EXCEED THE SPACEPROVIDED. A major unsolved problem in toxicologyhaving broad medical, economic, legal, and political consequences is the uncertainty of extrapolation to humans of the results of tests of high doses of xenobiotics given to laboratory animals. Our grant proposal will utilize advances in molecular and cellular biology to help define the genetic and environmental factors that may contribute to host resistance to risks for an adverse health effect to a given individual. We are focusing on the cytochromes P450 of the CYP3A gene subfamily, a group of microsomal hemoproteins prominent in human liver which are induced under conditions of """"""""stress"""""""" by glucocorticoids and also by such xenobiotics as phenobarbital, and polyhalogenated aromatic environmental chemicals. CYP3A are involved in the metabolism of clinically important drugs such as cyclosporin and nifedipine and also of numerous carcinogens and environmental pollutants. Taking advantage of new techniques in molecular biology and a well defined system for primary culture of nonproliferating adult rodent hepatocytes that maintain differentiated liver functions in culture and permit molecular analysis of functional core DNA elements that regulate CYP3A gene expression, we will characterize the relevant ligand dependent regulatory transcription protein factors. Accompanying this fundamental science is our clinical protocol to phenotype human volunteers for inducibility of CYP3A activity. The substantial variation we have already documented can be traced in part to hypoinduction in a carrier of a dysfunctional receptor allele of the PXRfamily. We will confirm and extend these findings to define the distribution of this and other genetic differences that may account for variation of induction and, hence, may serve as risk factors for disease. We will also use reverse genetic analysis to investigate a previously unrecognized assortment of genes, regulated under the same """"""""stress"""""""" controls, that extends beyond drug metabolism and even beyond the liver, possibly acting in concert to adapt to environmental stressors. Through a thoughtful combination of clinical and basic laboratory approaches, we expect to be able to quantitatively describe the molecular events that underlie changes in cellular expression of the CYP3A genes due to drugs, environmental chemicals, endocrine controls and other factors and to translate these data to the relevant genes in humans. We fully expect that refinements in understanding gene structure, gene expression, and disease outcome can be achieved by the proposed research program.
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