Metabolism of 3-hydroxychrysene by rat liver microsomal preparations.

3-Hydroxychrysene, a metabolite of the polycyclic aromatic hydrocarbon (PAH) chrysene, was metabolised by rat liver microsomal preparations obtained from Arochlor 1254-pretreated rats. Eight major metabolites were isolated by high performance liquid chromatography and characterised by u.v. spectroscopy and a variety of mass spectrometric techniques. The metabolites were unambiguously identified as 9-hydroxy-trans-1,2-dihydroxy-1,2-dihydrochrysene and 9-hydroxy-r-1,t-2,t-3,c-4-tetrahydroxy-1,2,3,4-tetrahydrochrysene and tentatively identified as 3-hydroxy-trans-5,6-dihydroxy-5,6-dihydrochrysene (since chrysene is a symmetrical molecule the 3- and 9-positions are equivalent), 9-hydroxy-trans-3,4-dihydroxy-3,4-dihydrochrysene, 1,2,3-trihydroxy-1,2,3,4-tetrahydrochrysene, an oxidised phenol and two diphenols. These results indicate that 3-hydroxychrysene can be further metabolised via a number of different pathways including those involving the formation of phenol- and triol-epoxides.

Metabolism of the bay-region diol-epoxide of chrysene to a triol-epoxide and the enzyme-catalysed conjugation of these epoxides with glutathione.

Metabolic activation of chrysene in mouse skin appears to involve r-1,t-2-dihydroxy-t-3,4-oxy-1,2,3,4-tetrahydrochrysene (anti-chrysene-1,2-diol 3,4-oxide) and 9-hydroxy-r-1,t-2-dihydroxy-t-3,4-oxy-1,2,3,4-tetrahydrochrysene (anti-9-OH-chrysene-1,2-diol 3,4-oxide). The enzyme-catalysed conjugation of these epoxides with [35S]glutathione has been studied in experiments in which the glutathione conjugates were separated by h.p.l.c. and examined by fluorescence spectrophotometry. Both anti-chrysene-1,2-diol 3,4-oxide and anti-9-OH-chrysene-1,2-diol 3,4-oxide formed conjugates nonenzymically and both were shown to be substrates for rat liver glutathione transferases. When anti-chrysene-1,2-diol 3,4-oxide was incubated with [35S]glutathione and a rat liver microsomal metabolizing system, glutathione conjugates with h.p.l.c. and fluorescence spectral characteristics identical to those of conjugates formed from both anti-chrysene-1,2-diol 3,4-oxide and anti-9-OH-chrysene-1,2-diol 3,4-oxide were detected. This finding provides evidence that anti-chrysene-1,2-diol 3,4-oxide can be further metabolized to the triol-epoxide, anti-9-OH-chrysene-1,2-diol 3,4-oxide by rat liver microsomal systems.

Mutagenic potential of DNA adducts formed by diol-epoxides, triol-epoxides and the K-region epoxide of chrysene in mammalian cells.

The syn- and anti-isomers of chrysene-1,2-diol-3,4-oxide (syn-diol-epoxide and anti-diol-epoxide) and of 9-hydroxychrysene-1,2-diol-3,4-oxide (syn-triol-epoxide and anti-triol-epoxide), and chrysene-5,6-oxide, the K-region epoxide, were tested for their ability to induce 6-thioguanine-resistant mutants in V79 Chinese hamster cells. The levels of DNA adducts formed by each compound in the V79 cells were determined by 32P-post-labelling analysis. The most potent mutagen, in terms of the mutation frequency/nmol compound administered, was the anti-triol-epoxide, which was 1.7 times as active as the anti-diol-epoxide. The anti-diol-epoxide was approximately 10 times more active than both the syn-triol-epoxide and the syn-diol-epoxide, which in turn were several times more active than the K-region epoxide. However, when the results were expressed as mutations/pmol total adducts formed, the anti-triol-epoxide and anti-diol-epoxide were shown to be of similar potency and approximately twice as active as the other three compounds. Thus differences in the conformation of adducts formed with DNA by syn- and anti-isomers may be responsible for their different mutagenic potentials; the presence of a phenolic OH-group at the 9-position of a chrysene-1,2-diol-3,4-oxide appears to increase its chemical reactivity.

Mutagenic and cell-transforming activities of triol-epoxides as compared to other chrysene metabolites.

The syn- and anti-isomers of the bay-region diol-epoxides of chrysene and of 3-hydroxychrysene and their metabolic precursors have been investigated for mutagenicity in Salmonella typhimurium (reversion to histidine prototrophy) and V79 Chinese hamster cells (acquirement of resistance to 6-thioguanine) and for transforming activity in M2 mouse prostate cells. Other known and potential chrysene metabolites have been included in mutagenicity experiments. Direct mutagenic activity in S. typhimurium TA 100 exhibited, in order of potency, anti-triol-epoxide greater than syn-triol-epoxide greater than anti-diol-epoxide greater than syn-diol-epoxide greater than chrysene 5,6-oxide much greater than chrysene-1,2-quinone, chrysene-3,4-quinone, and chrysene 5,6-quinone. Chrysene, the six isomeric chrysenols, and the trans-dihydrodiols [trans-1,2-dihydroxy-1,2-dihydrochrysene (chrysene-1,2-diol), trans-3,4-dihydroxy-3,4-dihydrochrysene, trans-5,6-dihydroxy-5,6-dihydrochrysene, and 9-hydroxy-trans-1,2-dihydroxy-1,2-dihydrochrysene (9-hydroxychrysene-1,2-diol)] were inactive per se but were activated to mutagens in the presence of reduced nicotinamide adenine dinucleotide phosphate-fortified postmitochondrial fraction (S9 mix) of liver homogenate from Arochlor 1254-treated rats. Chrysene, 3-hydroxychrysene, chrysene-1,2-diol, and 9-hydroxychrysene-1,2-diol were activated efficiently; the other compounds were activated weakly. In S. typhimurium TA 98, the mutagenic activities of the chrysene derivatives were weak in comparison with those in the strain TA 100. trans-3,4-Dihydroxy-3,4-dihydrochrysene (in the presence of S9 mix) was the most efficacious mutagen in strain TA 98. The relative mutagenic potencies of the directly active compounds differed from the results obtained in strain TA 100, in that in strain TA 98 the anti-diol-epoxide was more mutagenic than the triol-epoxides and chrysene 5,6-oxide was more mutagenic than syn-diol-epoxide and syn-triol-epoxide. In V79 cells, the order of mutagenic potency was: anti-triol-epoxide greater than anti-diol-epoxide greater than syn-triol-epoxide greater than syn-diol-epoxide greater than chyrsene 5,6-oxide greater than chrysene-1,2-diol (in the presence of S9 mix) greater than 9-hydroxychrysene-1,2-diol (in the presence of S9 mix) greater trans-3,4-dihydroxy-3,4-dihydrochrysene in the presence of S9 mix). Chrysene, 3-hydroxychrysene, 5-hydroxychrysene, and 6-hydroxychrysene showed no mutagenic effects in V79 cells, either in the presence or absence of S9 mix.(ABSTRACT TRUNCATED AT 400 WORDS)

The formation of 9-hydroxychrysene-1,2-diol as an intermediate in the metabolic activation of chrysene.

9-Hydroxy-trans-1,2-dihydro-1,2-dihydroxychrysene (9-hydroxychrysene-1,2-diol), which may be the triol involved in the formation of a chrysene triol-epoxide-DNA adduct in mouse skin, was not detected when chrysene was incubated with rat-liver microsomal preparations. In separate experiments an excess of synthetic 9-hydroxychrysene-1,2-diol was added during the incubation of 3H-labelled chrysene with rat-liver microsomes and was then re-isolated. The triol was found to contain a radioactive product that had chromatographic properties identical to those of 9-hydroxychrysene-1,2-diol when examined by reverse-phase h.p.l.c., both before and after acetylation, by normal-phase h.p.l.c. and by t.l.c. both before and after oxidation. When treated with m-chloroperoxybenzoic acid, the synthetic 9-hydroxychrysene-1,2-diol formed products that possessed alkylating activity and that reacted with DNA in vitro. Examination of the triol-epoxides produced by oxidation of a mixture of synthetic and metabolic 9-hydroxychrysene-1,2-diol by t.l.c. suggested that the anti-isomer was formed.