The formation of dihydrodiols from benzo[alpha]pyrene by oxidation with an ascorbic acid/ferrous sulphate/EDTA system.

In the oxidation of benzo[alpha]pyrene in an abscorbic acid-ferrous sulphate-EDTA system, four dihydrodiols were detected. Three, trans-4,5-dihydro-4,5-dihydroxybenzo[alpha]pyrene, trans-7,8-dihydro-7,8-dihydroxybenzo[alpha]pyrene and trans-9,10-dihydro-9,10-dihydroxybenzo[alpha]pyrene were identified by their UV spectra and by direct comparisons of their chromatographic properties, using HPLC, with those of the authentic compounds. The fourth compound appeared to be trans-11,12-dihydro-11,12-dihydroxybenzo[alpha]pyrene since its ultraviolet spectrum was identical to that of the cis-dihydrodiol. Time-course experiments showed that the maximum amounts of products were obtained after 8 h of oxidation. A re-examination of the dihydrodiols formed from benzo[alpha]pyrene by rat-liver microsomal fractions failed to show the formation of the trans-11,12-dihydrodiol.     

The metabolism of 3-methylcholanthrene and some related compounds by rat-liver homogenates

1. A chromatographic investigation of the products of the metabolism of 3-methylcholanthrene by rat-liver homogenates showed the formation of compounds with the properties of 1- and 2-hydroxy-3-methylcholanthrene, cis- and trans-1,2-dihydroxy-3-methylcholanthrene and 11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene. A glutathione conjugate that is probably S-(11,12-dihydro-12-hydroxy-3-methyl-11-cholanthrenyl)glutathione was also detected. 3-Methylcholanthrene-1- and -2-one and -1,2-quinone were also present, but these products may have arisen by the chemical oxidation of the corresponding hydroxy compounds. 2. Other metabolic products were tentatively identified as 9- and 10-hydroxy-3-methylcholanthrene, 4,5-dihydro-4,5-dihydroxy-3-methylcholanthrene and 3-hydroxymethylcholanthrene. 3. 1- and 2-Hydroxy-3-methylcholanthrene were converted by homogenates into the related ketones and into products with the properties of cis- and trans-1,2-dihydroxy-3-methylcholanthrene: 3-methylcholanthren-1- and -2-one were converted into their related hydroxy compounds and into the isomeric 1,2-dihydroxy compounds. The isomeric 1,2-dihydroxy compounds were each partly converted into the other isomer by these homogenates. All the above substrates also yielded products that appeared to be derivatives of 3-hydroxymethylcholanthrene. 4. 3-Methylcholanthrylene was converted by rat-liver homogenates into products with the properties of trans-1,2-dihydroxy-3-methylcholanthrene, 2-hydroxy-3-methylcholanthrene and 3-methylcholanthren-2-one. A small amount of the cis-1,2-dihydroxy compound was also formed, together with a glutathione conjugate that is possibly S-(2-hydroxy-3-methyl-1-cholanthrenyl)glutathione or its positional isomer. 5. An unidentified product was detected in the metabolism of 3-methylcholanthrene, the monohydroxy compounds, the ketones and the dihydroxy compounds, the formation of which appeared to involve metabolism at the 1,2-bond. 6. 11,12-Epoxy-11,12-dihydro-3-methylcholanthrene was converted by rat-liver homogenates into products with the properties of 11-hydroxy-3-methylcholanthrene (or, less likely, the 12-isomer), 11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene and the glutathione conjugate described above. Products with the properties of these compounds were formed when the epoxide was allowed to react with glutathione in an aqueous medium. 7. Mouse-liver homogenate converted 3-methylcholanthrene into products with the chromatographic properties of 1- and 2-hydroxy-3-methylcholanthrene, cis- and trans-1,2-dihydroxy-3-methylcholanthrene, 11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene, 3-methylcholanthrene-1- and -2-one and -1,2-quinone and the unidentified hydroxy-3-methylcholanthrenes. 8. The syntheses of cis- and trans-1,2-dihydroxy-3-methylcholanthrene, 3-methylcholanthren-2-one, 2-hydroxy-3-methylcholanthrene, 3-methylcholanthrylene, 11,12-epoxy-11,12-dihydro-3-methylcholanthrene and trans-11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene are described.     

The formation of dihydrodiols by the chemical or enzymic oxidation of benz[a] anthracene and 7,12-dimethylbenz[a] anthracene.

When benz[a] anthracene was oxidised in a reaction mixture containing ascorbic acid, ferrous sulphate and EDTA, the non-K-region dihydrodiols, trans-1,2-dihydro-1,2-dihydroxybenz[a] anthracene and trans-3,4-dihydro-3,4-dihydroxybenz[a] anthracene together with small amounts of the 8,9- and 10,11-dihydrodiols were formed. When oxidised in a similar system, 7,12-dimethylbenz[a] anthracene yielded the K-region dihydrodiol, trans-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz[a] anthracene and the non-K-region dihydrodiols, trans-3,4-dihydro-3,4-dihydroxy-7,12-dimethylbenz[a] anthracene, trans-8,9-dihydro-8,9-dihydroxy-7,12-dimethylbenz[a] anthracene, trans-10,11-dihydro-10,11-dihydroxy-7,12-dimethylbenz[a] anthracene and a trace of the 1,2-dihydrodiol. The structures and sterochemistry of the dihydrodiols were established by comparisons of their UV spectra and chromatographic characteristics using HPLC with those of authentic compounds or, when no authentic compounds were available, by UV, NMR and mass spectral analysis. An examination by HPLC of the dihydrodiols formed in the metabolism, by rat-liver microsomal fractions, of benz[a] anthracene and 7,12-dimethylbenz[a] anthracene was carried out. The metabolic dihydriols were identified by comparisons of their chromatographic and UV or fluorescence spectral characteristics with compounds of known structures. The principle metabolic dihydriols formed from both benz[a] anthracene and 7,12-dimethylbenz[a] anthracene were the trans-5,6- and trans-8,9-dihydrodiols. The 1,2- and 10,11-dihydrodiols were identified as minor products of the metabolism of benz [a] anthracene and the tentative identification of the trans-3,4-dihydriol as a metabolite was made from fluorescence and chromatographic data. The minor metabolic dihydriols formed from 7,12-dimethylbenz[a] anthracene were the trans-3,4-dihydrodiol and the trans-10,11-dihydriol but the trans-1,2-dihydrodiol was not detected in the present study.     

Degradation of benzo[a]pyrene by Mycobacterium vanbaalenii PYR-1

Metabolism of the environmental pollutant benzo[a]pyrene in the bacterium Mycobacterium vanbaalenii PYR-1 was examined. This organism initially oxidized benzo[a]pyrene with dioxygenases and monooxygenases at C-4,5, C-9,10, and C-11,12. The metabolites were separated by reversed-phase high-performance liquid chromatography (HPLC) and characterized by UV-visible, mass, nuclear magnetic resonance, and circular dichroism spectral analyses. The major intermediates of benzo[a]pyrene metabolism that had accumulated in the culture media after 96 h of incubation were cis-4,5-dihydro-4,5-dihydroxybenzo[a]pyrene (benzo[a]pyrene cis-4,5-dihydrodiol), cis-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (benzo[a]pyrene cis-11,12-dihydrodiol), trans-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (benzo[a]pyrene trans-11,12-dihydrodiol), 10-oxabenzo[def]chrysen-9-one, and hydroxymethoxy and dimethoxy derivatives of benzo[a]pyrene. The ortho-ring fission products 4-formylchrysene-5-carboxylic acid and 4,5-chrysene-dicarboxylic acid and a monocarboxylated chrysene product were formed when replacement culture experiments were conducted with benzo[a]pyrene cis-4,5-dihydrodiol. Chiral stationary-phase HPLC analysis of the dihydrodiols indicated that benzo[a]pyrene cis-4,5-dihydrodiol had 30% 4S,5R and 70% 4R,5S absolute stereochemistry. Benzo[a]pyrene cis-11,12-dihydrodiol adopted an 11S,12R conformation with 100% optical purity. The enantiomeric composition of benzo[a]pyrene trans-11,12-dihydrodiol was an equal mixture of 11S,12S and 11R,12R molecules. The results of this study, in conjunction with those of previously reported studies, extend the pathways proposed for the bacterial metabolism of benzo[a]pyrene. Our study also provides evidence of the stereo- and regioselectivity of the oxygenases that catalyze the metabolism of benzo[a]pyrene in M. vanbaalenii PYR-1.     

Epoxy derivatives of aromatic polycyclic hydrocarbons. The preparation and metabolism of epoxides related to benzo[a]pyrene and to 7,8- and 9,10-dihydrobenzo[a]pyrene

Products that appeared to be mainly benzo[a]pyrene 7,8-oxide and benzo[a]pyrene 9,10-oxide were synthesized and their chemical and biochemical properties were investigated. The oxides were unstable and readily rearranged to phenols. They were converted by rat liver homogenates and microsomal preparations into phenols and dihydrodiols, but glutathione conjugates were not formed in appreciable amounts. The dihydrodiols formed from benzo[a]pyrene 7,8- and 9,10-oxide by rat liver microsomal preparations were identical in their chromatographic and spectrographic properties with dihydrodiols formed when benzo[a]pyrene was metabolized by rat liver homogenates. 9,10-Dihydrobenzo[a]pyrene 7,8-oxide and 7,8-dihydrobenzo[a]pyrene 9,10-oxide were also synthesized. They were converted by rat liver homogenates and microsomal preparations into the related cis- and trans-dihydroxy compounds. Glutathione conjugates were formed from the oxides by rat liver homogenates. Both 7,8- and 9,10-dihydrobenzo[a]pyrene were metabolized by rat liver homogenates to mainly the trans-isomers of the related dihydroxy compounds. In experiments with boiled homogenates, the benzo[a]pyrene oxides were converted into phenols, whereas the dihydrobenzo[a]pyrene oxides yielded small amounts of the related dihydroxy compounds.     

Identification of metabolites of benzo[j]fluoranthene formed in vitro in rat liver homogenate

The metabolites of benzo[j]fluoranthene (BjF) as formed in vitro using the 9000 X g supernatant from Aroclor-pretreated rats have been identified. Two dihydrodiols, trans-4,5-dihydro-4,5-dihydroxyBjF and trans-9,10-dihydro-9,10-dihydroxyBjF have been identified as major metabolites by comparison of their spectral and chromatographic properties with those of pure synthetic standards. There was no evidence that any of the isomeric 2,3-dihydrodiol was formed as a metabolite of BjF under these incubation conditions. Neither of the metabolic dihydrodiols of BjF were formed with a high degree of stereoselectivity. The enantiomeric purity of the 4,5-dihydrodiol was 20% while that of the 9,10-dihydrodiol was 46%. At least four phenols were detected among the metabolites of BjF. These were identified as 3-, 4-, 6- and 10-hydroxyBjF based upon comparison of their UV spectra and HPLC retention times with those of synthetic reference standards. BjF-4,5-dione was also identified as a metabolite under these incubation conditions.     

Metabolism of benzo(a)pyrene by a dioxygenase enzyme system of the freshwater green alga Selenastrum capricornutum

The green alga Selenastrum capricornutum was incubated with benzo(a)-pyrene under an atmosphere of 20% (18)O2: 80% N2. The cis-11,12-dihydro-11,12-dihydroxybenzo(a)pyrene, cis-7,8-dihydro-7,8-dihydroxybenzo(a)pyrene and cis-4,5-dihydro-4,5-dihydroxybenzo(a)pyrene, were isolated by HPLC and analyzed by mass spectrometry. The metabolites produced molecular ions at m/z 290 and 286. Elemental analysis of the ion at m/z 290 gave an elemental composition of C20H14(18)O2 with 13% (18)O2 incorporation. The results indicate that S. capricornutum produces cis vicinal dihydrodiols from molecular oxygen via a dioxygenase enzyme pathway. The dioxygenase enzymes are characteristic of the bacterial metabolic pathway and unlike those of eukaryotic organisms which involve monooxygenase enzymes.     

Fluorescence study of DNA-complexes formed after metabolic activation of benzo(a)pyrene derivatives

Benzo(a)pyrene derivatives (1-, 2-, 3-, 7-, and 9-hydroxy-benzo(a)pyrene and trans-9,10-dihydro-9,10-dihydroxy-, -4,5-dihydro-4,5-dihydroxy-, and -7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene) were metabolized by liver microsomes isolated from 3-methylcholanthrene-treated rats in the presence of calf thymus DNA. The isolated DNA was then assayed by fluorescence for bound metabolic products. Only 2-hydroxy-benzo(a)pyrene, 9-hydroxy-benzo(a)pyrene and trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene yielded detectable amounts of DNA-bound products. In contrast to the product(s) from 9-hydroxy-benzo(a)pyrene, the metabolites of 2-hydroxy-benzo(a)pyrene and trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene, both strong carcinogens, had similar excitation spectra and gave considerably increased fluorescence intensities when the DNA was denatured. These data indicate structural similarities in the DNA complexes formed after metabolic activation of 2-hydroxy-benzo(a)pyrene and trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene.     

Metabolic activation of 3-methylcholanthrene: mutagenic and transforming activities of the 9,10-dihydrodiol.

3-Methylcholanthrene and five related dihydrodiols have been tested for microsome-mediated mutagenicity towards $\text{Salmonella}\text{typhimurium}$ TA100 and for the induction of mutation to 8-azaguanine resistance in V79 Chinese hamster cells and malignant transformation in M2 mouse fibroblasts. In both mutagenicity test systems, the 9,10-diol was considerably more active than either the parent hydrocarbon, the related $\text{cis}$-2α,3-diol, the $\text{trans}$-4,5-, the $\text{trans}$-7,8- or the $\text{trans}$-11,12-dihydrodiols. At a non-toxic concentration (1μg/ml medium), the 9,10-diol induced the formation of more transformed malignant foci in cultures of M2 cells than 3-methylcholanthrene and the other diols were either inactive or only weakly active in this test system. The results obtained indicate that the 9,10-dihydrodiol derived from 3-methylcholanthrene is involved, presumably following conversion into the corresponding vicinal diol-epoxide, 9,10-dihydro-9,10-dihydroxy-3-methylcholanthrene 7,8-oxide, in the metabolic activation of this carcinogenic polycyclic hydrocarbon.     

Effects of a 6-fluoro substituent on the metabolism of benzo(a)pyrene 7,8-dihydrodiol to bay-region diol epoxides by rat liver enzymes

Metabolism of trans-7,8-dihydroxy-7,8-dihydro-6-fluorobenzo(a)pyrene by liver microsomes from 3-methylcholanthrene-treated rats and by a highly purified monooxygenase system, reconstituted with cytochrome P-450c, has been examined. Although both the fluorinated and unfluorinated 7,8-dihydrodiol formed from benzo(a)pyrene by liver microsomes share (R,R)-absolute configuration, the fluorinated dihydrodiol prefers the conformation in which the hydroxyl groups are pseudodiaxial due to the proximate fluorine. The fluorinated 4,5- and 9,10-dihydrodiols are also greater than 97% the (R,R)-enantiomers. For benzo(a)pyrene, metabolism of the (7R,8R)-dihydrodiol to a bay-region 7,8-diol-9,10-epoxide in which the benzylic hydroxyl group and epoxide oxygen are trans constitutes the only known pathway to an ultimate carcinogen. With the microsomal and the purified monooxygenase system, this pathway accounts for 76-82% of the total metabolites from the 7,8-dihydrodiol. In contrast, only 32-49% of the corresponding diol epoxide is obtained from the fluorinated dihydrodiol and this fluorinated diol epoxide has altered conformation in that its hydroxyl groups prefer to be pseudodiaxial. Much smaller amounts of the diastereomeric 7,8-diol-9,10-epoxides in which the benzylic hydroxyl groups and the epoxide oxygen are cis are formed from both dihydrodiols. As the fluorinated diol epoxides are weaker mutagens toward bacteria and mammalian cells relative to the unfluorinated diol epoxides, conformation appears to be an important determinant in modulating the biological activity of diol epoxides. One of the more interesting metabolites of 6-fluorinated 7,8-dihydrodiol was a relatively stable arene oxide, probably the 4,5-oxide, which is resistant to the action of epoxide hydrolase.     

Metabolic activation of benzo[a]pyrene in human skin maintained in short-term organ culture

The metabolic activation of benzo[a]pyrene (BP) was examined in six samples of human skin after topical application of the hydrocarbon to the skin in short-term organ culture. The results show that all of the samples were capable of metabolizing BP to water-soluble products and to ether-soluble products that included the 4,5-, 7,8- and 9,10-dihydrodiols and a product which had chromatographic properties identical with those of authentic trans-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (BP-11,12-diol). The major BP-deoxyribonucleoside adduct detected in each skin sample appeared to be formed from the reaction of r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BP-7,8-diol 9,10-oxide) with deoxyguanosine residues in DNA.     

Enantioselective aliphatic hydroxylations of racemic 1-hydroxy-3-methylcholanthrene by rat liver microsomes

Enantiomeric pairs of 1-hydroxy-3-hydroxymethylcholanthrene (1-OH-3-OHMC), 3-methylcholanthrene (3MC) trans- and cis-1,2-diols, and 1-hydroxy-3-methylcholanthrene (1-OH-3MC) were resolved by HPLC using a covalently bonded (R)-N-(3,5-dinitrobenzoyl)phenylglycine chiral stationary phase (Pirkle type 1A) column. The absolute configuration of an enantiomeric 3MC trans-1,2-diol was established by the exciton chirality CD method following conversion to a bis-p-N,N-dimethylaminobenzoate. Incubation of an enantiomeric 1-OH-3MC with rat liver microsomes resulted in the formation of enantiomeric 3MC trans- and cis-1,2-diols; the absolute configurations of the enantiomeric 1-OH-3MC and 3MC cis-1,2-diol were established on the basis of the absolute configuration of an enantiomeric 3MC trans-1,2-diol. Absolute configurations of enantiomeric 1-OH-3-OHMC were determined by comparing their CD spectra with those of enantiomeric 1-OH-3MC. The relative amount of three aliphatic hydroxylation products formed by rat liver microsomal metabolism of racemic 1-OH-3MC was 1-OH-3-OHMC greater than 3MC cis-1,2-diol greater than 3MC trans-1,2-diol. Enzymatic hydroxylation at C2 of racemic 1-OH-3MC was enantioselective toward the 1S-enantiomer over the 1R-enantiomer (approximately 3/1); hydroxylation at the C3-methyl group was enantioselective toward the 1R-enantiomer over the 1S-enantiomer (approximately 58/42). Rat liver microsomal C2-hydroxylation of racemic 1-OH-3MC resulted in a 3MC trans-1,2-diol with a (1S,2S)/(1R,2R) ratio of 63/37 and a 3MC cis-1,2-diol with a (1S,2R)/(1R,2S) ratio of 12/88, respectively.     

Stereoselective metabolism of anthracene and phenanthrene by the fungus Cunninghamella elegans.

The fungus Cunninghamella elegans oxidized anthracene and phenanthrene to form predominately trans-dihydrodiols. The metabolites were isolated by reversed-phase high-pressure liquid chromatography for structural and conformational analyses. Comparison of the circular dichroism spectrum of the fungal trans-1,2-dihydroxy-1,2-dihydroanthracene to that formed by rat liver microsomes indicated that the major enantiomer of the trans-1,2-dihydroxy-1,2-dihydroanthracene formed by C. elegans had an S,S absolute stereochemistry, which is opposite to the predominately 1R,2R dihydrodiol formed by rat liver microsomes. C. elegans oxidized phenanthrene primarily in the 1,2-positions to form trans-1,2-dihydroxy-1,2-dihydrophenanthrene. In addition, a minor amount of trans-3,4-dihydroxy-3,4-dihydrophenanthrene was detected. Metabolism at the K-region (9,10-positions) of phenanthrene was not detected. Comparison of the circular dichroism spectra of the phenanthrene trans-1,2- and trans-3,4-dihydrodiols formed by C. elegans to those formed by mammalian enzymes indicated that each of the dihydrodiols formed by C. elegans had an S,S absolute configuration. The results indicate that there are differences in both the regio- and stereoselective metabolism of anthracene and phenanthrene between the fungus C. elegans and rat liver microsomes.     

Regio- and stereoselective metabolism of 7,12-dimethylbenz[a]anthracene by Mycobacterium vanbaalenii PYR-1

The degradation of 7,12-dimethylbenz[a]anthracene (DMBA), a carcinogenic polycyclic aromatic hydrocarbon, by cultures of Mycobacterium vanbaalenii PYR-1 was studied. When M. vanbaalenii PYR-1 was grown in the presence of DMBA for 136 h, high-pressure liquid chromatography (HPLC) analysis showed the presence of four ethyl acetate-extractable compounds and unutilized substrate. Characterization of the metabolites by mass and nuclear magnetic resonance spectrometry indicated initial attack at the C-5 and C-6 positions and on the methyl group attached to C-7 of DMBA. The metabolites were identified as cis-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz[a]anthracene (DMBA cis-5,6-dihydrodiol), trans-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz[a]anthracene (DMBA trans-5,6-dihydrodiol), and 7-hydroxymethyl-12-methylbenz[a]anthracene, suggesting dioxygenation and monooxygenation reactions. Chiral stationary-phase HPLC analysis of the dihydrodiols showed that DMBA cis-5,6-dihydrodiol had 95% 5S,6R and 5% 5R,6S absolute stereochemistry. On the other hand, the DMBA trans-5,6-dihydrodiol was a 100% 5S,6S enantiomer. A minor photooxidation product, 7,12-epidioxy-7,12-dimethylbenz[a]anthracene, was also formed. The results demonstrate that M. vanbaalenii PYR-1 is highly regio- and stereoselective in the degradation of DMBA.     

Morphological transforming activity and metabolism of cyclopenta-fused isomers of benz[a]anthracene in mammalian cells

4 isomeric cyclopenta-derivatives of benz[e]anthracene (benz[a]aceanthrylene, benz[j]aceanthrylene, benz[l]aceanthrylene, and benz[k]acephenanthrylene) were examined for their ability to morphologically transform C3H10T1/2CL8 mouse-embryo fibroblasts. All of these polycyclic aromatic hydrocarbons studied except benz[k]acephenanthrylene transformed C3H10T1/2CL8 cells to both type II and type III foci in a concentration-dependent fashion. Benz[j]aceanthrylene was the most active, equivalent in activity to benzo[a]pyrene on a molar basis, in producing dishes of cells with transformed foci (94% at 1.0 microgram/ml). Benz[e]aceanthrylene, and benz[l]aceanthrylene produced 58% and 85% of the dishes with foci respectively at 10 micrograms/ml. Metabolism studies with [3H]benz[j]aceanthrylene in C3H10T1/2CL8 cells in which unconjugated, glucuronic acid conjugated, and sulfate conjugated metabolites were measured indicated that the dihydrodiol precursor to the bay-region diol-epoxide, 9,10-dihydroxy-9,10-dihydrobenz[j]aceanthrylene, was the major dihydrodiol formed (55%). Smaller quantities of the cyclopenta-ring dihydrodiol, 1,2-dihydroxy-1,2-dihydrobenz[j]aceanthrylene (14%), and the k-region dihydrodiol, 11,12-dihydroxy-11,12-dihydrobenz[j]aceanthrylene (5%) were also formed. Similar studies with [14C]benz[l]aceanthrylene indicated that the k-region dihydrodiol, 7,8-dihydroxy-7,8-dihydrobenz[l]aceanthrylene was the major metabolite formed (45%). The cyclopenta-ring dihydrodiol, 1,2-dihydroxy-1,2-dihydrobenz[l]aceanthrylene and 4,5-dihydroxy-4,5-dihydrobenz[l]aceanthrylene were formed in minor amounts (less than 6%). Therefore, metabolism at the cyclopenta-ring of B(j)A and B(l)A is a minor pathway in C3H10T1/2CL8 cells in contrast to previously reported studies with cyclopenta[cd]pyrene in which the cyclopenta-ring dihydrodiol was the major metabolite. These results suggest that routes of metabolic activation other than oxidation at the cyclopenta-ring such as bay region or k-region activation may play an important role with these unique polycyclic aromatic hydrocarbons in C3H10T1/2CL8 cells.     

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.     

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.     

Biotransformation of various substituted aromatic compounds to chiral dihydrodihydroxy derivatives

The biotransformation of four different classes of aromatic compounds by the Escherichia coli strain DH5alpha(pTCB 144), which contained the chlorobenzene dioxygenase (CDO) from Pseudomonas sp. strain P51, was examined. CDO oxidized biphenyl as well as monochlorobiphenyls to the corresponding cis-2,3-dihydro-2,3-dihydroxy derivatives, whereby oxidation occurred on the unsubstituted ring. No higher substituted biphenyls were oxidized. The absolute configurations of several monosubstituted cis-benzene dihydrodiols formed by CDO were determined. All had an S configuration at the carbon atom in meta position to the substituent on the benzene nucleus. With one exception, the enantiomeric excess of several 1,4-disubstituted cis-benzene dihydrodiols formed by CDO was higher than that of the products formed by two toluene dioxygenases. Naphthalene was oxidized to enantiomerically pure (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. All absolute configurations were identical to those of the products formed by toluene dioxygenases of Pseudomonas putida UV4 and P. putida F39/D. The formation rate of (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene was significantly higher (about 45 to 200%) than those of several monosubstituted cis-benzene dihydrodiols and more than four times higher than the formation rate of cis-benzene dihydrodiol. A new gas chromatographic method was developed to determine the enantiomeric excess of the oxidation products.     

Dual-label high-performance liquid chromatographic assay for femtomole levels of benzo[a]pyrene metabolites

A dual-label HPLC assay to measure femtomole quantities of ethyl acetate-extractable [3H]benzo[a]pyrene metabolites was developed. 14C-labeled metabolites of benzo[a]pyrene formed by rat liver 9000g supernatant were used as both internal standards and chromatographic markers. The percentage deviation between assays was determined to be between 11 and 13% for 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene, 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, benzo[a]pyrene-3,6-quinone, benzo[a]pyrene-1,6-quinone, and 9-hydroxybenzo[a]pyrene, 22% for 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene, and less than 5% for 3-hydroxybenzo[a]pyrene. The detection limit of this assay was between 3 and 10 fmol per metabolite. The application of this technique to the metabolism of [3H]benzo[a]pyrene by microsomes of hamster and human oral cavity tissue is described.     

Effects of a fluoro substituent on the fungal metabolism of 1-fluoronaphthalene.

The metabolism of 1-fluoronaphthalene by Cunninghamella elegans ATCC 36112 was studied. The metabolites were isolated by reverse-phase high-pressure liquid chromatography and characterized by the application of UV absorption, 1H nuclear magnetic resonance, and mass spectral techniques. C. elegans oxidized 1-fluoronaphthalene predominantly at the 3,4- and 5,6-positions to form trans-3,4-dihydroxy-3,4-dihydro-1-fluoronaphthalene and trans-5,6-dihydroxy-5,6-dihydro-1-fluoronaphthalene. In addition, 1-fluoro-8-hydroxy-5-tetralone, 5-hydroxy-1-fluoronaphthalene, and 4-hydroxy-1-fluoronaphthalene as well as glucoside, sulfate, and glucuronic acid conjugates of these phenols were formed. Circular dichroism spectra of the trans-3,4- and trans-5,6-dihydrodiols formed from 1-fluoronaphthalene indicated that the major enantiomers of the dihydrodiols have S,S absolute stereochemistries. In contrast, the trans-5,6-dihydrodiol formed from 1-fluoronaphthalene from 3-methylcholanthrene-treated rats had Cotton effects that are opposite in sign (R,R) to those formed by C. elegans. The results indicate that the fungal monooxygenase-epoxide hydrolase systems are highly stereoselective in the metabolism of 1-fluoronaphthalene and that a fluoro substituent blocks epoxidation at the fluoro-substituted double bond, decreases oxidation at the aromatic double bond that is peri to the fluoro substituent, and enhances metabolism at the 3,4- and 5,6-positions of 1-fluoronaphthalene.