Glutathione S-Transferase Functions in Chemoprevention

描述（由申请人提供）：本实验室和其他实验室的研究提供了证据，证明谷胱甘肽S-转移酶（GSTs）（II期解毒）在正常细胞防御致癌物反应性亲电代谢物中的重要作用，以及作为化学预防剂诱导的生物反应的一部分。我们采用了转基因细胞建模的方法来研究GST的具体贡献，单独表达和相关基因，包括细胞色素P450（第一阶段激活）和多药耐药蛋白（第三阶段外排）多基因家族的成员结合。我们已经发现，对细胞毒性和/或遗传毒性（DNA加合物或诱变）的保护可能是完全不同的，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，以及与GST共表达的P450激活酶。我们建议用我们现有的单转染和双转染的V79细胞系继续这些研究，这些细胞系单独稳定表达人GSTP 1、GSTM 1或GSTA 1，也与人P450- 1A 1或-1B1组合，并添加-1A2。我们将重点关注这些P450和GST的有限的多环芳烃（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）;和5-甲基芘。我们还将研究雌二醇的代谢和毒性，据报道，在这些细胞系中，这些β同工酶激活和hGSTP 1解毒。在目标#1中，我们将检查每种P450和GST组合之间的独特代谢相互作用，细胞毒性/细胞凋亡、DNA加合物和致突变性作为终点。目标#2将模拟细胞-细胞相互作用，询问这些细胞系的二元混合物是否在多环芳烃的存在下显示简单的加和性，或协同作用或拮抗作用（例如通过稳定中间体的交换），以及GST表达是否与共培养的两种不同细胞系中的一种或另一种P450协同作用更有效。目的#3将确定GST对某些致癌物的细胞毒性与遗传毒性效应的保护作用存在巨大差异的机制。在目标#4中，我们将确定有趣观察的机制，即对4-硝基喹啉氧化物或4-羟基壬烯醛缀合具有高效率的GST表达赋予矛盾的敏感性而不是保护。这些研究将提高我们的知识的因素，管理化学保护功能的GST。

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.