Glutathione S-Transferase Functions in Chemoprevention

描述(申请人提供)：本实验室和其他实验室的研究表明，谷胱甘肽S转移酶(GSTs)的表达(第二阶段解毒)在正常细胞对致癌物的反应性亲电代谢产物的防御中发挥重要作用，并作为化学预防药物诱导的生物反应的一部分。我们使用转基因细胞模型的方法来检测单独表达的GST和与相关基因结合表达的特异性贡献，包括细胞色素P450(I期激活)和多药耐药蛋白(III期外排)多基因家族的成员。我们发现，对细胞毒性和/或遗传毒性(DNA加合物或突变)的保护可能有很大的不同，在某些情况下主要取决于GST的特性和表达水平，或在其他情况下取决于致癌物的性质，是否需要激活，以及P450激活酶与GST共同表达。我们建议继续对我们现有的稳定表达人GSTP1、GSTM1或GSTA1的单转染和双转染V79细胞株进行这些研究，并将其与人P450-1A1或-1B1结合，添加-1A2。我们将专注于这些P450和GSTs的一组有限的多环芳烃(PAH)底物，苯并[a]芘(及其两个7，8-二氢二醇对映体)；更有效的二苯并[a，i]芘(及其中间体11，12-二醇代谢物)；以及5-甲基大黄烯。我们还将检测雌二醇在这些细胞系中的代谢和毒性，据报道，雌二醇由这些CYP同工酶激活，并被hGSTP1解毒。在目标1中，我们将以细胞毒性/凋亡、DNA加合物和致突变性为终点，研究每种P450和GST组合之间独特的代谢相互作用。Aim#2将模拟细胞间的相互作用，询问这些细胞系的二元混合物在多环芳烃存在时是简单的相加，还是协同或拮抗(例如，通过交换稳定的中间产物)，以及在两个共同培养的不同细胞系中，GST的表达是否与其中一个或另一个P450更有效。目的#3将确定在GST对某些致癌物的细胞毒性和遗传毒性效应的保护作用下观察到的巨大差异的机制。在目标#4中，我们将确定这一有趣观察的机制，即高效表达GSTs对4-硝基喹啉氧化物或4-羟基壬烯醛结合提供矛盾的敏感性而不是保护。这些研究将增强我们对GST化学保护功能的影响因素的了解。

DESCRIPTION (provided by applicant): Research in this lab and others has provided evidence for an important role for expression of glutathione S-transferases (GSTs) (Phase II detoxification) in normal cellular defenses against reactive electrophilic metabolites of carcinogens, and as part of the biological response induced by chemopreventive agents. We have employed a transgenic cell modeling approach to examine the specific contributions of GSTs expressed individually and in combination with relevant genes, including members of the cytochrome P450 (Phase I activation) and the multidrug resistance protein (Phase III efflux) multigene families. We have found that protection against cytotoxicity and/or genotoxicity (DNA adducts or mutagenesis) can be quite different, depending in some cases primarily on GST characteristics and expression level, or in other cases on the nature of the carcinogen, whether it requires activation, and the P450 activation enzyme co-expressed with the GST. We propose to continue these investigations with our existing single-and dual-transfected V79 cell lines that stably express human GSTP1, GSTM1, or GSTA1, alone and also in combination with human P450-1A1 or-1B1, with addition of -1A2. We will focus on a limited set of polycyclic aromatic hydrocarbon (PAH) substrates for these P450s and GSTs, Benzo[a]Pyrene (and its two 7,8-dihydrodiol enantiomers); the more potent DiBenzo[a,I]Pyrene (and its intermediate 11,12-diol metabolites); and 5-methylchrysene. We will also examine metabolism and toxicities of estradiol, reportedly activated by these CYP isozymes and detoxified by hGSTP1, in these cell lines. In Aim #1 we will examine the unique metabolic interactions between each P450 and GST combination, with both cytotoxicity/apoptosis, DNA adducts, and mutagenicity as endpoints. Aim # 2 will model cell-cell interactions, asking whether binary mixtures of these cell lines show simple additivity, or synergy or antagonism in the presence of PAHs (e.g. via exchange of stable intermediates), and if GST expression is more effective in concert with one or the other P450 in the two different cell lines co-cultured. Aim #3 will determine the mechanisms that underlie the large differences observed in GST protection against the cytotoxic vs. genotoxic effects of certain carcinogens. In Aim #4 we will determine the mechanism for the intriguing observation that expression of GSTs with high efficiency for 4-nitroquinoline oxide or 4-hydroxynonenal conjugation confer paradoxical sensitivity instead of protection. These studies will enhance our knowledge of the factors that govern chemoprotective functions of GSTs.