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

Three experiments using 20 muM roxyamino)-1-methyl-6-phenylimidazo[4,5 -b]pyridine (N-OH-PhIP) were performed to induce mutations in the dihy drofolate reductase (DHFR) gene of a hemizygous Chinese hamster ovary (CHO) cell line (UA21). Metabolized forms of this chemical primarily b ind at the C-8 position of guanine in DNA. In total, 21 independent in duced mutants were isolated and 20 were characterized. DNA sequencing showed that the preferred mutation type found in 75% of the induced DH FR- 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 change s affected purine targets on the nontranscribed strand of the gene in nearly all of the mutants sequenced (18/19). At the time that the firs t two experiments were performed, the initial adduct levels were quant itated in treated cells at the mutagenic dose by P-32-postlabeling. Wh ile the induced frequency of mutation was relatively low (approximatel y 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) nucleotide s). This latter method was then employed to learn if the induced mutat ion frequency correlated with rapid overall genome repair of PhIP-DNA adducts. Total adduct levels, determined using DNA samples from treate d cells collected after intervals of time, were reduced by about 50% a fter 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 be tter understand the mutational and repair results, we performed comput ational modeling to determine the lowest energy structure for the majo r dG-C8-PhIP adduct in a repetitively mutated duplex sequence opposite dA. Results of this analysis indicate that the PhIP-modified base res embles previous structural determinations of (deoxyguanosin-8-yl)-amin ofluorene; the carcinogen is in the B-DNA minor groove and it adopts a syn conformation mispaired with an anti A. The implications of this c onformational distortion in DNA structure for damage recognition by ce llular repair enzymes are discussed.