The proposed studies will evaluate the mechanisms by which dehydroepiandrosterone (DHEA) and its metabolites alter the metabolic activation of and block early steps in the promotion phase of chemical carcinogenesis and toxicity. Currently, DHEA, a potent peroxisome proliferator (PP) is being used in a number of human trials to enhance immune function and ameliorate several metabolic disorders, but humans appear to be a species which is not highly responsive to PP. Our preliminary results, correlated with the literature, suggest that DHEA and its metabolite function in both Peroxisome Proliferator Activated Receptor Alpha (PPARalpha)-dependent and independent pathways. Our goals to characterize the PPARalpha-independent actions of DHEA or one of its metabolites are encapsulated in four specific aims: 1. Establish whether metabolites of DHEA serve as proximal inducers of peroxisome proliferation on other processes specific for DHEA action using cell-based reporter assays. The metabolites of DHEA formed by rat, hamster and human liver microsomes will be characterized by GC/MS and the enzymology of their formation deducted using specific inhibitors. Cell-based reporter assays with PPARalpha expression constructs and fatty acyl-CoA oxidase reporter constructs will test their role is ligand/activators for PPARalpha. 2. Characterize the properties of DHEA or any active metabolites involving PPARalpha or the non-classical glucorticoid induction mechanism described by Guzelian and Kasper. Our preliminary data suggests that CYP2Bl and CYP3A are induced by DHEA in vivo, but not by other peroxisome proliferators. Since the CYP4 enzyme subfamily are not good catalysts of PAH metabolism, CYP2Bl and 3A23 must account for enhanced metabolism possibly due to the non-classical mechanism of induction. The effects of DHEA in rat and human hepatocytes will address this possibility; PPARalpha knock-out mice will be used to dissect PPARalpha-dependent and independent pathways of regulation. 3. Utilize the technique of Differential Display to elucidate gene products induced or suppressed by DHEA in rats in vivo or cultured human hepatocytes, relative to other PP. The technique of Differential Display will be utilized to observe genes whose expression is altered (induced or repressed) by DHEA, but not by a prototype PP, nafenopin. This will allow us to ascertain large differences in mechanism between DHEA and other PP, in addition to regulation of CYP2B, CYP3A and CYP2C11 expression and related human genes. 4. The goal of Aim 4 is to characterize the PPARalpha-independent molecular regulation of P450s and other genes. Genes which we show are not regulated through PPAR will be studied by preparing reporter constructs with 5'-flanking regions of those genes to evaluate PPAR- independent pathways of DHEA action.
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