Cytochrome P450s (P450s or CYPs) are a superfamily of heme-containing enzyme that catalyze Phase I oxidative, reductive and peroxidative reactions of both endogenous (steroids, fatty acids, vitamins, prostaglandins, retinoids, etc.) and exogenous substrates (chemical pollutants, drugs and carcinogen). The zebrafish (Danio rerio) has become a widely used animal model for studying embryogenesis and developmental genetics and as an animal model to investigate mechanisms of chemical carcinogenesis. Compared to rainbow trout (Oncorhynchus mykiss), however, zebrafish are relatively resistant to certain carcinogens such as aflatoxin B1 (AFB1) and dibenzo[a,1]pyrene (DBP), thus reducing their utility as carcinogenesis models. Their is considerable interest, therefore, in finding zebrafish strains that are more sensitive to carcinogens. Because of their central roles in bioactivation and metabolism processes, it also has become important to establish the contribution of different CYP forms to carcinogenesis and tumor tissue selectivity in the zebrafish and to utilize bioactivating or detoxifying CYP expression as a tool to help select sensitive zebrafish strains. Therefore, the objectives of this project are to: 1) clone, sequence, heterologously express, enzymatically and kinetically characterize and map major zebrafish CYPs potentially involved in carcinogen activation and detoxification; 2) use zebrafish CYP isozyme specific oligonucleotide probes to determine their tissue and cell-specific expression patterns in zebrafish mutant lines used to assess the sensitivity of the fish to selected carcinogens; 3) select mutant zebrafish line on the basis of inactivating mutations of tumor suppressor genes and/or high carcinogen bioactivating CYP isoform expression; and 4) determine the sensitivity of wild-type and mutant zebrafish lines to AFB1 and DBP. This proposed work should add significantly to the foundation of CYP and carcinogenesis research in zebrafish.
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