The long-term objective of this project is to establish screening systems for chemicals that produce mutations in important regulatory DNA sequences of mammalian cells at low levels of exposure. During the Phase I study, the ability of different classes of toxic chemicals to induce mutations in ras proto-oncogenes in rat liver epithelial cell cultures, a well-established model for carcinogenesis was documented. The specific objectives for the Phase II of this grant will include: 1. Adaptation of the liver cell model for quantitative measurement of mutated ras proto-oncogenes, and refinement of the PCR technology for error-free amplification of the oncogene. 2. Determination of the temporal relationship between mutations of ras gene in rat liver epithelial cells exposed to genotoxic chemicals and the emergence of an altered tumor suppressor gene, p53. 3. Extension of new system to include direct-acting and non-genotoxic chemicals as additional positive controls for the proposed large-scale screening. 4. Classification of many diverse chemicals based on their mutagenic action on the ras proto-oncogene and the p53 tumor suppressor gene. The salient technical innovation in these studies is the use of PCR amplification of the very few mutations induced in a oncogenes and suppressor genes in an in vitro liver cell model of chemical carcinogenesis that simulate human exposure in vivo.
