GENE-SPECIFIC DNA ADDUCTION, REPAIR, AND MUTAGENICITY OF HETEROCYCLIC AMINES

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DNA adducts formed by several heterocyclic amines (HAs) have been extensively studied by our laboratory. Studies examining the mutation spectra and repair of the DNA adducts of 2-amino-1-methyl-6- phenylimidazo[4,5-b]pyridine (PhIP) in the dihydrofolate reductase (DHFR) gene of Chinese hamster ovary cells were completed. The studies showed that PhIP produces predominantly G:T transversions in the nontranscribed strand, which is consistent with the notion that repair is higher in the transcribed than the nontranscribed strand of active genes. Conformational modeling of the major adduct of PhIP, the C8-guanine adduct, has been completed. We found that the PhIP-C8-guanine adduct is in the syn conformation when mispaired with an adenine base. These studies suggest that the conformation of the adduct may influence mutation spectra and recognition by repair enzymes. In separate studies, we compared the mutation spectra of 2-amino-3-methylimidazo[4,5-f]- quinoline (IQ) and PhIP adducts in the Sup-F shuttle vector system using repair deficient and proficient human fibroblasts. In parallel with studies in the DHFR gene, studies in the Sup-F shuttle vector system show that IQ and PhIP produce predominantly G:T transversion mutations in this system. Preferred sites of base substitution mutations were determined. Interestingly, there were differences between the two HAs in the mutation spectra induced. Repair capacity of the host cells also appeared to influence mutation spectra. The results from both the DHFR and sup-F mutation studies have led us to conclude that a guanine base in the four- base sequence CAG(Pu) is one preferred target site for PhIP-induced mutagenesis in repair proficient cells. The effects of HA adducts on gene expression were studied in mammalian expression vectors containing the human growth hormone reporter gene. The results showed that IQ and PhIP DNA adducts have different potencies with regard to inhibiting gene expression and, further, that promoter strength did not influence the degree of inhibition with either carcinogen adduct.