DHEA--PPAR DEPENDENT & INDEPENDENT MECHANISMS OF ACTION

拟议的研究将评估通过哪些机制 脱氢表雄酮(DHEA)及其代谢产物改变代谢 激活和阻止化学物质推广阶段的早期步骤 致癌和毒性。目前，脱氢表雄酮是一种强有力的过氧酶体 增殖剂(PP)正被用于一些人体试验，以增强 免疫功能和改善几种代谢紊乱，但人类 似乎是一种对PP反应不强的物种。我们的 与文献相关的初步结果表明，脱氢表雄酮 及其代谢产物在两种过氧化物体增殖物激活中的作用 受体α(PPARpha)依赖和独立的通路。我们的目标 表征脱氢表雄酮或其一种的PPARα非依赖作用 代谢物被封装在四个特定的目标中： 1.确定脱氢表雄酮的代谢物是否为近端诱导剂 脱氢表雄酮作用所特有的其他过程中的过氧化物酶体增殖 使用基于细胞的报告分析。脱氢表雄酮的代谢物由大鼠形成， 仓鼠和人肝微粒体将通过GC/MS和 使用特定的抑制剂推断其形成的酶学。 基于细胞的报告基因分析与PPARpha表达构建和 脂肪酰-辅酶A氧化酶报告结构将测试它们的作用 PPARα的配体/激活剂。 2.表征脱氢表雄酮或任何活性代谢物的性质 涉及PPARα或非经典糖皮质激素诱导 Guzelian和Kasper描述的机制。我们的初步数据 提示脱氢表雄酮在体内可诱导细胞色素P450_2β_1和细胞色素P450_3A的表达，而不是由 其他过氧化物酶增殖物。因为细胞色素P4酶亚家族不是 多环芳烃代谢的良好催化剂，CYP2B1和3A23必须解释 新陈代谢增强可能是由于非经典的 归纳法。脱氢表雄酮对大鼠和人肝细胞的影响 这种可能性；PPARpha基因敲除小鼠将被用来解剖 依赖于PPARα的和独立的调节途径。 3.利用差异显示技术阐明基因 脱氢表雄酮在大鼠体内或培养人体内的诱导或抑制产物 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 或被DHEA抑制)，但不是由原型PP，那非诺平。这将是 使我们能够确定DHEA和DHEA在机制上的巨大差异 其他PP，除对细胞色素P450 B、细胞色素P3A和细胞色素C11的调控外 表达和相关的人类基因。 4.目标4的目标是描述PPARα无关的特征 P450等基因的分子调控。我们展示的基因是 不受PPAR监管的将通过准备记者进行研究 带有这些基因5‘侧翼区的构建体以评估PPAR- 脱氢表雄酮作用的独立途径。

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.