The applicant proposes an integrated program to investigate characteristics of normal and neoplastic growth, growth control, carcinogen metabolism, DNA repair and tumor promotion in two fish model systems. The development of new, faster eukaryotic models for bioassay to identify potential carcinogens and promoters of carcinogenesis is a pressing need for research. A team of experimental pathologists and biochemists will address this research need using the most promising aspects of four years of continuous cooperation. The Japanese medaka (Oryzias latipes), a small aquarium teleost species, develops tumors of liver after exposure to proven mammalian genotoxic carcinogens. Brief latency period, sensitivity to a variety of procarcinogens and decreased cost while maintaining large numbers of exposed individuals argue for further development of this in vivo model. Morphological characteristics of foci, areas and tumors in this teleost model resemble those seen in mammalian models. A large proportion of the hepatic neoplasms of fishes are mixed tumors of hepatocytes and biliary epithelial cells. This work has shown that livers of fishes are enriched in biliary epithelial cells. Using the rainbow trout (Oncorhynchus mykiss), the applicant has developed methods for isolating and maintaining biliary epithelial cells and hepatocytes in serum free culture providing a unique opportunity for studying events related to carcinogen initiation and promotion. The applicant hypothesizes that in vitro and in vivo analyses of biliary epithelial cells and hepatocytes and their interactions will further our understanding of these important cells in health and environmentally-associated neoplastic disease. Three broad specific aims will be addressed. They are: 1) refine medaka in vivo hepatocarcinogenesis model by quantitatively determining dynamics of normal and neoplastic liver growth; 2) determine the efficiency of carcinogen activation, DNA adduct formation and DNA repair in trout hepatocytes and biliary epithelial cells and in medaka liver; and 3) establish biochemical actions of growth factors and tumor promoters in hepatocytes and biliary epithelial cells. In vitro analysis will be done to rapidly screen and identify those environmental compounds capable of acting as tumor promoters. Bioassay of these in the medaka model will determine their in vivo relevance. This molecular and cellular screening will be linked to in vivo bioassay to validate in vitro findings.
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