The genetic effects of metal compounds have been difficult to study. Altering exposure protocols and genetic endpoints enabled this laboratory to detect the mutagenicity of some metal compounds. Other compounds, while not mutagenic per se, could enhance the mutagenicity of ultraviolet light (UV). This proposal will address the mechanisms for these two phenomena. Since the effects of chromium and nickel on DNA have been well studied by others, the focus of this project will be to elucidate the effects on DNA by mutagenic or comutagenic compounds of copper, lead, iron, manganese, arsenic, molybdenum, tungsten and zinc (in order of priority) either alone or with UV irradiation. The ability of metal compounds to damage DNA, or to enhance UV- induced damage, will be studied using sequencing gel techniques. Sites of strand breaks, alkali-lability, DNA repair enzyme recognition and polymerase inhibition will be determined on at least one restriction fragment within the E. coli gpt coding sequence. Preliminary data suggests that in some cases (e.g. Cu2+), the comutagenic effect may be mediated by hydroxyl radicals formed by a Fenton reaction causing random strand breaks which can be inhibited by KI. In cases where specific sites of damage are identified, the nature of the lesions will be determined by physical chemical means. The effects on cellular DNA will be studied for those metal compounds which do not damage plasmid DNA or enhance UV-induced damage, using alkaline elution techniques. The ability of metal compounds to damage DNA will also be studied by genetic means. The plasmid pSV2 gpt will be treated, and the transfection frequency and mutation frequency (to gpt-) will be determined using Chinese hamster V79 hprt- cells as a host. In addition, mutagenesis of a stable V79 (gpt) transfectant will be carried out, and the frequency of large deletions will be measured. It is expected that mutagenesis at this locus will be more sensitive to multi-locus deletions (known to be caused by oxidative damage) compared to the endogenous hprt locus. Using the same genetic target (E. coli gpt) for the studies on DNA damage sites and mutagenesis will ultimately enable the determination of the mutagenic consequences of specific types of damage.
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