyrene (BP) was studied using liver microsomes from mature male Wistar rats induced with phenobarbital (PB), 3-methylcholanthrene (MC), Aroclor 1254 or olive oil (uninduced). In contrast to NADPH-supported metabolism, these inducers did not increase the CHP-dependent metabolism. Total BP metabolism was dependent on CHP concentration and was maximal at 0.15 mM, except for PB-induced microsomes, which had a maximum at 0.5 mM CHP. At 0.05 mM CHP, the major metabolites were phenols. However, increasing CHP concentration enhanced the formation of dihydrodiols, quinones and protein-bound BP but reduced phenol production. At and above 0.15 mM CHP, the profile of BP metabolites was essentially constant, with at least 66% quinones but no more than 10% phenols. The effect of CHP on inhibition of phenol formation and enhancement of quinone formation was reversed by preincubation of microsomes with BP or by increasing BP concentration. These results suggest that CHP-dependent metabolism of BP is selectively mediated by constitutive cytochrome P-450 isozyme(s) and that two forms of BP binding sites exist in cytochrome P-450 isozymes and are responsible for the hydroxylation of BP at C-3 and C-6.     

Radical cations as precursors in the metabolic formation of quinones from benzo[a]pyrene and 6-fluorobenzo[a]pyrene. Fluoro substitution as a probe for one-electron oxidation in aromatic substrates

Three classes of products are formed when benzo[a]pyrene (BP) is metabolized by cytochrome P-450: dihydrodiols, phenols and the quinones, BP 1,6-, 3,6- and 6,12-dione. These products have been thought to arise from attack of a catalytically-activated electrophilic oxygen atom. In this paper we report chemical and biochemical experiments which demonstrate that BP quinones arise from an initial one-electron oxidation of BP to form its radical cation. BP, 6-fluorobenzo[a]pyrene (6-FBP), 6-chlorobenzo[a]pyrene (6-ClBP), and 6-bromobenzo[a]pyrene (6-BrBP) were metabolized by uninduced and 3-methylcholanthrene-induced rat liver microsomes in the presence of NADPH or cumene hydroperoxide (CHP) as cofactor. BP and 6-FBP produced similar metabolic profiles with induced microsomes in the presence of NADPH or 2 mM CHP. With NADPH both compounds produced dihydrodiols, phenols and quinones, whereas with CHP, they yielded only quinones. Metabolism of BP and 6-FBP was also similar with uninduced microsomes and 2 mM CHP, yielding the same BP quinones. With uninduced microsomes in the presence of NADPH, BP produced all three classes of metabolites, whereas 6-FBP afforded only quinones. At a low concentration of CHP (0.10 mM), BP was metabolized to phenols and quinones, whereas 6-FBP gave only quinones. 6-ClBP and 6-BrBP were poor substrates, forming metabolites only with induced microsomes and NADPH. One-electron oxidation of BP by Mn(OAc)3 occurred exclusively at C-6 with predominant formation of 6-acetoxyBP and small amounts of BP quinones. In the one-electron oxidation of 6-FBP by Mn(OAc)3, the major products obtained were 6-acetoxyBP, a mixture of 1,6- and 3,6-diacetoxyBP, and BP quinones. Reaction of BP and 6-FBP radical cation perchlorates with water produced the same BP quinones. Conversely, electrophilic substitution of 6-FBP with bromine or deuterium ion afforded C-1 and/or C-3 derivatives with retention of the fluoro substituent at C-6. These results indicate that metabolic formation of BP quinones from BP and 6-FBP can only derive from their intermediate radical cation.     

Positional metabolism of benzo(a)pyrene in rat placenta and maternal liver. Comparison of induction effects

The biotransformation of [14C]benzo(a)pyrene (BP) was studied in vitro in the presence of microsomes prepared from isolated labyrinth and basal zone tissues of the rat placenta, as well as from maternal liver. Pregnant rats, day 14 of gestation, received beta-naphthoflavone (beta NF; 15 mg/kg, ip) or 3-methyl-cholanthrene (3MC; 30 mg/kg, ip). On day 15, placentae were dissected and microsomes were incubated with 17 microM [14C]BP and 2 mM NADPH. Metabolites formed in the incubation flasks were extracted and separated by HPLC utilizing a reverse phase column. Only trace BP metabolism occurred in basal zone microsomes from control, beta NF-, or 3MC-pretreated animals, as well as in labyrinth microsomes from control animals. In contrast, the preadministration of beta NF and 3MC increased labyrinth microsomal BP metabolism by 10- to 15-fold. Labyrinth and maternal liver microsomes from beta NF- and 3MC-treated animals actively converted BP to eight separate metabolites which co-chromatographed primarily with quinones and phenols. The overall formation of BP diol and phenolic metabolites by labyrinth microsomes was appreciably less than was observed for liver preparations. The very low activity of BP-4,5-oxide hydrolase in labyrinth microsomes compared to liver may in part explain the low level of formation of BP diols in placental microsomes. Labyrinth microsomes catalyzed the covalent binding of [3H]BP to calf thymus DNA, and this activity increased 5-fold following beta NF pretreatment. A comparison of induced tissues indicates that the amount of DNA binding in labyrinth microsomes is more extensive than would be expected by the level of total BP metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of harman and norharman on the metabolism of benzo(a)pyrene in isolated perfused rat lung and in rat lung microsomes.

The effects of harman and norharman, nitrogen-containing pyrolysis products of amino acids present in cigarette smoke, on the metabolism of benzo(a)pyrene in rat lung microsomes in vitro and in isolated perfused rat lung were studied. In rat lung microsomes, both harman and norharman inhibited the metabolism of benzo(a)pyrene (BP) to dihydrodiols, phenols and quinones at concentrations over approximately 0.05 mM. The formation of BP-7, 8-dihydrodiol and BP-9, 10-dihydrodiol was inhibited more than that of BP-4, 5-dihydrodiol. No appreciable differences in inhibition were seen between microsomes from control or 3-methylcholanthrene-pretreated rats. In isolated perfused rat lung, 1 mM of harman in the perfusion fluid inhibited the formation of ethyl acetate-soluble metabolites of benzo(a)pyrene except BP-9, 10-dihydrodiol, and inhibited the total covalent binding of benzo(a)pyrene metabolites to lung tissue macromolecules. 0.03 mM of harman seemed to increase other metabolites than BP-7,8-dihydrodiol without changing the total covalent binding. These results suggest that at most concentrations both β-carboline derivatives, harman and norharman, inhibit benzo(a)pyrene metabolism and covalent binding both in lung microsomes in vitro and in isolated perfused rat lung.     

Metabolism of benzo[a]pyrene by guinea pig adrenal and hepatic microsomes

Studies were carried out to compare the metabolism of benzo[a]pyrene (BP) by adrenal and hepatic microsomes obtained from adult male guinea pigs. Adrenal microsomes produced fluorescent metabolites (primarily phenols) approximately three to four times more rapidly than hepatic microsomes, but the differences in the rates were considerably smaller when total BP metabolism was assessed using an isotopic assay. The apparent discrepancy between the two assays is attributable to differences in the profiles of BP metabolites produced by adrenal and liver. Separation of metabolites by high pressure liquid chromatography revealed that adrenal microsomes converted BP to primarily a phenolic metabolite with a retention time identical to that of 3-hydroxy-BP. Liver microsomes, by contrast, produced approximately equal amounts of compounds co-chromatographing with 3-hydroxy-BP and BP-4,5-dihydrodiol. Small amounts of other metabolites were also produced by adrenal and hepatic microsomes. Liver microsomes catalyzed the conversion of BP to metabolites that became covalently bound to exogenous DNA. The amount of binding was dependent upon the duration of incubation and concentration of microsomal protein. Adrenal microsomes, by contrast, did not promote BP binding to DNA. Inhibition of microsomal epoxide hydratase activity with trichloropropene oxide (TCPO) blocked the formation of dihydrodiol metabolites of BP by adrenal and liver microsomes. In the presence of TCPO, liver microsomes produced large amounts of a BP metabolite co-chromatographing with BP-4,5-oxide. TCPO also increased the rate of production of DNA-binding roetabolites by liver microsomes but had no effect on the formation of DNA-binding metabolites by adrenal microsomes. The results demonstrate major differences in the pathways of BP metabolism by guinea pig adrenal and hepatic microsomes. Although adrenal microsomes metabolize BP more rapidly than hepatic microsomes, far greater amounts of reactive metabolites are produced by the liver. Thus, adrenal metabolism of BP may be of little toxicological significance.     

Alterations of pulmonary benzo[a]pyrene metabolism by reactive oxygen metabolites.

Superoxide anion radical and hydrogen peroxide (H2O2) are reactive oxygen metabolites which are thought to be involved in oxidant-induced lung injuries. Therefore, we studied their effects on the pulmonary metabolism of benzo[a]pyrene (BP) in rat lung microsomes. The microsomes were incubated with xanthine and xanthine oxidase to generate superoxide anion (effects verified with superoxide dismutase) or H2O2 and then the products formed during the metabolism of BP were measured. Both oxygen metabolites inhibit BP hydroxylase activity, i.e., the production of 3- and 9-hydroxybenzo[a]pyrene (phenols) in a concentration-dependent manner. The phenols account for approximately 75% of metabolite formation and are the major products of BP metabolism. Two components of the monooxygenase system responsible for BP metabolism, cytochrome P-450 and NADPH-cytochrome P-450 reductase, are also inhibited by the two oxygen metabolites in a similar manner. Superoxide anion is more effective than H2O2 in the inhibition of both BP hydroxylase and the monooxygenase components. Neither oxygen metabolite has any effect on the formation of minor metabolites of benzo[a]pyrene, i.e., BP-quinones and BP-dihydrodiols. These are the BP metabolites thought to produce toxic effects and which may lead to the formation of carcinogens and/or mutagens. The results of all these experiments suggest that exposure of lung microsomes to oxygen metabolites can lead to a slowing of overall BP metabolism and the increased accumulation of potentially toxic BP metabolites.     

Effects of organic solvent vehicles on benzo[a]pyrene metabolism in rabbit lung microsomes

In order to study the metabolism of benzo[a]pyrene (BP), it must be dissolved in an organic solvent vehicle for delivery to the tissue. We studied the effects of five organic solvent vehicles, i.e. dimethyl sulfoxide (DMSO), acetone, methanol, ethanol, and ethyl acetate, on benzo[a]pyrene hydroxylase activity and the BP metabolite profile in rabbit lung microsomes. Fluorescence detection of 3- and 9-OH-BP was used to evaluate benzo[a]pyrene hydroxylase activity, and the BP metabolite profile was obtained by HPLC analysis. All solvent vehicles inhibited benzo[a]pyrene hydroxylase in a dose-dependent manner. When the smallest volume of each solvent (10 microliter/ml reaction mixture) was employed, the resulting enzyme activities as related to solvent type, from highest to lowest, were DMSO greater than or equal to methanol greater than ethanol greater than or equal to acetone greater than ethyl acetate. HPLC analysis of BP metabolites formed in the presence of the five solvent vehicles showed that production of all metabolites was greatest when DMSO was used and that linearity of product formation was retained longer with DMSO. The metabolites produced when DMSO was used as the solvent were BP-9,10-diol, BP-4,5-diol, BP-7,8-diol, BP-1,6-quinone, BP-3,6-quinone and 3-OH-BP. A similar metabolite profile was obtained when reactions were carried out with methanol as the solvent vehicle, although the magnitude of production was less than with DMSO. When acetone was used, there were greater amounts of BP-4,5-diol and BP quinone formation and lesser amounts of 3-OH-BP formed than with DMSO or methanol. When ethanol or ethyl acetate was used as a solvent, BP-9,10-diol and 3-OH-BP were the only metabolites produced. These results indicate that all solvent vehicles studied inhibit benzo[a]pyrene hydroxylase from rabbit lung microsomes in a dose-dependent manner and that the magnitudes and types of metabolites formed are highly dependent upon the specific solvent used as the vehicle. The study also indicates that DMSO is probably the solvent vehicle of choice for study of BP metabolism in rabbit lung microsomes.     

Central role of radical cations in metabolic activation of polycyclic aromatic hydrocarbons

1. Development of the chemistry of polycyclic aromatic hydrocarbon (PAH) radical cations has provided evidence that these intermediates play a major role in the metabolism of PAHs by P450 and in their binding to DNA.

2. Fluoro substitution of benzo[a]pyrene (BP) represents a suitable probe for studying mechanisms of oxygen transfer in the P450-catalysed formation of quinones and phenols from BP. Formation of BP-1,6-, -3,6- and -6,12-dione from the metabolism of 6-fluoroBP (6-FBP) is mediated by the intermediate 6-FBP⁺. Similarly, metabolism of 1-FBP and 3-FBP by rat liver microsomes produces BP-1,6-dione and BP-3,6-dione respectively. These results demonstrate that formation of quinones and phenols occurs via an initial electron transfer from BP to P450 and subsequent transfer of oxygen from the iron-oxo complex of P450 to BP.

3. Radical cations also play a major role in the formation of DNA adducts by the potent carcinogens 7,12-dimethylbenz[a]anthracene (DMBA), BP and dibenzo[a, l]pyrene (DB[a, l]P). In the binding of BP both in vitro and in vivo, 80% of the adducts are formed by one-electron oxidation, namely, 8-(BP-6-yl)guanine (BP-6-C8Gua), BP-6-N7Gua and BP-6-N7adenine (Ade), and are lost from the DNA by depurination. For DB[a, l]P, depurinating adducts formed from the radical cation, DB[a, l]P-10-C8Gua, DB[a, l]P-10-N7Gua, DB[a, l]P-10-N7Ade, and DB[a, l]P-10-N3Ade comprise 50% of the total DNA adducts. For DMBA, 99% of the adducts are depurinating adducts formed from the radical cation, 7-CH₃BA-12-CH₂-N7Gua and 7-CH₃BA-12-CH₂-N7Ade.

4. In summary, radical cations of PAHs play a major role in both the metabolism and metabolic activation leading to formation of DNA adducts that are critical in the mechanism of tumour initiation.     

Radical cations in the horseradish peroxidase and prostaglandin H synthase mediated metabolism and binding of benzo[a]pyrene to deoxyribonucleic acid

Metabolism and DNA binding studies are used to investigate mechanisms of activation for carcinogens. In this paper we describe metabolism of benzo[a]pyrene (BP) and 6-fluorobenzo[a]pyrene (6-FBP) by two peroxidases, horseradish peroxidase (HRP) and prostaglandin H synthase (PHS), which are known to catalyze one-electron oxidation. In addition, binding of BP and BP quinones to DNA was compared in the two enzyme systems. The only metabolites formed from BP or 6-FBP by either enzyme were the quinones, BP 1,6-, 3,6- and 6,12-dione. HRP metabolized BP and 6-FBP to the same extent and produced the same proportion of each dione from both compounds, approximately 40% each of BP 1,6- and 3,6-dione and 20% BP 6,12-dione. PHS formed twice as much quinones from BP as from 6-FBP and produced relatively more BP 3,6-dione from 6-FBP (46%) compared to BP (30%) and relatively less BP 6,12-dione from 6-FBP (16%) compared to BP (33%). Removal of the fluoro substituent in the metabolism of 6-FBP is consistent only with an initial one-electron oxidation of the substrate. Since BP quinones were the only products formed in HRP- and PHS-catalyzed activation of BP, their possible binding to DNA was compared to that of BP. No significant binding of BP quinones to DNA occurred with either HRP or PHS. These results, coupled with those from other chemical and biochemical experiments, demonstrate that HRP- and PHS-catalyzed one-electron oxidation of BP to its radical cation is the mechanism of formation of quinones and binding of BP to DNA.     

Microsomal metabolism and covalent binding of [3H/14C]-bromobenzene. Evidence for quinones as reactive metabolites

1. The metabolism and covalent binding of [3H/14C]bromobenzene has been investigated using liver microsomes from untreated and phenobarbital (PB)-pretreated rats. A model has been developed to relate the observed 3H/14C ratios in the covalently bound residues to the type of metabolite (epoxide versus quinone) responsible for their formation. 2. With control microsomes metabolism was linear for 60 minutes, but with PB microsomes the time course showed a short-lived burst of rapid metabolism followed by a long phase with an overall rate comparable to control. With both types of microsomes covalent binding was synchronous with metabolism. 3. The normalized 3H/14C ratios of recovered substrate and water-soluble metabolites was 1.0, whereas that of the covalently bound material was only 0.5. Such extensive loss of tritium implies that a considerable portion of the covalent binding arises from bromobenzene metabolites more highly oxidized than an epoxide (e.g. quinones). 4. The normalized 3H/14C ratios for bromobenzene metabolites covalently bound to liver proteins in vivo (total and microsomal) was the same as with microsomes in vitro (0.5). However, for the lung and kidney the 3H/14C ratios were considerably higher (0.71 and 0.62), indicating that differences between tissues in vivo may be greater than between liver microsomes in vitro and in vivo.     

Tissue and sex differences in the activation of aromatic hydrocarbon hydroxylases in rats

Betamethasone and α-naphthoflavone produced similar activation of biphenyl 2-hydroxylase and benzo[a]pyrene 3-hydroxylase in control male rat liver microsomes. In small intestinal epithelial microsomes, betamethasone had no effect whereas α-naphthoflavone caused a pronounced activation of benzo[a]pyrene hydroxylation and a lesser activation of biphenyl 2-hydroxylation. In lung microsomes, betamethasone had no effect on either enzyme activity whereas α-naphthoflavone had no effect on biphenyl 2-hydroxylase but inhibited benzo[a]pyrene hydroxylase. In kidney cortex microsomes from male rats both compounds caused inhibition or had no effect whereas in kidney cortex microsomes from female rats betamethasone activated whereas α-naphthoflavone had no effect.Activation also occurred in isolated viable hepatocytes from male rats. The response of biphenyl 2-hydroxylase was very similar to that found in male rat liver microsomes but benzo[a]pyrene hydroxylase was more sensitive to activation and less sensitive to inhibition than in microsomes. The findings are interpreted as demonstrating the presence of more than one ‘latent’ aromatic hydrocarbon hydroxylase in rodents.     

The mechanistic plurality of cytochrome P-450 and its biological ramifications

The mechanistic plurality of the microsomal cytochrome P-450 enzyme system is illustrated by studies of the oxidative metabolism of benzo[a]pyrene, 3-hydroxybenzo[a]pyrene and arachidonic acid. Rat liver microsomal metabolism of benzo[a]pyrene or 3-hydroxy-benzo[a]pyrene, supported by cumene hydroperoxide, generates benzo[a]pyrene quinones via molecular oxygen-dependent and -independent pathways. Arachidonic acid is metabolized by rat liver microsomal fractions to a variety of oxygenated products, including cis-trans diene conjugated monohydroxy-acids, epoxy-acids as well as omega- and omega-1-oxidation products. The chemistry of the different reaction products is discussed in terms of the possible mechanisms responsible for their formation and the role of the haemoprotein during catalysis. An integrated view for the reaction cycle of cytochrome P-450 is presented.     

Metabolism and covalent binding of [14C]toluene by human and rat liver microsomal fractions and liver slices

The in vitro metabolism of [14C]toluene by liver microsomes and liver slices from male Fischer F344 rats and human subjects has been compared. Rat liver microsomes produced only benzyl alcohol from toluene. Liver microsomes from human subjects metabolized toluene to benzyl alcohol, benzaldehyde, and benzoic acid. Liver microsomes from one human donor also produced p-cresol and o-cresol. The overall rate of toluene metabolism by human liver microsomes was 9-fold greater than by rat liver microsomes. Human liver microsomal metabolism of benzyl alcohol to benzaldehyde required NADPH and was inhibited by carbon monoxide and high pH (pH 10). but was not inhibited by ADP-ribose or sodium azide. These results suggest that cytochrome P-450, rather than alcohol dehydrogenase, was responsible for the metabolism of benzyl alcohol to benzaldehyde. Human and rat liver slices metabolized toluene to hippuric acid and benzoic acid. The overall rate of toluene metabolism by human liver slices was 1.3-fold greater than by rat liver slices. Cresols and cresol conjugates were not detected in human or rat liver slice incubations. Covalent binding of [14C]toluene to human liver microsomes and slices was 21-fold and 4-fold greater than to the comparable rat liver preparations. Covalent binding did not occur in the absence of NADPH, was significantly decreased by coincubation with cysteine, glutathione, or superoxide dismutase, and was unaffected by coincubation with lysine. Protease and ribonuclease digestion decreased the amount of toluene covalently bound to human liver microsomes by 78% and 27% respectively. Acid washing of human liver microsomes had no effect on covalent binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies related to the mechanism of 3-BHA-induced neoplasia of the rat forestomach

The major objective of this investigation has been to determine the mechanism by which 3-BHA induces forestomach tumours in rodents. In vitro studies of liver microsomal metabolism of [14C]-3-BHA show binding of metabolites to microsomal protein which could be markedly decreased by addition of L-cysteine. p-Toluenesulphonic acid hydrolysis of the labelled microsomes showed a radioactive peak that co-chromatographed with the major product of the reaction of tert-butylquinone (tert-BuQ) and L-cysteine. In vivo binding of metabolites of [14C]-3-BHA to microsomal protein of the forestomach, glandular stomach and liver was determined. Forestomach microsomal protein contained 14 times as much bound radioactivity as glandular stomach and 12 times as much as liver. HPLC studies showed marked qualitative differences in the distribution of labelled compounds in hydrolysates of microsomes from the three tissues. The forestomach contained peaks not present in the other two tissues. In other studies it was shown that the 3-tert-butyl-5-methoxy-1,2-benzoquinone reacted rapidly with NADPH and NADH. tert-BuQ did so more slowly. Current data suggest that two factors may be of importance for 3-BHA carcinogenesis. The first is thiol depletion resulting from direct binding of quinone metabolites of 3-BHA to tissue thiols. Secondary reactions due to the presence of the quinones could also deplete -SH groups. Such thiol depletion could account for the threshold level existing for 3-BHA carcinogenesis. The second factor is an attack on tissue constituents from reactive metabolites of 3-BHA, as is evident from protein binding, and possibly also from oxygen radicals produced as a result of redox cycling of quinone and hydroquinone metabolites of 3-BHA.     

CYP1A1 is a major enzyme responsible for the metabolism of granisetron in human liver microsomes

Granisetron, a potent 5-HT3 receptor antagonist, has been reported to be mainly metabolized to 7-hydroxygranisetron and a lesser extent to 9'-desmethylgranisetron in humans. A previous study indicated that cytochrome P450 (CYP)3A4 is a major catalyst of 9'-demethylation, although the major CYP isoform(s) responsible for 7-hydroxylation are unknown. To clarify granisetron 7-hydroxylase, the in vitro metabolism of granisetron using expressed human CYPs and human liver microsomes was investigated. 7-Hydroxygranisetron was produced almost exclusively by CYP1A1, while, apparently, 9'-desmethylgranisetron was preferentially produced by CYP3A4. Marked inter-individual differences in the ratio of the formation of 7-hydroxygranisetron and 9'-desmethylgranisetron in human liver microsomes was observed. Granisetron 7-hydroxylase activity was strongly correlated with benzo[a]pyrene 3-hydroxylase activity (p<0.0001), but not with testosterone 6beta-hydroxylase activity in human liver microsomes. Furthermore, an anti-human CYP1A1 antibody completely inhibited 7-hydroxylation in human liver microsomes, however, the reaction was not inhibited at all by an anti-CYP3A4 antibody. On the other hand, granisetron 9'-demethylase activity correlated significantly not only with testosterone 6beta-hydroxylase activity (p<0.0001) but also with benzo[a]pyrene 3-hydroxylase activity (p<0.01). Consistent with this, both the anti-CYP1A1 and anti-human CYP3A4 antibodies inhibited the 9'-demethylase activity. These data indicate that CYP1A1 is a major enzyme responsible for the metabolism of granisetron via a main 7-hydroxylation pathway and an alternative 9'-demethylation route. This is the first report demonstrating the substantial contribution of CYP1A1 to the metabolism of a drug, although its role in the metabolism of environmental compounds is well established.     

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.     

Metabolism and mutagenicity of dibenzo[a,e]pyrene and the very potent environmental carcinogen dibenzo[a,l]pyrene

Dibenzo[a,l]pyrene (DB[a,l]P) is one of the most potent carcinogens ever tested in mouse skin and rat mammary gland. DB[a,l]P is present in cigarette smoke and, presumably, in other environmental pollutants. Metabolism and mutagenicity studies of this compound compared to the weak carcinogen dibenzo[a,e]pyrene (DB[a,e]P) can provide preliminary evidence on its mechanism of carcinogenesis. The mutagenicity of DB[a,l]P, DB[a,e]P, and benzo[a]pyrene (BP) was compared in the Ames assay with Aroclor-induced rat liver S-9. BP was the strongest mutagen. In strain TA100, DB[a,l]P and DB[a,e]P were marginally mutagenic. In strain TA98 both compounds were mutagenic, and DB[a,l]P induced more than twice as many revertants as DB[a,e]P. The mutagenicity of DB[a,l]P does not correlate with its carcinogenicity, since DB[a,l]P is a much stronger carcinogen, but a much weaker mutagen, than BP. The NADPH-supported metabolism of DB[a,e]P and DB[a,l]P was conducted with uninduced and 3-methylcholanthrene-induced rat liver microsomes. Metabolites were analyzed by reverse-phase HPLC and identified by NMR, UV, and mass spectrometry. Uninduced microsomes produced only traces of metabolites with either compound. The major metabolites of DB[a,l]P with induced microsomes were DB[a,l]P 8,9-dihydrodiol, DB[a,l]P 11,12-dihydrodiol, 7-hydroxyDB[a,l]P, and a DB[a,l]P dione. The metabolites of DB[a,e]P with induced microsomes were DB[a,e]P 3,4-dihydrodiol, 3-hydroxyDB[a,e]P, 7-hydroxyDB[a,e]P, and 9-hydroxyDB[a,e]P. Some of these metabolites are very useful in assessing possible pathways of activation in the initiation of cancer.     

Cell-catalyzed binding of 3H-(-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene to cellular and exogenous DNA and the role of purified human liver epoxide hydrolase

Cultured human monocytes, lymphocytes, Fischer rat liver (TRL-2) cells, and Buffalo rat liver (BRL) cells catalyzed the conversion of 3H(-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene [3H(-)t-7,8-dihydrodiol BP] to r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide I) and r-7,t-7-8-dihydroxy-c-9,10-oxy-7,8,9,10- tetrahydrobenzo[a]pyrene (diol epoxide II; r-7 indicates that the substituent at the 7-position is the reference, and t and c indicate that the substituents trans and cis, respectively, to the reference substituent). These appear to be the most reactive metabolites of benzo[a]pyrene (BP) and were covalently bound to both exogenous and intact cellular DNA in tissue culture media. The cells induced by benzanthracene (BA) exhibited greater levels of DNA binding than the controls and this binding was linear with increasing cell content in human monocytes, in TRL-2 cells and in Buffalo rat liver cells. The binding to DNA was greater than controls in BA-preinduced lymphocytes, but was not linear. The DNA binding in control cells showed a nonlinear increase with increasing cell concentration in all experiments. The addition of human liver epoxide hydrolase (EC 3.3.2.3) to the incubation medium reduced the amount of reactive metabolites binding to DNA by 12-15% in control and by 23-41% in BA-induced monocytes. Thus, with whole cell systems of either human monocytes or lymphocytes, the addition of purified human liver epoxide hydrolase reduced the binding of 3H(-)t-7,8-dihydrodiol BP metabolites to DNA. Human monocytes and lymphocytes also catalyzed the covalent binding of 3H(-)t-7,8-dihydrodiol BP to intact cellular DNA. The addition of 3H(-)t-7,8-dihydrodiol BA to tissue culture media caused the inhibition of covalent DNA binding in BA-preinduced monocyte by 58% and lymphocytes by 25%. Previous work has shown that BA is metabolized and converted to BA-diol epoxides by microsomes. These results indicate that BA-diol epoxides and BP diol epoxides are competing for the same binding sites on DNA. On the other hand, the addition of 10 nmol of 3H(-)t-7,8-dihydrodiol BP to the incubation of control and BA-preinduced cell homogenate and further incubation at 37 degrees C for 25 min showed that the DNA binding in BA-preinduced cell homogenates was much greater than controls. Homogenates of cells induced by BA exhibited a greater level of DNA binding than controls.(ABSTRACT TRUNCATED AT 400 WORDS)     

Epidermis: the major site of cutaneous benzo(a)pyrene and benzo(a)pyrene 7,8-diol metabolism in neonatal BALB/c mice

The metabolism of benzo(a)pyrene (BP) and benzo(a)pyrene-7,8-diol (BP-7,8-diol) by microsomes prepared from whole skin, dermis, and epidermis of neonatal BALB/c mice pretreated with topically applied 3-methylcholanthrene (MCA) was compared. In control animals, microsomes prepared from epidermis showed higher rates of metabolism of BP and BP-7,8-diol (1.4-2.6-fold) than did microsomes prepared from whole skin or dermis. A single topical application of MCA increased the rate of metabolism of BP and BP-7,8-diol in microsomes prepared from whole skin, dermis, and epidermis. The greatest increase occurred in the epidermis. The in vivo covalent binding of [3H]BP, [3H]BP-7,8-diol, and 7,12-[3H]dimethylbenz(a)anthracene ([3H]DMBA) to DNA was found to be greater in epidermis (8.7-15.4-fold) than in whole skin or in dermis. A single topical application of MCA to BALB/c mice enhanced the in vivo binding of [3H]BP, [3H]BP-7,8-diol and [3H]DMBA to DNA of whole skin, dermis, and epidermis more than 2-fold. Exposure of Salmonella tester strains TA98 and TA100 to 2-aminoanthracene, a skin carcinogen, in the presence of an epidermal metabolic activation mixture resulted in a greater mutagenic response when compared to activation mixtures derived from whole skin or dermis. These results indicate that epidermis is the major site of polycyclic aromatic hydrocarbon metabolism and of enzyme-mediated covalent binding of polycyclic aromatic hydrocarbon carcinogens to DNA in skin of BALB/c mice and that topically applied MCA has maximum enzyme induction effects in this skin compartment.     

Metabolism of chrysene by brown bullhead liver microsomes.

We have investigated the regio- and stereoselective metabolism of chrysene, a four-ring symmetrical carcinogenic polycyclic aromatic hydrocarbon (PAH), by the liver microsomes of brown bullhead (Ameriurus nebulosus), a bottom-dwelling fish species. The liver microsomes from untreated and 3-methylcholanthrene (3-MC)-treated brown bullheads metabolized chrysene at the rate of 30.1 and 82.2 pmol/mg protein/min, respectively. Benzo-ring diols (1,2-diol and 3,4-diol) were the major chrysene metabolites formed by liver microsomes from control and 3-MC-treated fish. However, the control microsomes produced a considerably higher proportion of chrysene 1,2-diol (benzo-ring diol with a bay region double bond) plus 1-hydroxychrysene, than 3,4-diol plus 3-hydroxychrysene, indicating that these microsomes are selective in attacking the 1,2- position of the benzo-ring. On the other hand, 3-MC-induced microsomes did not show such a regioselectivity in the metabolism of chrysene. Control bullhead liver microsomes, compared to control rat liver microsomes, produced a considerably higher proportion of chrysene 1,2-diol, the putative proximate carcinogenic metabolite of chrysene. Like rat liver microsomes, bullhead liver microsomes produced only trace amounts of the K-region diol. Chrysene 1,2-diol and 3,4-diol formed by the liver microsomes from both control and 3-MC-treated bullheads consisted predominantly of their R,R-enantiomers. Chrysene is metabolized by bullhead liver microsomal enzymes to its benzo-ring diols with a relatively lower degree of stereoselectivity compared to benzo[a]pyrene (a five-ring PAH), but with a higher degree of stereoselectivity compared to phenanthrene (a three-ring PAH). The data of this study, together with those from our previous studies with phenanthrene, benzo[a]pyrene and dibenzo[a,l]pyrene (a six-ring PAH), indicate that the regioselectivity in the metabolism of PAHs by brown bullhead and rainbow trout liver microsomes does not vary greatly with the size and shape of the molecule, whereas the degree of stereoselectivity in the metabolism of PAHs to benzo-ring dihydrodiols does.