MUTATION AND REPAIR INDUCED BY THE CARCINOGEN 2-(HYDROXYAMINO)-1-METHYL-6-PHENYLIMIDAZO[4,5-B]PYRIDINE (N-OH-PHIP) IN THE DIHYDROFOLATE-REDUCTASE GENE OF CHINESE-HAMSTER OVARY CELLS AND CONFORMATIONAL MODELING OF THE DG-C8-PHIP ADDUCT IN DNA

Three experiments using 20 muM 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) were performed to induce mutations in the dihydrofolate reductase (DHFR) gene of a hemizygous Chinese hamster ovary (CHO) cell line (UA21). Metabolized forms of this chemical primarily bind at the C-8 position of guanine in DNA. In total, 21 independent induced mutants were isolated and 20 were characterized. DNA sequencing showed that the preferred mutation type found in 75% of the induced DHFR- clones was G.C --> T.A single and tandem double transversions. In addition to base substitutions, one mutant carried a -1 frameshift and another one had lost the entire locus by deletion. The induced changes affected purine targets on the nontranscribed strand of the gene in nearly all of the mutants sequenced (18/19). At the time that the first two experiments were performed, the initial adduct levels were quantitated in treated cells at the mutagenic dose by P-32-postlabeling. While the induced frequency of mutation was relatively low (approximately 5 X 10(-6)), the adduct levels after a 1-h exposure of UA21 cells to 20 muM N-OH-PhIP were relatively high (13 adducts x 10(-6) nucleotides). This latter method was then employed to learn if the induced mutation frequency correlated with rapid overall genome repair of PhIP-DNA adducts. Total adduct levels, determined using DNA samples from treated cells collected after intervals of time, were reduced by about 50% after 6 h, and about 70% after 24 h. Since overall genome repair in CHO cells is relatively slow compared with preferential gene repair, the removal of dG-C8-PhIP adducts was apparently efficient. In order to better understand the mutational and repair results, we performed computational modeling to determine the lowest energy structure for the major dG-C8-PhIP adduct in a repetitively mutated duplex sequence opposite dA. Results of this analysis indicate that the PhIP-modified base resembles previous structural determinations of (deoxyguanosin-8-yl)-aminofluorene; the carcinogen is in the B-DNA minor groove and it adopts a syn conformation mispaired with an anti A. The implications of this conformational distortion in DNA structure for damage recognition by cellular repair enzymes are discussed.