DEHP-INDUCIBLE CYTOCHROME P450 FORMS IN KIDNEY AND LIVER

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Members of the cytochrome P450 4A (CYP4A) subfamily catalyze the omega - or penultimate -hydroxylation of saturated and unsaturated fatty acids and prostaglandins. P450 4A forms are induced by peroxisome proliferators and their induction represents one of the earliest cellular events following exposure to these agents. It is thought that P450 4A induction and formation of oxidized fatty acids may be essential for induction of peroxisomal enzymes. There is considerable interest in the cellular effects of peroxisome proliferators, because these chemicals have been shown to cause hepatic tumors and testicular atrophy in rodents. In addition, 4A forms synthesize products which mediate vasoconstriction of renal vessels and an increase in P450-mediated fatty acid omega- hydroxylase activity is reported prior to the elevation in blood pressure in genetically-bred hypertensive rats. P450 4A forms have been identified in many species and one human 4A form has been sequenced. In rats, three members of the 4A family, 4A1, 4A2, and 4A3 have been identified and form specific oligonucleotide probes and an antibody which recognizes the three forms on western blots have been developed. Certain P450 4A forms exhibit organ specific expression and are regulated by sex hormones. The objectives of this competitive grant renewal are: 1) To localize specific 4A forms in distinct segments of the rat nephron by western blot analysis, because there is considerable uncertainty over the identity of renal forms. The localization of renal 4A forms will be compared in male and female rats because expression of renal 4A forms is sex hormone-dependent; 2) To identify 4A forms which are induced by two immunosuppressive drugs, cyclosporine and FK 506. Cyclosporine is an important therapeutic agent used in transplantation surgery and in treatment of autoimmune diseases, but nephrotoxicity is a major side effect. Studies indicate there is a correlation between induction of fatty acid omega-hydroxylase activity by cyclosporine and "the onset of nephrotoxicity; 3) To examine the mechanisms by which calcium channel blockers suppress induction of hepatic enzymes in DEHP (diethylhexyl phthalate), MEHP (monoethylhexyl phthalate) or clofibrate treated rats or isolated hepatocytes and to examine whether 20-hydroxyeicosatetraenoic acid (20-HETE) and other omega- or penultimate- oxidized fatty acids regulate intracellular calcium concentrations in isolated rat hepatocytes; and 4) To examine the interferon-induced suppression of hepatic 4A forms in phthalate or clofibrate treated rats or isolated hepatocytes. Interferon has been shown to inhibit the induction of P450 4A forms and its suppressive effects on peroxisome fatty acyl CoA oxidase will be examined. The long-term goals of this proposal are to determine the function of P450-mediated fatty acid omega-hydroxylases, its role in peroxisome proliferation, and its physiological function in the kidney.