HUMAN FETAL LIVER GST--HYDROXYNONENAL CONJUGATION

4-羟基壬烯醛(4HNE，C9H16O2)是脂质过氧化过程中形成的主要α，β-不饱和羰基，也是迄今测试过的最具细胞毒性和致突变性的醛之一。4HNE被认为是许多与环境有关的疾病的病原体，包括肝纤维化、胎儿酒精中毒、癌症、动脉粥样硬化和阿尔茨海默病。4HNE在人体组织中被乙醇脱氢酶、乙醛脱氢酶和谷胱甘肽S转移酶解毒。在这些酶中，人GST同工酶A4(hGSTA4-4)对4HNE具有独特的、特别高的活性。我们发现，一些个体和中期妊娠的胎儿捐献者没有检测到肝脏GST-4HNE活性。此外，怀孕中期的胎儿不能有效地通过ADH解毒4HNE，ADH是4HNE清除的竞争途径。因此，未出生的婴儿可能代表着对宫内促氧化剂敏感的亚群。这笔赠款的具体目标和方法将是：1)利用人肝切片的体外分离和培养来表征和比较4HNE在成人和胎儿肝脏中的代谢；2)利用定量聚合酶链式反应和定量免疫印迹技术充分表征成人和胎儿肝脏和胎盘中GST-4HNE活性和hGSTA4同工酶表达的变化；3)通过GST蛋白纯化、测序和免疫印迹鉴定胎儿肝脏和胎盘中潜在的代谢4HNE的新的GST亚型；4)使用转基因细胞系和精密肝切片的动态培养来确定不良的4HNE接合子和胎儿是否会增加4HNE相关的细胞毒性、5)确定hGSTA4在成人和胎儿肝脏中是否可诱导表达，如果是，是否具有原位保护性。这个项目的结果将扩大我们对人类接触环境化学品产生的剧毒代谢物的风险程度和变化的理解，这种代谢物与许多疾病状态有关。此外，该项目对孕妇接触的促氧化药物或化学物质的宫内影响也有重要影响。

4-hydroxynonenal (4HNE, C9H16O2) is the major alpha,beta- unsaturated carbonyl formed during lipid peroxidation and one of the most cytotoxic and mutagenic aldehydes ever tested. 4HNE has been implicated as a causative agent in a number of environmentally-related diseases including liver fibrosis, fetal alcohol toxicity, cancer, atherosclerosis, and Alzheimer's disease. 4HNE is detoxified in human tissues by alcohol dehydrogenases (ADH), aldehyde dehydrogenases (ALDH), and glutathione S-transferases (GST). Of these enzymes, human GST isozyme A4 (hGSTA4-4) has a unique and particularly high activity toward 4HNE. We have found that some individuals and second trimester fetal donors do not have detectable hepatic GST-4HNE activity. In addition, second trimester fetuses do not effectively detoxify 4HNE by ADH, a competing pathway for 4HNE removal. Thus, unborn children may represent a sensitive subpopulation to in utero pro-oxidants. The specific aims and approaches of this grant will be to 1) characterize and compare 4HNE metabolism in human adult and fetal liver using in vitro fractions and culture of human liver slices, 2) fully characterize the variation in hepatic GST-4HNE activities and hGSTA4-4 isozyme expression in human adult and fetal liver, and placenta using quantitative PCR and quantitative western blotting, 3) identify potential novel GST isoforms in fetal liver and placenta that metabolize 4HNE by GST protein purification, sequencing, and immunoblotting, 4) use transfected cell lines and dynamic culture of precision liver slices to determine if poor 4HNE conjugators and fetuses are at increased risk to 4HNE- associated cellular toxicities, and 5) determine if hGSTA4 mRNA expression is inducible in human adult and fetal liver and if so, if inducibility is protective in situ. The results of this project will extend our understanding of the extent and variation of human risk to a highly toxic metabolite generated on exposure to environmental chemicals that is linked to a number of disease states. In addition, this project has important implications in terms of the in utero effects of pro-oxidant drugs or chemicals to which pregnant women are exposed.