Cancer is now regarded as a genetic disease caused by accumulated alterations of genes in somatic cells by various mutagenic processes. Of the many potential mutagenic compounds found in the environment, several metals have been identified as mutagens as well as carcinogens. The physiological effects of exposure to trace quantities of metals, ubiquitous in nature as a result of natural processes and environmental contamination, are well documented. Notably, Mn, Co, Cd, Be, Ag, Cr, Pb, Ni, and Cu cations have been shown to increase mutagenicity in an assay designed to measure DNA base substitution errors. The goal of this research project is to complete a comprehensive analysis of the mutagenic effects of thirteen metal salts on the fidelity of DNA synthesis. The research uses an in vitro fidelity assay which allows detection of a variety of different errors occurring during synthesis of a 250 nucleotide target sequence contained within a natural M13mp2 DNA template. To date, a detailed analysis of the mutagenic effects of Mn2plus and Ni2plus is nearly completed, and studies on Cr3plus, and Pb2plus are well underway. During the requested funding period, the remaining selected metal cations (Zn2plus, Co2plus, Be2plus Cu2plus Hg2plus Sr2plus, Fe2plus, and Se4plus) will be analyzed for their mutagenic effects. For each of the selected metals, measurement of dose effects on in vitro DNA synthesis and fidelity, determination of mutation frequencies and error rates for specific types of mutations, and construction of mutational spectra will be undertaken. The results of this research project will provide valuable information on mutagenic specificity and dose levels for the selected metals and will be used to elucidate the mechanism(s) of metal-induced mutagenesis. This project includes students in every aspect. Students learn fundamentals of DNA manipulations, plaque assays, in vitro DNA synthesis, microbiological techniques, DNA sequence analysis, data management, and safe use of hazardous reagents.
