DOSE-DEPENDENT DIFFERENCES IN THE PROFILE OF MUTATIONS INDUCED BY OXY-9S,10R-EPOXY-7,8,9,10-TETRAHYDROBENZO(A)PYRENE IN THE CODING REGION OF THE HYPOXANTHINE (GUANINE) PHOSPHORIBOSYLTRANSFERASE GENE IN CHINESE-HAMSTER V-79 CELLS())

Chinese hamster V-79 cells were exposed to a high dose (0.30-0.48 muM; 32% cell survival), an intermediate dose (0.04-0.10 muM; 100% cell su rvival) or a low dose (0.01-0.02 muM; 97% cell survival) of oxy-9S,10R -epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene [(+)-BPDE] which is the ultim ate carcinogenic metabolite of benzo(a)pyrene. The mutation frequency for cells treated with dimethyl sulfoxide vehicle or with low, interme diate or high doses of (+)-BPDE were 1, 10, 52 or 514 8-azaguanine-res istant colonies/10(5) survivors, respectively. Independent 8-azaguanin e-resistant clones were isolated, and complementary DNAs were prepared by reverse transcription. The coding region of the hypoxanthine (guan ine) phosphoribosyltransferase (HPRT) gene was amplified by the polyme rase chain reaction and sequenced. Altogether, 368 (+)-BPDE-induced mu tant clones were examined. At all doses, base substitutions were the m ost prevalent mutations observed (about 72% of the mutant clones), fol lowed by exon deletions (about 26% of the mutant clones) and frame-shi ft mutations (about 6% of the mutant clones). At the high cytotoxic do se, 7 of 120 base substitutions occurred at AT base pairs (6%) and 113 at GC base pairs (94%). At the intermediate noncytotoxic dose, 20 of 82 base substitutions occurred at AT base pairs (24%) and 62 at GC bas e pairs (76%). At the low noncytotoxic dose, 27 of 76 base substitutio ns were at AT base pairs (36%) and 49 were at GC base pairs (64%). The results indicated that decreasing the dose of (+)-BPDE decreased the proportion of mutations at GC base pairs and increased the proportion of mutations at AT base pairs. As the dose of (+)-BPDE was decreased, there was a dose-dependent decrease in the proportion of GC-->TA trans versions from 69% to 42% of the base substitutions) and a dose-depende nt increase in the proportion of AT-->CG transversions (from 1% to 25% of the base substitutions). The data also indicated dose-dependent di fferences in (+)-BPDE-induced exon deletions and hot spots for base su bstitutions at GC and AT base pairs. Although more than 99% of the (+) -BPDE-induced mutations at guanine occurred on the nontranscribed stra nd of DNA, (+)-BPDE-induced mutations at adenine occurred on both the transcribed and nontranscribed strands. The ratio of mutations at aden ine on the transcribed strand to mutations at adenine on the nontransc ribed strand was 35:19 in (+)-BPDE-treated V-79 cells. These observati ons suggest different mechanisms of mutation induction at GC and AT ba se pairs and/or differences in repair mechanisms for premutagenic lesi ons at GC and AT base pairs. Several nucleotide sequences with frequen t (+)-BPDE-induced mutations targeted at guanines in the coding region of the HPRT gene were identified. These included AGGGGGGC, TGGA, AGGA , TGGT, AGGC, TGGGA, AGGGA, and TGGGGA. Seventy % of all base substitu tion mutations targeted at guanine were on these sequences. (+)-BPDE-i nduced mutations in ras motifs (corresponding to ras codons 12, 13, 61 ) in the coding region of the HPRT gene were more commonly observed th an by chance.