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）的表达在正常细胞防御致癌物的反应性亲电代谢物中起重要作用，并作为化学预防剂诱导的生物反应的一部分。我们采用了一种转基因细胞建模方法来研究GSTs单独表达和与相关基因联合表达的具体贡献，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加合物或诱变）的保护可能完全不同，这主要取决于在某些情况下主要取决于GST的特征和表达水平，或者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共表达。我们建议用我们现有的单和双转染的V79细胞系继续这些研究，这些细胞系可以稳定地单独表达人GSTP1， GSTM1或GSTA1，也可以与人P450-1A1或1b1联合表达，并添加-1A2。我们将重点关注这些p450和gst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（及其中间的11,12-二醇代谢物）；和5-methylchrysene。我们还将研究雌二醇在这些细胞系中的代谢和毒性，据报道雌二醇是由这些CYP同工酶激活并由hGSTP1解毒的。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-细胞相互作用，询问这些细胞系的双重混合物在PAHs存在下是否表现出简单的可加性，或协同作用或拮抗作用（例如，通过交换稳定的中间体），以及在两种不同的细胞系中，GST与一种或另一种P450的表达是否更有效。目标#3将确定在GST对某些致癌物的细胞毒性和基因毒性作用的保护中观察到的巨大差异背后的机制。在目标#4中，我们将确定有趣观察的机制，即高效表达4-硝基喹啉氧化物或4-羟基壬烯醛偶联的GSTs赋予矛盾的敏感性而不是保护。这些研究将增强我们对控制GSTs化学保护功能的因素的认识。

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.