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
- Columbia Univ., New York, NY (United States)
- Oak Ridge National Lab., TN (United States)
- New York Univ., NY (United States)
- National Institutes of Health, Bethesda, MD (United States)
Three experiments using 20 [mu]M 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[sup [minus]] clones was G[center dot]C [yields] T[center dot]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[sup 32]P-postlabeling. While the induced frequency of mutation was relatively low ([approximately]5 x 10[sup [minus]6]), the adduct levels after a 1-h exposure of UA21 cells to 20 [mu]M N-OH-PhIP were relatively high (13 adducts x 10[sup [minus]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 sample from treated cells collected after intervals of time, were reduced by about 50% after 6 h, and about 70% after 26 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. 63 refs., 8 figs., 5 tabs.
- DOE Contract Number:
- FG02-90ER60931; AC05-84OR21400
- OSTI ID:
- 6955816
- Journal Information:
- Chemical Research in Toxicology; (United States), Vol. 7:2; ISSN 0893-228X
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
CHO CELLS
MUTATION FREQUENCY
MUTANTS
DNA SEQUENCING
PICOLINES
BIOCHEMICAL REACTION KINETICS
MUTAGENESIS
BIOLOGICAL PATHWAYS
DAMAGE
DNA
DNA ADDUCTS
GUANINE
NUCLEOTIDES
ADDUCTS
AMINES
ANIMAL CELLS
AROMATICS
AZAARENES
AZINES
HETEROCYCLIC COMPOUNDS
HYDROXY COMPOUNDS
KINETICS
NUCLEIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PURINES
PYRIDINES
REACTION KINETICS
STRUCTURAL CHEMICAL ANALYSIS
550200* - Biochemistry
560300 - Chemicals Metabolism & Toxicology