A major unsolved problem in toxicology having 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 the host's 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 the bioactivation of such carcinogens as aflatoxins and benzo[a]pyrene. Taking advantage of a newly defined system primary culture of rat and human hepatocytes on Matrigel, a reconstituted basement membrane and permits retention of the differentiated state in vitro, we will make proximate comparisons of the regulation of the controlling DNA segments of the CYP3A structural genes and their products between animal and human cells under defined, rigorously controlled conditions. Molecular isolation and characterization techniques will be used to specify the core DNA elements that regulate CYP3A gene expression in the liver and the igand dependent regulatory transcription protein factors that interact with these DNA response elements. We will also use reverse genetic analysis to investigate a previously unrecognized assortment of genes regulated under the same """"""""stress"""""""" controls that extends beyond the liver acting in concert to adapt to environmental stressors. Facilitating this work is a Cell Culture and Tissue Bank which provides the animal and human material needed for these investigations. Through a thoughtful combination of these 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. We will investigate the question of how these effects may differ among individuals. 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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| Brown, S E; Quattrochi, L C; Guzelian, P S (1997) Characterization of a pretranscriptional pathway for induction by phenobarbital of cytochrome P450 3A23 in primary cultures of adult rat hepatocytes. Arch Biochem Biophys 342:134-42 |
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| Brown, S E; Guzelian, C P; Schuetz, E et al. (1995) Critical role of extracellular matrix on induction by phenobarbital of cytochrome P450 2B1/2 in primary cultures of adult rat hepatocytes. Lab Invest 73:818-27 |
