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 microM) or a high cytotoxic dose (2.0 microM) 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 approximately 7% of the mutant clones), followed by exon deletions (present in approximately 22% of the mutant clones) and frame shift mutations (present in approximately 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.(ABSTRACT TRUNCATED AT 400 WORDS)
Wei
S J
SJ
Department of Chemical Biology and Pharmacognosy, College of Pharmacy, Rutgers, State University of New Jersey, Piscataway 08855.
Chang
R L
RL
Hennig
E
E
Cui
X X
XX
Merkler
K A
KA
Wong
C Q
CQ
Yagi
H
H
Jerina
D M
DM
Conney
A H
AH
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000633:toxicity*; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D013237:Stereoisomerism
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D007041:Hypoxanthine Phosphoribosyltransferase