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

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Most of our knowledge about the processing of damaged DNA has been derived from examination of the entire genome. Recent evidence has suggested, however, that DNA adducts of chemical carcinogens are heterogeneously distributed throughout the genome and that repair of the adducts will depend on the location in the genome, the transcriptional activity of the gene sequence, and whether the adduct is on the transcribed or non-transcribed strand. In addition, the location of adducts and the existence of gene- and strand-specific preferential repair are expected to play critical roles in the spectrum of mutations induced by carcinogens. In these studies we have been developing methods to measure heterocyclic arylamine (HAA) adducts and repair in select genes. We are applying these techniques to measure adducts in different genomic regions, and in transcribed and non-transcribed strands of genes. In addition, the spectrum of PhIP-induced mutations is being examined in the same gene in which studies on the initial adduct levels and repair are being conducted. Southern hybridization analysis and probing for the sequence of interest following treatment of HAA-adducted DNA with agents that incise DNA at adduct sites has been used. We have found that purified uvrABC excinuclease, a normal repair enzyme in E. coli, recognizes and incises DNA of IQ, MeIQx, and PhIP adducts. In addition, heat, alkali, and piperidine have also been found to cause incisions at HAA adduct sites and to enable the quantitation of these adducts in specific genes. Using this technique, we have found that 4-nitro- quinoline 1-oxide, a compound structurally related to the HAAs, is not preferentially repaired in active genes, nor selectively repaired in the transcribed strand over the non-transcribed strand of the active DHFR gene of Chinese hamster ovary cells. Studies with PhIP reveals that these adducts are repaired in the DHFR gene and the transcribed strand of this gene. Analysis of PhIP-induced mutations in the DHFR gene are underway as a means of determining the kinds of mutations induced by this agent and relating aspects of gene- and strand-specific repair to the spectrum of mutations observed.