DHEA--PPAR DEPENDENT & INDEPENDENT MECHANISMS OF ACTION

拟议的研究将评估 脱氢表雄酮（DHEA）及其代谢产物改变代谢 激活和阻断化学品促进阶段的早期步骤 致癌性和毒性。目前，脱氢表雄酮，一种有效的过氧化物酶体 增殖剂（PP）正在用于许多人体试验，以提高 免疫功能并改善多种代谢紊乱，但人类 似乎是一个物种，这是不高度响应PP。我们 与文献相关的初步结果表明，DHEA 及其代谢产物在过氧化物酶体激活 受体α（PPARalpha）依赖性和独立性途径。我们的目标 为了表征DHEA或其一种的PPARalpha独立作用， 代谢物被封装在四个具体目标中： 1.确定DHEA的代谢产物是否作为 过氧化物酶体增殖对DHEA作用的其他特异性过程 使用基于细胞的报告分析。DHEA在大鼠体内的代谢产物， 仓鼠和人肝微粒体将通过GC/MS表征， 其形成的酶学使用特定的抑制剂来推断。 使用PPARalpha表达构建体的基于细胞的报告基因测定和 脂肪酰辅酶A氧化酶报告构建体将测试它们的作用， PPARalpha的配体/活化剂。 2.表征DHEA或任何活性代谢产物的特性 涉及PPARalpha或非经典类糖皮质激素诱导 Guzelian和Kasper描述的机制。我们的初步数据 表明CYP 2B 1和CYP 3A在体内由DHEA诱导，而不是由 其他过氧化物酶体增殖物。由于CYP 4酶亚家族不是 CYP 2B 1和3A 23是PAH代谢的良好催化剂， 代谢增强，可能是由于非经典机制， 诱导DHEA在大鼠和人肝细胞中的作用将解决 这种可能性; PPARalpha敲除小鼠将用于解剖 PPARalpha依赖和独立的调节途径。 3.利用差异显示技术阐明基因 DHEA在大鼠体内或培养的人中诱导或抑制的产物 肝细胞，相对于其他PP。差异显示技术 将用于观察其表达被改变（诱导）的基因 或抑制），但不被原型PP，那非诺平。这将 使我们能够确定DHEA和 除调节CYP 2B、CYP 3A和CYP 2C 11外，其他PP 表达和相关的人类基因。 4.目标4的目标是表征PPARalpha非依赖性 P450和其他基因的分子调控。我们展示的基因是 将由准备报告者研究未通过PPAR调节的 构建具有这些基因的5 '-侧翼区的构建体，以评估PPAR- DHEA作用的独立途径。

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.