Effect of various chemicals on the metabolism of benzo(a)pyrene by cultured rat colon

The effect of various co- and anti-carcinogens of colon carcinogenesis on the metabolism of benzo(a)pyrene (BP) in cultured rat colon is reported. Rat colon enzymatically converted BP into metabolites which bind to cellular macromolecules i.e., DNA and protein. Activity of aryl hydrocarbon hydroxylase (AHH) activity and binding levels of BP to macromolecules were higher in the descending colon when compared to other segments. The major metabolites of BP, extractable with ethylacetate, were quinones, tetrols, 7,8-diol and a peak containing 9,10-dihydroxy-9,10-dihydrobenzo(a)pyrene and 7,8,9-trihydroxy-7,8-dihydrobenzo(a)pyrene. The binding levels of BP to DNA and protein in the explant was lowered by co-incubation with 7,8-benzoflavone (7,8-BF) (3.6 and 18.0 μM), a known inhibitor of AHH, and with disulfiram (100 μM), an anti-oxidant. The absence of vitamin A in the media also resulted in a lower level of BP binding to DNA and protein and in lower activity of AHH. Pretreatment with known inducers of AHH such as phenobarbital (PB) or benz(a)anthracene (BA), did not have any significant effect on the binding levels of BP to DNA or on the AHH activity. of the bile acids investigated only taurodeoxycholic acid significantly increased the binding level of BP to DNA.     

Inhibition of hepatic aryl hydrocarbon hydroxylase by 3-methylcholanthrene, 7,8-benzoflavone and other inducers added in vitro.

A close correlation has been observed between the ability of aromatic polycyclic hydrocarbons and 7,8-benzoflavone (7,8-BF) to induce hepatic aryl hydrocarbon hydroxylase (AHH) in vivo and to inhibit the induced enzyme system in vitro. The activity of this mono-oxygenase was measured by the conversion of 14C-labeled dimethylbenz(a)anthracene (DMBA) or benzo(a)pyrene (BP) to water-soluble products by rat liver preparations (8000 X g supernatant). DMBA as substrate had the advantage over BP in giving a wider range of ethyl acetate-soluble metabolites and allowing the observation of changes in the pattern of these products following injection or addition of the inducing agents. This property was used to detect low concentration (0.1 muM) of polycyclic hydrocarbons which are strong AHH inducers and which may also be carcinogenic. The liver preparation was active for several months when stored at --20 degrees. A possible mechanism of action for the in vitro behaviour of polycyclic hydrocarbons and 7,8-BF towards AHH is proposed.     

Unusual patterns of benzo[a]pyrene metabolites and DNA-benzo[a]pyrene adducts produced by human placental microsomes in vitro

Human placental microsomes were incubated with [³H]benzo[a]pyrene (BP) and Salmon sperm DNA and the resulting metabolite-nucleoside complexes resolved by Sephadex LH-20 chromatography. The metabolite pattern was analyzed by high-pressure liquid chromatography (HPLC). The incubates were also co-chromatographed with extracts obtained from incubates with rat liver microsomes and [¹⁴C]BP. Phenols, quinones and 7,8-dihydrodiol were detected in the placental incubates. Both 9,10- and 4,5-dihydrodiols were very low as compared with control rat liver samples. Placental microsomes catalyzed the binding of BP metabolites to DNA in vitro, giving rise to two main complexes which co-chromatographed with rat liver-produced peaks attributable to 7,8-diol-9,10-epoxide and 7,8-oxide and/or quinones when metabolized further. The nucleoside metabolite peaks attributable to 4,5-oxide and 9-phenol-4,5-oxide were lacking when compared with the binding pattern catalyzed by rat liver. Both the total binding and specific metabolite-nucleoside adducts in the placenta correlated with fluorometrically measured aryl hydrocarbon hydroxylase (AHH) activity and with the amount of dihydrodiol formed. The results demonstrate that both the metabolite pattern and the nucleoside-metabolite complexes formed by the placental microsomes in vitro differed greatly from those produced by rat liver microsomes. These studies also suggest that it is not possible to predict specific patterns of DNA binding from AHH measurements or even from BP metabolite patterns, especially when comparing different tissues and species.     

Metabolism of benzo[A]pyrene and the related enzyme activities in hamster embryo cells.

The rate of metabolism of benzo[a]pyrene (BP) and changes in related enzyme activities in cultured hamster embryo cells during successive subculture were studied. The activity of aryl hydrocarbon hydroxylase (AHH) was the highest when embryo cells were first dispersed in tissue culture flasks and decreased during subsequent passages. On the other hand, UDP-glucuronyl transferase activity increased gradually during successive subculture. Treatment of the cells with 13 nmol/ml of benz[a]anthracene (BA) for 24 h increased the activity of AHH but not that of UDP-glucuronyl transferase. The metabolism of BP was measured in cells of the passages 1, 3 and 7; metabolism of BP was most efficient in cells in passage 3 and their formation of glucuronic acid conjugates of BP, one of the major metabolites found in the medium, was 3- and 10-fold more than those of cells in passages 1 and 7, respectively. Analysis of BP-metabolites extracted from the medium with ethylacetate showed that the main metabolites were 9,10-diol and 7,8-diol. Phenols and quinones were released by treatment of the medium with beta- glucuronidase and their amounts were larger than those of diols at all passages. These results show that in hamster embryo cells in early passage, BP is metabolized to conjugates of phenols with glucuronic acid.     

Metabolism of benzo[A]pyrene in hamster embryo cells. Effect of the concentration of benzo[A]pyrene on its metabolism.

The metabolism of benzo[a]pyrene (BP) by hamster embryo cells was studied. The production of water-soluble metabolites, shown to be conjugates with glucuronic acid, depended on BP concentration. With increased BP concentration the amount of glucuronic acid conjugates increased, but the proportion of conjugates in BP or its metabolites present in the medium decreased. The metabolites extracted with ethylacetate were trans-7,8-dihydrodiol-BP (7,8-dihydrodiol) and trans-9,10-dihydrodiol-BP (9,10-dihydrodiol), but large peaks of phenolic metabolites were found by high pressure liquid chromatography (HPLC) after digesting the medium with beta-glucuronidase. Therefore, BP is metabolized to oxygenated forms, and of these, most of the phenolic metabolites and parts of the dihydrodiols are conjugated with glucuronic acid. The proportions of dihydrodiols to phenols, estimated by HPLC after beta-glucuronidase digestion, decreased when the BP concentration was decreased. The results suggest that dihydrodiols are less readily glucuronidated than phenols and so may be metabolized further to metabolites other than glucuronic acid conjugates.     

Metabolic activation of promutagens, detectable in Ames' Salmonella assay, by 5000 X g supernatant of rat ventral prostate

Induction of aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin-O-deethylase (7-EOD) activities as well as of benzo[a]pyrene (BP) metabolite formation in rat prostatic microsomes has been demonstrated after treatment with beta-naphthoflavone (BNF). The capacity to convert promutagenic compounds to ultimate mutagenic metabolites in the Ames' Salmonella assay by 5000 X g supernatant of rat ventral prostate was investigated. Male rats were treated with BNF, polychlorinated biphenyls (PCB; Arochlor 1254), phenobarbital (PB) and the vehicle, corn oil. PCB or BNF pretreatment increased the AHH- and 7-EOD activities 100-200-fold in the rat prostate 5000 X g supernatant (S-5 fraction). Epoxide hydrolase (EH) and glutathione-S-transferase (GST) activities were not affected while UDP-glucuronosyltransferase (UDP-GT) was increased 2.2- and 2.5-fold by PCB and BNF, respectively. PB did not significantly affect any of the enzyme activities measured. A dose-dependent increase in mutagenic response versus amount of 5000 X g supernatant and promutagen (aflatoxin B1 (AFB), 2-aminofluorene (2-AF), BP) was observed. The most pronounced activation was obtained with S-5 fraction from BNF- or PCB-treated rats. The great sensitivity of prostatic AHH to certain inducers and the capacity of the prostate to produce mutagenic metabolites might be of importance for initiation of prostatic cancer by environmental factors.     

Nuclear metabolism of benzo(a)pyrene and of (±)-trans-7,8-dihydroxy-7,8,-dihydrobenzo(a)pyrene: Comparative chromatographic analysis of alkylated DNA

Rat liver nuclei were incubated with [¹⁴C]benzo(a)pyrene (BP) or [³H](±)-trans-7,8-dihydrodiol of BP (³H-BP-7,8-diol) in the presence of a NADPH-generating system. The nuclei were able to form from BP the 9,10-, 4,5- and 7,8-dihydrodiols, the 3,6- and 1,6-quinones as well as the 3- and 9-phenols. The total nuclear metabolism was stimulated 11-fold by prior administration to the rats of 3-methylcholanthrene (3MC). BP-7,8-dihydrodiol formation, under these circumstances, was enhanced 29-fold. The rat liver nuclei were also able to form from [³H]BP-7,8-diol, (±)-7β,8α-dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydro BP (diol epoxide 1), (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydro BP (diol epoxide 2), as well as three unknown metabolites. Diol epoxides 1 and 2 represented 23 and 65% of the total metabolites produced during the control nuclear incubation. Pretreatment of the rats with 3MC resulted in 4-fold increase in nuclear metabolic activity. Under the latter circumstances, the diol epoxides 1 and 2 represented 43 and 38%, respectively, of the total nuclear metabolites. Incubation of liver nuclei with labeled BP or BP-7,8-diol in the presence of NADPH resulted in alkylation of DNA. The alkylated deoxyribonucleosides were separated by Sephadex LH-20 chromatography. Two peaks of radioactivity were noted after incubation with the parent polycyclic hydrocarbon while only one peak was seen after incubation with the diol derivative. These results emphasize the importance of nuclei in the metabolism of BP and in the subsequent alkylation of DNA, reactions which may be related to mutagenesis or carcinogenesis.     

Effects of various inducers on diethylstilbestrol metabolism, drug-metabolizing enzyme activities and the aromatic hydrocarbon (Ah) receptor in male Syrian golden hamster liver

In order to elucidate the role of metabolic activation of the synthetic estrogen, diethylstilbestrol (DES), in the mechanism of liver tumor formation in male Syrian golden hamsters observed after combined treatment with DES and 7,8-benzoflavone (7,8-BF), the metabolism of DES and the concentrations and activities of various drug-metabolizing enzymes were studied in hamster liver microsomes after various pretreatments. The levels of the hepatic aromatic hydrocarbon (Ah) receptor were also determined. Pretreatment with 7,8-BF increased both P450 and cytochrome b5 levels, whereas phenobarbital (PB) and 3-methylcholanthrene (MC) induced P450 but not cytochrome b5. 7,8-BF pretreatment increased 7-ethoxyresorufin-O-deethylase (EROD) 3-fold and 7-pentoxyresorufin-O-dealkylase (PROD) 2.5-fold, whereas aromatic hydrocarbon hydroxylase (AHH) and 7-ethoxycoumarin-O-deethylase (ECOD) activities were only slightly induced by 7,8-BF. MC pretreatment increased EROD 8-fold and PROD activity 7-fold, whereas PB pretreatment enhanced AHH 4.5-fold and PROD activity 4-fold. In contrast to PB, pretreatment with 7,8-BF and MC reduced the oxidative metabolism of DES in hepatic microsomes, but the pattern of metabolites was identical with that in untreated controls. Treatment of hamsters with the inducers changed the hepatic Ah receptor level. PB and MC-pretreatment resulted in an increase of the receptor level 1.5-fold and 1.3-fold, respectively, whereas 7,8-BF-pretreatment leads to a 1.5-fold decrease. The dissociation constant Kd is 170 nM for the reaction of 7,8-BF with the hamster Ah receptor compared to 70 nM for 5,6-BF and 38 nM for 2,3,7,8-tetrachlorodibenzofuran (TCDF). The Kd-value is 3.6 nM for TCDF with the rat receptor protein. It is concluded from these data that metabolic activation of DES is not involved in the mechanism of hepatocarcinogenesis in this animal tumor model.     

Benzo[a]pyrene-DNA-adducts and monooxygenase activities in mice treated with benzo[a]pyrene, cigarette smoke or cigarette smoke condensate

Synchronous fluorescence spectrophotometry (SFS), developed to study benzo[a]pyrene-7,8-diol-9,10-epoxide(BPDE)-DNA, was used to measure the in vivo formation of DNA-adducts in genetically responsive C57BL/6 (B6) and non-responsive DBA/2 (D2) mice. Treatment with cigarette smoke by inhalation for 3-16 days, or i.p. injection of cigarette smoke condensate or neutral fraction did not lead to detectable levels of BPDE-DNA-adducts in either lungs or liver, although aryl hydrocarbon hydroxylase (AHH) activity, an indicator of benzo[a]pyrene (BP) metabolism, was clearly induced in lungs of B6 mouse. A dose-dependent amount of BPDE-DNA-adducts in lung and somewhat less in liver was found after i.p. injection with BP (20-80 mg/kg). Mice treated with vehicle or 4 mg/kg of BP were negative for adducts by SFS. In B6 mice AHH was induced both in lungs and livers while there was no AHH induction in D2 mice although the levels of BPDE-DNA-adducts were somewhat higher than in B6 mice. Thus, no clear correlation seems to exist between AHH activity and the formation of BPDE-DNA-adducts. Also, according to our results SFS can be used to quantitate adduct-formation in in vivo animal studies.     

Glucuronidation of benzo[a]pyrene in hamster embryo cells

The formation of water-soluble metabolites of tritium-labeled benzo[a]pyrene (BP) by cultured hamster embryo cells was studied. The ratio of the radioactivity in the aqueous phase to that in the organic phase increased with the incubation period. After incubation for 48 h with 3.75 nmol/ml of [3H] BP in the medium more than 90% of the 3H-radioactivity was found in the aqueous phase, whereas with 10-fold more BP about half the radioactivity remained in the organic phase. The main metabolites extracted from the medium at 37.5 nmol/ml BP with ethyl acetate by high pressure liquid chromatography (HPLC) were 9,10-diol and 7,8-diol; but after treatment of the medium with beta-glucuronidase the main oxygenated metabolites were phenols, the amount of 9-OH BP being more than that of 3-OH BP. beta-Glucuronidase also released 9,10-diol and 7,8-diol, but most of these diols were in the free form in the medium. The medium from cells treated with 3.75 nmol/ml BP has a quantitatively different profile, and most of the radioactivity obtained by extraction with organic solvent and digestion with beta-glucuronidase was eluted in the regions of phenols. These results show that in hamster embryo cells BP is mainly metabolised to conjugates of phenols with glucuronic acid.     

The DNA binding of benzo[alpha]pyrene metabolites catalysed by rat lung microsomes in vitro and in isolated perfused rat lung.

When [³H]benzo[a]pyrene is incubated in vitro together with DNA, NADPH and rat lung microsomes, covalent binding of benzo[a]pyrene (BP) metabolites to DNA occurs. These metabolite-nucleoside complexes can be resolved into several distinct peaks by elution of a Sephadex LH-20 column with a water-methanol gradient. 3-Methylcholanthrene (MC) pretreatment of animals induces the total covalent binding in vitro several-fold and increases the amounts of at least five metabolite-nucleoside complexes associated with the 7,8-diol-9,10-epoxidcs, the 7,8-oxide or quinones oxygenated further, the 4,5-oxide and phenols oxygenated further. These increases correspond well with the increases in the production of both non-K-region and K-region metabolites of BP by lung microsomes, as determined by highpressure liquid chromatography (HPLC). On the other hand, when [³H]BP is metabolized in isolated perfused rat lung, only the peak representing the 7,8-diol-9,10-epoxide bound to nucleoside(s) is readily detectable and then only in lungs from MC-treated animals. The extent of binding of BP metabolites to lung DNA is very low, about 0.0004% of the total dose applied to the perfusion medium; more than 60% of this can be accounted for by the binding of the 7,8-diol-9,10-epoxides to nucleoside(s). It is suggested that the further metabolism leading to metabolites not available to covalent binding, (e.g. conjugation) of primary BP metabolites in the intact tissue is responsible for the differences in the metabolite-nucleoside patterns observed in vivo, as compared with microsomal metabolism in vitro.     

The effect of manganese and ferric ions on the in vitro formation of dihydrodihydroxy metabolites of benzo(a)pyrene

The effect of ferric and manganese ions on the in vitro metabolism of benzo(a)pyrene (BP) to dihydrodihydroxy (diol) metabolites by rat liver microsomal preparations was studied. Of the 3 diols separated by high-pressure liquid chromatography (HPLC) and called diols 1, 2 and 3 in order of elution, diol 1 was identified by its U.V. spectrum as the 9,10-diol; diols 2 and 3 have not yet been identified positively but are probably the 4,5- and 7,8-diols respectively. Higher concentrations of both metals altered the diol profile; 10 and 50 mumol Fe3+ per incubation caused the disappearance of diols 1 and 2 and an increase in diol 3; 10 mumol Mn2+ caused a significant decrease in diol 2 while 50 mumol reduced diol 2 to a negligible amount and inhibited the formation of diol 1; both concentrations caused a relative increase in diol 3. If the tentative identification of diol 3 as the 7,8-diol is correct, manganese and ferric ions could be significant in the metabolism of BP to the active metabolite, the 7,8-diol-9,10-epoxide.     

Benzo(a)pyrene activation to 7,8-dihydrodiol 9,10-oxide by rat liver microsomes. Control by selective product inhibition

Metabolism of benzo(a)pyrene (BP) and 7,8-dihydrodiol by 3-methylcholanthrene (MC)-induced rat liver microsomes are both subject to severe inhibition by primary metabolites of BP, which was analyzed by determining individual inhibition constants for all primary BP metabolites for both BP and 7,8-dihydrodiol metabolism. Monooxygenation of 7,8-dihydrodiol was, surprisingly, 5 to 10 times more sensitive than monooxygenation of BP to inhibition by all primary metabolites, even though both reactions require the same enzyme, cytochrome P-450c. Two representative products, 1,6-quinone and 9-phenol, were both strong, competitive inhibitors of BP metabolism with Ki values of 0.12 and 0.74 microM, respectively. The total effect of product inhibition on the overall reactions was determined by fitting progress curves of BP, 7,8-dihydrodiol, and anti-7,8-dihydrodiol 9,10-oxide (determined as 7,10/8,9-tetrol) over a range of BP concentrations to integrated steady-state equations using experimental Vmax and Km values. The effective product inhibition factors for BP and 7,8-dihydrodiol metabolism, determined from progress curve fits, were only 2-fold higher than the corresponding calculated theoretical values. The effective product inhibition factors, obtained from progress curve analysis, confirmed that 7,8-dihydrodiol metabolism was substantially more sensitive to inhibition by primary BP metabolites than BP metabolism itself. This difference probably reflects the much higher affinity of cytochrome P-450c for BP (Kd = 6 nM), as compared to 7,8-dihydrodiol (Kd = 175 nM) that was established spectrophotometrically both for the purified cytochrome and for MC microsomes. The Km for BP metabolism is 50 to 100 times higher than the Kd, while the Km is similar to the Kd for 7,8-dihydrodiol metabolism. The discrepancy for BP between Km and Kd suggests that standard Michaelis-Menten kinetics may be perturbed by either slow substrate or product dissociation.     

Product inhibition of benzo[a]pyrene metabolism in uninduced rat liver microsomes: Effect of diol epoxide formation

Conversion of benzo[a]pyrene (BP) to BP 7,8-dihydrodiol 9,10-oxides (DE) (measured as 7,10/8,9-tetrols) by untreated (UT) rat liver microsomes is over 10 times slower than following 3-methylcholanthrene (MC) induction. Time courses have been subjected to a kinetic analysis analogous to that previously reported for metabolism by MC-induced microsomes (J. Biol. Chem., 259 (1984) 13770–13776). Competition between BP and 7,8-dihydrodiol for P-450 is the major determinant of the rate of DE formation. Glucuronidation of quinones and phenols only increases the isolated BP metabolites including DE by 40%. This indicates far less inhibition by these products than for metabolism in MC-microsomes (4–6-fold). Thus stimulation may result from a decreased quinone-mediated oxidation of metabolites. In the presence of DNA, UT-microsomes metabolize BP to approximately equal amounts of 9-phenol-4,5-oxide (9-PO) and DE/DNA adducts. Addition of uridine diphosphoglucuronic acid (UDPGA) fails to enhance modification of DNA by DE, but formation of the 9-PO adduct is reduced as a result of lower free 9-phenol levels. The kinetic characteristics of BP metabolism by UT-microsomes are highly sensitive to the presence of very small but variable amounts (2–25 pmol/mg) of the very active cytochrome P-450_c, which is the predominant form in MC-microsomes. The major effect of elevated levels of P-450_c is an 8-fold increase in DE formation at low concentrations of BP due to a lowering of K_m (7.9–2.6 μM) and an increase in the regioselectivity for DE formation from 7,8-dihydrodiol (5–15% of total BP metabolites). The formation of DE was directly correlated with the content of P-450_c (r = 0.94). The presence of increased levels of P-450_c in UT-microsomes is probably due to previous exposure of the animals to environmental inducers and is minimized by controlled housing and feeding.     

Simple vs. complex inheritance of inducible aryl hydrocarbon hydroxylase in mouse tissues

The genetics of induction of hepatic and lung aryl hydrocarbon hydroxylase (AHH) have been studied in Af/Ki and AKR/Ki mice and in their F1 and F2 progeny after administration of 3-methylcholanthrene (3MC). Furthermore, the induction of AHH was investigated using the fetal liver explant model system with 3MC, trans-1,2-dihydroxy-3MC, and 4'-bromoflavone as the inducers. The results obtained with the above strains were contrasted with those from the C57BL/6Ki, DBA/2+Ki, and their crosses. The present investigation revealed a complex pattern of inheritance of basal and inducible AHH in lung and liver of AKR/Ki and Af/Ki, with a poor correlation between lung and liver. Hepatic AHH was not fully inducible in the F1 hybrids, while the frequency distribution function in the F2 mice was suggestive of more than two distinct classes.     

Mutagenicity of benzo[a]pyrene 7,8-dihydrodiol and 7,12-dimethylbenz[a]anthracene 3,4-dihydrodiol in S. typhimurium mediated by microsomes from rat liver and mouse skin

The mutagenic activities of trans-7,8-dihydro-7,8-dihydroxybenzo[a]-pyrene (BP 7,8-diol) and of trans-3,4-dihydroxy-7,12-dimethylbenz[a]-anthracene (DMBA 3,4-diol) towards S. typhimurium TA100 were measured in assays that were carried out on a micro-scale in liquid medium in the presence of microsomal fractions prepared from mouse skin or rat liver. In the presence of an NADPH-generating system, microsomal enzymes converted both diols into mutagens that were probably the respective 'bay-region' diol-epoxides. The rate of the enzyme-catalysed conversion of the BP 7,8-diol into mutagens by microsomal preparations from mouse epidermis was similar to that occurring with microsomes from rat liver. Pretreatment of mice by the topical application of benz[a]anthracene (BA) or 7,12-dimethylbenz[a]-anthracene (DMBA) increased the mutagenic activity of BP 7,8-diol mediated by mouse skin microsomal preparations by 2-fold and this was paralleled by a 4-fold increase in epidermal aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) activity. The results are discussed in relation to the high susceptibility of mouse skin to polycyclic aromatic hydrocarbon (PAH) carcinogenesis.     

Binding of metabolically activated benzo(a)pyrene to DNA and histones of rat liver,lung and regenerating liver

Binding of metabolically activated benzo(a)pyrene (BP) to the DNA and histones of nuclei isolated from rat liver, lung, and regenerating liver was examined. Separation of enzymically degraded DNA by Sephadex LH-20 chromatography resulted in several peaks of radioactivity and the major peak was probably derived from the binding of 7,8-diol-9,10-epoxide of BP with DNA. A high level of BP metabolites was also bound to the Hl histone fraction. The patterns of BP binding with the components of nuclei from the different cell types were similar. Implications of these observations as related to carcinogenic tissue susceptibility are discussed.     

Chiral stationary phase high-performance liquid chromatographic resolution and absolute configuration of enantiomeric benzo[a]pyrene diol-epoxides and tetrols

Enantiomers of diastereomeric benzo[a]pyrene (BP) diol-epoxides, r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro-BP (BP 7,8-diol-anti-9,10-epoxide), r-7,t-8-dihydroxy-c-9,10-epoxy-7,8,9,10-tetrahydro-BP (BP 7,8-diol-syn-9,10-epoxide), r-9,t-10-dihydroxy-t-7,8-epoxy-7,8,9,10-tetrahydro-BP (BP 9,10-diol-anti-7,8-epoxide), and several 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrenes (BP tetrols) were resolved by high-performance liquid chromatography (HPLC) using columns packed with either (R)-N-(3,5-dinitrobenzoyl)phenylglycine[(R)-DNBPG] or (S)-N-(3,5-dinitrobenzoyl)leucine [(S)-DNBL], which is either ionically or covalently bonded to gamma-aminopropylsilanized silica. Resolution of enantiomers was confirmed by ultraviolet-visible absorption and circular dichroism spectral analyses. Resolved enantiomers of BP diol-epoxides were each hydrolyzed in acidic solution to a pair of diastereomeric tetrols which were separated by reversed-phase HPLC. Absolute stereochemistries of enantiomeric diol-epoxides were deduced by the absolute configuration of their hydrolysis products.     

Biochemical basis for the acquisition of resistance to benzo[a]pyrene in clones of mouse liver cells in culture

In a Namru mouse liver epithelial cell strain designated NMuLi, aryl hydrocarbon hydroxylase (AHH) activity peaked at 12 h post-induction with 1 μg/ml of benzo(a)pyrene (BaP) in both confluent and growing cells. Maximal levels of AHH activity were reached on day two post-plating. This induced activity was inhibited in vitro 78% by gassing the incubation mixture with carbon monoxide for 15 s, and inhibited 93% by addition of 40 μg/ml of 7,8 benzoflavone(BF).Induced AHH levels were higher in epithelial clones that were sensitive to the toxicity of BaP than in resistant clones. The survival fraction of clones from NMuLi and of subclones derived from a sensitive clone of NMuLi after BaP treatment was a negative exponential function of the maximal induced AHH activity in the clones.One of the clones, NMuLi cl 8, was extremely susceptible to the toxic effects of BaP, the ±(trans)-7α, 8β-dihydroxy-7,8-dihydro-BaP(7,8-diol), and the (±)-7α,8β-dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydro-BaP (diolepoxide), known metabolites of BaP. The toxicity of BaP and the 7,8 diol to this clone was inhibited by BF, suggesting that these cells possessed an enzyme activity inhibitable by BF that could epoxidize BaP to the 7,8 oxide and then epoxidize the resultant 7,8 diol to the diol-epoxide. Another clone derived from NMuLi, clone 7, was relatively resistant to the toxic effects of BaP and the 7,8-diol, but still extremely susceptible to the toxic effects of the diol-epoxide. The slight toxicity to BaP in this clone was inhibited by BF, but the toxicity of the 7,8-diol to this clone was not inhibited by BF. A typical cytochrome P450 inhibitor, metyrapone, had no effect on the toxicity of BaP, the 7,8-diol, or the diol-epoxide to either clone 7 or clone 8.The results suggest that these liver cells possess two enzymes that play some role in polycyclic hydrocarbon-induced toxicity. Enzyme A, a BaP-inducible enzyme that is inhibitable by BF, efficiently metabolizes BaP to the 7,8-diol and the 7,8-diol to the diol-epoxide. It is responsible for most of the hydrocarbon toxicity. Enzyme B is not inhibitable by BF and metabolizes the 7,8-diol less efficiently to the diol-epoxide or efficiently to other, less toxic products.     

Stereoselective hydroxylation at the aliphatic carbons of 7,8- and 9,10-dihydrobenzo[a]pyrenes by rat liver microsomes

Optically active 7-hydroxy-7,8-dihydrobenzo[a]pyrene and 8-hydroxy-7,8-dihydrobenzo[a]pyrene were identified as two of the major metabolites formed by incubation of 7,8-dihydrobenzo[a]pyrene with rat liver microsomes. Optically active 9-hydroxy-9,10-dihydrobenzo[a]pyrene and 10-hydroxy-9,10-dihydrobenzo[a]pyrene were similarly identified as two of the minor metabolites of 9,10-dihydrobenzo[a]pyrene. The formation of these metabolites was abolished either by prior treatment of liver microsomes with carbon monoxide or the absence of NADPH, but was not inhibited by an epoxide hydrolase inhibitor. The results indicate that the aliphatic carbons of dihydro polycyclic aromatic hydrocarbons may undergo stereoselective hydroxylation reactions catalyzed by the cytochrome P-450 system of rat liver microsomes.