NOVEL GST AND DETOXICATION OF DIOL EPOXIDES

本项目的总体目标是定义 功能独特的α类小鼠谷胱甘肽转移酶 (GST)(记为~小鼠GST 9.5“)及其人类同源物(S) 致癌二醇环氧化物(DES)的细胞保护作用 广泛存在于环境中的多环芳烃 污染物。我们实验室最近的研究表明，GST 9.5 是A1和A2型小鼠GST亚基的异源二聚体。这个 本项目的假设是小鼠GST 9.5(mGSTA1-2)，它， 与其他阿尔法类哺乳动物不同的是，GST异常活跃 对DES，及其人类同源基因(S)可能在 DES的解毒作用。这一假设将通过以下方式进行检验：(I) 测定组织分离的小鼠GSTs的动力学常数， 包括GST 9.5，以及重组的mGSTA1-1和mGSTA2-2 我们最近生产的同源二聚体，在GSH中 各种海湾和峡湾地区DES的共轭，(Ii)识别 关键氨基酸(S)可能占相对较高的 GST 9.5 A1型亚基对DES AS的催化效率 与A2型亚基(通过定点突变)相比 和突变体的动力学特征)，(Iii)确定 GSH-1诱导的GST 9.5亚基在A/J小鼠其他组织中的表达 亲和层析-反相高效液相色谱法，(Iv) 确定GST 9.5亚单位在HEPA-1中表达的影响 稳定转染细胞对(+)-抗BPDE细胞毒作用的影响 (反式N2dG-BPD加合物)，(V)体内相关性的测定 六种抗癌药物化学预防效果的比较 大蒜有机硫化物对BP诱发的前胃癌变的抑制作用 A/J小鼠和肺组织中GST水平的变化。 包括GST 9.5，在肝脏和靶器官中，(Vi)提纯 来自小鼠GST的人同源基因(S)的胃和/或肝 9.5其结构、免疫学和动力学 特征，以及(Vii)确定人的表达 GST9.5在人类其他组织中的同源基因(S)。这些研究将 不仅提高了我们对保护机制的理解 对抗致癌的DES，也阐明了小鼠GST的作用 9.5及其同源物(S)对这些致癌物的解毒作用。 从长远来看，操纵这一组GST的战略将 细胞/器官的优势对癌症来说非常重要 预防。

The overall objective of this project is to define the role of a functionally unique alpha class murine glutathione (GSH) transferase (GST) (designated as ~mouse GST 9.5") and its human orthologue(s) in cellular protection against carcinogenic diol epoxides (Des) of polycyclic aromatic hydrocarbons, which are widespread environmental pollutants. Recent studies from our laboratory indicate that GST 9.5 is a heterodimer of A1 and A2 type murine GST subunits. The hypothesis of this project is that mouse GST 9.5 (mGSTA1-2), which, unlike other alpha class mammalian GSTs, is exceptionally active toward Des, and its human orthologue(s) may play an important role in the detoxication of Des. This hypothesis will be tested by: (I) determining the kinetic constants for tissue-isolated murine GSTs, including GST 9.5, as well as the recombinant mGSTA1-1 and m GSTA2-2 homodimers, which were recently produced by us, in the GSH conjugation of various bay-and fjord-region Des, (ii) identifying the critical amino acids(s) which may account for the relatively higher catalytic efficiency of the A1 type subunit of GST 9.5 toward Des as compared with the A2 type subunit (through site-directed mutagenesis and kinetic characterization of the mutants), (iii) determining the expression of GST 9.5 subunits in other A/J mouse tissues by GSH- affinity chromatography followed by reverse-phase HPLC, (iv) determine the effects of expression of GST 9.5 subunits in Hepa-1 cells, through stable transfection, on cytotoxicity of (+)-anti-BPDE (trans-N2dG-BPD adduct), (v) determining the correlation in vivo between chemopreventive efficacies of six anti-carcinogenic organosulfides from garlic against BP-induced cancer of forestomach and lung in A/J mice wit their effects on the levels of various GSTs, including GST 9.5, in the liver and target organs, (vi) purifying from stomach and/or liver of the human orthologue(s) of murine GST 9.5 for its/their structural, immunological and kinetic characterization, and (vii) determining the expression of human orthologue(s) of GST 9.5 in other human tissues. These studies will not only enhance our understanding of the protective mechanisms against carcinogenic Des but also shed light on the role of mouse GST 9.5 and its orthologue(s) in the detoxication of these carcinogens. In the long term, strategies to manipulate this group of GSTs to the advantage of cells/organ should be extremely important for cancer prevention.