This project involves the development and testing of new assays for in vivo exposure to toxic agents, based on aberrant gene expression and/or amplification. Circulating lymphocytes form rats exposed to CdCl2, NiCl2, Benzene, or benzo(a)pyrene will be cultured in microtiter well plates and transferred to nitrocellulose filters. Alternatively, pooled lymphocytes form animals will be used for extraction of mRNA and DNA. Northern and Southern blot hybridization analysis will be done using RNA and DNA from lymphocytes of treated and control animals using cloned metallothionein, cytochrome P-450, and other genes as probes. monoclonal or polyclonal lymphocyte cultures transferred to nitrocellulose filters will be hybridized using the cell colony hybridization method developed by Dr. Rossman It is anticipated that these assays of gene expression at the RNA level will be much more sensitive than previously published methods using functional assays or radioimmuneassays for protein products. We will also examine a number of genes, including oncogenes, for gene amplification and/or rearrangements as a potential indication of genotoxic exposure. If increases in gene expression or amplification are seen in lymphocytes for animals receiving high doses of toxicants using a particular assay, the assay will be further tested by performing dose response and time course experiments. Finally, any assay which has proven to be reproducible, highly sensitive and relatively simple to perform will be further developed for testing in human populations.
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