This proposal investigates mechanisms of genotoxicity in rainbow trout as a central theme, with a primary emphasis on the genetics, molecular genetics, and biochemistry of carcinogenesis and tumor promotion. Many attributes justify use of this aquatic model in environmental health research. Trout reduce dependence on mammals for cancer research, provide comparative mechanism information, show high sensitivity (nanogram tumor response), have such low cost that many thousands of animals per tumor study are reasonable, have zero and near-zero background incidence, and are readily manipulated to produce clones or polyploids. An emphasis on mechanisms will broaden our comparative base for extrapolating cancer studies from surrogates to humans, while also increasing understanding of tumor development in feral fish species. The four interactive projects within this theme are: 1. Investigate the molecular basis for tumor initiation and development in trout, including: mechanisms that determine the incidence, site-specific generation and selective propagation of Ki-ras p21 allelomorphs; the influence of carcinogen dose, proliferative regeneration, DNA adduction, and repair capacity on the incidence of Ki-ras activation, down to 10-3 tumor risk (ED0.1% tumor study); and the influence of triploid as a selective environment for oncogenesis involving dominant p53 point mutations. 2. Examine the genetic control of variation in tumor response in outbred, clonal, and triploid populations of trout. Utilize ploidy manipulation to assess tumor suppressor gene involvement in trout carcinogenesis, and establish the use of RFLP markers and known suppressor gene probes to investigate deletional events in diploid and triploid populations of isogenic trout. Initiate development of a genetic map of DNA markers for rainbow trout. 3. Establish the metabolic capacity of early trout life stages for procarcinogen bioactivation and alkylation repair. Clone, sequence, and establish the expression of three new trout cytochrome P450 genes. Establish the time- and tissue-dependent expression of these and two additional trout P450s, their inductive response to selected environmental agents, and their potential to bioactivate selected procarcinogens. Establish the levels, developmental expression, and tissue distribution of 06- alkyltransferase. 4. For clinical application, establish the mechanisms through which the naturally occurring compound indole-3-carbinol, a putative """"""""anticarcinogen"""""""" currently proposed for human intervention trials, also behaves as a tumor promoter. Investigate if the promotional mechanism is mediated in trout and mice through Ah receptor agonism. Rigorously quantify its dose-responsive promotional potency and the potential presence of a threshold dose for promotion, in order to assess promotional risk vs. chemopreventive benefit.
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