MUTAGENIC SELECTIVITY AT THE HPRT LOCUS IN V-79 CELLS - COMPARISON OF MUTATIONS CAUSED BY BAY-REGION BENZO[A]PYRENE 7,8-DIOL-9,10-EPOXIDE ENANTIOMERS WITH HIGH AND LOW CARCINOGENIC ACTIVITY

Earlier studies from our laboratories characterized the mutation profile of the optically active (+)-7R,8S-dihydroxy-9S,10R -epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-BPDE-the ultimate carcinogenic metabolite of benzo[a]pyrene] in the coding region of the hypoxanthine (guanine) phosphoribosyltransferase (HPRT) gene of Chinese hamster V-79 cells. In the present study, we evaluated the mutation profile of (-)-7S,8R-dihydroxy-9R, 10S-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-BPDE-a weakly carcinogenic or inactive enantiomer] and compared its mutation profile with that of (+)-BPDE. In both diol epoxide enantiomers, the benzylic 7-hydroxy group and epoxide oxygen are trans. The mutation frequency for V-79 cells treated with DMSO vehicle or with a low non-cytotoxic dose (0.5 mu M) or a high cytotoxic dose (2.0 mu M) of (-)-BPDE was 1,25 or 185 8-azaguanine-resistant colonies/10(5) survivors, respectively. Independent 8-azaguanine-resistant clones were isolated, and complementary DNAs were prepared by reverse transcription. The coding region of the HPRT gene was amplified by the polymerase chain reaction and sequenced. Altogether, 92 (-)-BPDE-induced mutant clones were examined. At both doses, base substitutions were the most prevalent mutations observed (present in similar to 70% of the mutant clones), followed by exon deletions (present in similar to 22% of the mutant clones) and frame shift mutations (present in similar to 6% of the mutant clones) in the cDNAs analyzed. At the high cytotoxic dose, 5 out of 36 base substitutions occurred at AT base pairs (14%) and 31 at GC base pairs (86%). At the low non-cytotoxic dose, 7 out of 34 base substitutions were at AT base pairs (21%) and 27 were at GC base pairs (79%). Although there was a trend towards an increase in the proportion of mutations at AT base pairs when the dose of (-)-BPDE was decreased, this trend was not statistically significant. The data also indicated no dose-dependent differences in the kinds of base substitutions or exon deletions in cDNAs induced by (-)-BPDE. Ninety-one per cent of the (-)-BPDE-induced mutations that occurred at guanine were on the non-transcribed strand of DNA and 9% were on the transcribed strand. In contrast to these results, 50% of the (-)-BPDE-induced mutations that occurred at adenine were on the transcribed strand and 50% on the non-transcribed strand. A comparison of the mutation profiles of (-)- and (+)-BPDE revealed that (i) (+)-BPDE is more mutagenic than (-)-BPDE, (ii) (+)-BPDE caused a significantly higher proportion of mutations at AT base pairs as the dose decreased, but this was not observed for (-)-BPDE, (iii) (+)-BPDE showed high selectivity for GC --> TA transversions, but considerably less selectivity was observed for (-)-BPDE, and (iv) (-)-BPDE had different hot spots for base substitutions than did (+)-BPDE.