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

Abstract

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)

The p53 tumor suppressor protein reduces point mutation frequency of a shuttle vector modified by the chemical mutagens (±)7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, aflatoxin B1 and meta-chloroperoxybenzoic acid

“…Frameshifts (deletions of 1 ± 2 bp plus insertions) tended to be less frequent at the higher dose (10%) than at the lower dose (18%) and there was a signi®cant increase in the proportion of tandem base mutations at the higher dose (45%) compared to the lower dose (28%). For AFB1 and BPDE, the relatively high proportions of tandem mutations as compared to previous reports (Courtemanche and Anderson, 1994;Yang et al, 1987;Wei et al, 1994) may be related to the use of the pYZ289 shuttle vector or the 10(3) cells or both.…”

“…The second possibility is favored because p53-dependent MFR was observed at low doses but not at high doses of CPBA, a result hard to reconcile with a requirement for a threshold of DNA damage to obtain p53-dependent MFR. In addition, there are dose-dependent di erences in the pro®le of BPDEinduced mutations in the hprt gene of V79 cells (Wei et al, 1991(Wei et al, , 1993, suggesting dose-dependent di erences in the type of BPDE-induced DNA damage. Furthermore, it is certainly not unreasonable to suspect the existence of (dose-dependent) di erences in the type of damage caused by diverse DNA-damaging agents.…”

“…AFB1 and BPDE are well-studied genotoxic carcinogens that are known to cause point mutations (Foster et al, 1983;Trottier et al, 1992b;Levy et al, 1992;Yang et al, 1987;Wei et al, 1991Wei et al, , 1993. Although CPBA is a known DNA-damaging agent (Jacobsen and Humayun, 1986;Rosenthal et al, 1990), its mutagenic properties seem not to have been reported previously.…”

“…Most guanine substitutions (84%) a ected only a single base pair. BPDE, a mutagen known to exhibit dose-dependent di erences in the pro®le of mutations (Wei et al, 1993), was tested at two doses. The distribution of mutations followed those obtained previously (Wei et al, 1993(Wei et al, , 1994 for low and high doses of (+)-BPDE and (7)-BPDE in that there was a tendency for G:C to T:A transversions to be more frequent at the higher dose (51%) than at the lower dose (43%).…”

“…BPDE, a mutagen known to exhibit dose-dependent di erences in the pro®le of mutations (Wei et al, 1993), was tested at two doses. The distribution of mutations followed those obtained previously (Wei et al, 1993(Wei et al, , 1994 for low and high doses of (+)-BPDE and (7)-BPDE in that there was a tendency for G:C to T:A transversions to be more frequent at the higher dose (51%) than at the lower dose (43%). Frameshifts (deletions of 1 ± 2 bp plus insertions) tended to be less frequent at the higher dose (10%) than at the lower dose (18%) and there was a signi®cant increase in the proportion of tandem base mutations at the higher dose (45%) compared to the lower dose (28%).…”