Characterization of the mutational profile of (+)-7 R ,8 S -dihydroxy-9 S ,10 R -epoxy-7,8,9,10-tetrahydrobenzo[ a ]pyrene at the hypoxanthine (guanine) phosphoribosyltransferase gene in repair-deficient Chinese hamster V-H1 cells

Abstract

Earlier studies have shown that the profile of mutations induced by (+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (+)-BPDE at the hypoxanthine (guanine) phosphoribosyltransferase (hprt) gene of Chinese hamster V79 cells was dependent on the concentration of (+)-BPDE. In the present study, we examined the effect of the concentration of (+)-BPDE on its mutational profile at the hprt gene in repair-deficient V-H1 cells (a derivative of V79 cells) to explore the role of DNA repair in the dose-dependent mutational profile of (+)-BPDE. Independent hprt mutant clones were isolated after exposing V-H1 cells to dimethylsulfoxide (DMSO) or to low (4-6 nM; 95% cell survival) or high (40-48 nM; 31% cell survival) concentrations of (+)-BPDE in DMSO. The mutation frequencies for the DMSO control and for the low and high concentration groups were 0.1, 2.1 and 32.9 mutant colonies/10(5) survivors, respectively. The profile of mutations at the hprt gene was characterized for 148 (+)-BPDE-induced mutant clones and the results from the present study were compared with those obtained earlier with V79 cells. The data indicated that: (i) V-H1 cells were approximately 9-fold more sensitive to the cytotoxic effects of (+)-BPDE than V79 cells; (ii) the mutation frequency in V-H1 cells was similar to that observed in V79 cells following exposure to similar concentrations of (+)-BPDE; (iii) (+)-BPDE-induced mutations at guanine on the transcribed strand of the hprt gene were common in V-H1 cells but were extraordinarily rare in V79 cells; (iv) (+)-BPDE-induced mutations at adenine on the transcribed strand of the hprt gene were common in both V-H1 and V79 cells; (v) although exposure of V79 cells to different doses of (+)-BPDE resulted in a dose-dependent mutational profile at the hprt gene, this was not observed in V-H1 cells. Our observations indicate a defect in the transcription-coupled repair of (+)-BPDE-DNA adducts in V-H1 cells and that the repair activity deficient in V-H1 cells is essential for the dose-dependent mutational profile observed with (+)-BPDE in V79 cells.

S-(2-Chloroethyl)glutathione-generated p53 Mutation Spectra Are Influenced by Differential Repair Rates More than Sites of Initial DNA Damage

“…However, transcription-coupled repair is an issue in terms of which strand is repaired. In other work by Wei and Conney (22,(73)(74)(75), the mutation spectrum (with other genes) resulting from benzo[a]pyrene diol epoxide treatment of mammalian cells was dependent upon the dose of the carcinogen, and saturation of DNA repair has been proposed as a likely mechanism underlying this phenomenon. Work by Tang's group on human ras K gene codons 12 and 14 has also been interpreted in terms of effects of C methylation on binding, and rates of repair also appear to be a factor (25) (although these ras K codon 12 and 14 adducts have not been extensively evaluated in terms of producing mutations).…”

A Single Site-specific trans-Opened 7,8,9,10-Tetrahydrobenzo[a]pyrene 7,8-Diol 9,10-EpoxideN2-Deoxyguanosine Adduct Induces Mutations at Multiple Sites in DNA

“…However, the level of mutation induced by both adducts was substantially higher than in our study (13 versus 1.8 -2.5%), and low levels (Ͻ1%) of G 3 C and G 3 A mutations were also detected. These differences may stem mainly from differences in the experimental systems; the single-stranded vector system used by Page et al (31) and Moriya et al (37) eliminates the involvement of DNA repair, whereas the double-stranded vector system used in our study is sensitive to DNA repair (both in V79 and in V-H1 cells, which have ϳ50% remaining capacity to remove adducts derived from the (ϩ)-(7R,8S,9S,10R)-enantiomer of BPDE from DNA compared with V79 cells (27)). Consequently, the results of our study show substantially lower mutagenicity of the examined adducts and reflect both DNA repair of the adducts and fidelity of their bypass.…”

“…It is possible that the defect of V-H1 cells in the xeroderma pigmentosum complementation group D/ERCC2 gene (25) influences mainly transcriptional coupled repair (18) and causes a decreased ability of the cells to preferentially repair BPDE adducts from the transcribed strand of active genes (27), whereas the efficiency of global genomic repair (18), which is responsible for the repair of the non-transcribed strand (location of the adducts in this study), may be affected only marginally. Both lesions in both adducted G sites generated the same kind of mutation (G 3 T), and the 10S adduct induced identical secondary mutations in these two cell lines.…”

“…These cells are defective in the xeroderma pigmentosum complementation group D/ERCC2 gene encoding for an ATP-dependent DNA helicase (25), an essential subunit of transcription and nucleotide excision repair complex TFIIH (26). They are 9-fold more sensitive to cytotoxic effects of (ϩ)-BPDE-2 than V79 cells and have ϳ50% lower capacity for removal of (ϩ)-BPDE-2-induced adducts from DNA compared with V79 cells (27). We qualitatively and quantitatively evaluated mutagenic effects of both 10R and 10S adducts in a sequence of seven nucleotides by a novel quantitative minisequencing method, and we compared their differences with respect to 10R/10S stereochemistry, position of the adducts at two adjacent sites, and also cellular DNA repair status.…”

Molecular modeling benzo[a]pyrene N2-dG adducts in the two overlapping active sites of the Y-family DNA polymerase Dpo4

“…[8][9][10][11][12]), and may in fact be important in human cancer (e.g., [13] and references therein). B[a]P mutagenesis has been extensively studied, and mutational spectra with the biologically relevant metabolite (+)-anti-B[a]PDE have been determined in Escherichia coli [14,15] and in mammalian (CHO) cells ( [16,17] and references therein).…”