Many of the available assays for genotoxicity depend upon the persistence of DNA damage and therefore, they fail to detect exposure to low levels of toxic or carcinogenic chemicals. In this proposal we have designed an assay to monitor competence of DNA excision repair and an assay to monitor replication in two genomic domains. Since many cellular functions are required for the regulation of DNA repair and DNA replication, chemicals that target sites other than DNA may be detected in these assays as well as those that damage DNA. In addition the sensitivity of detection of DNA damaging agents may be increased in these new assays. This expectation is based on our observations that HgCl2 is a very effective inhibitor of x-ray-induced strand break repair at low, nontoxic levels that do not produce measurable DNA lesions. Preliminary results with Ni(II) and Cr(VI) indicate that nontoxic levels of these metals also alter strand break repair. This assay is expected to detect exposure to these metals and needs to be tested for other types of compounds. Peripheral blood lymphocytes will be used to test the repair assay following in vivo exposure of rats to a single chemical. Environmentally exposed human populations will be tested using similar protocols. The extent of DNA damage due to exposure will be measured as unscheduled DNA synthesis (UDS). For chemicals that produce positive results in the assay for excision repair we will compare the sensitivity and the persistence of the response to the yield of sister chromatid exchanges (SCE). The new assay for replication will be developed only for human lymphocytes and will monitor the extent and temporal relation ship of synthesis of satellite and ribosomal DNA. The potential advantages of this assay over conventional measures of replication is the possibility that numerous agents may perturb the timing of synthesis for the sequences selected by effects on rate of synthesis, frequency of initiation events, or less direct effects such as reduced levels of ATP. The unifying theme for this proposal is to develop sensitive assays for DNA replication and DNA repair that are cellular processes requiring a complex assortment of cellular structures and enzymes. This approach should increase the probability of detecting agents that target sites other than DNA or that produce DNA lesions beneath the sensitivity of detection.
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