HUMAN FETAL LIVER GST--HYDROXYNONENAL CONJUGATION

4-羟基壬烯醛（4HNE, C9H16O2）是在脂质过氧化过程中形成的主要的α， β -不饱和羰基，也是迄今为止测试过的最具细胞毒性和诱变性的醛之一。4HNE已被认为是许多环境相关疾病的致病因子，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在人体组织中通过乙醇脱氢酶（ADH）、醛脱氢酶（ALDH）和谷胱甘肽s -转移酶（GST）解毒。在这些酶中，人类GST同工酶A4 （hGSTA4-4）对4HNE具有独特且特别高的活性。我们发现一些个体和妊娠中期胎儿供体没有可检测到的肝GST-4HNE活性。此外，妊娠中期胎儿不能通过抗利尿激素有效解毒4HNE，而抗利尿激素是清除4HNE的竞争途径。因此，未出生的孩子可能是对子宫内促氧化剂敏感的亚群。该资助的具体目的和方法将是：1)利用体外培养和人肝切片表征和比较成人和胎儿肝脏中的4HNE代谢；2)利用定量PCR和定量western blotting全面表征成人和胎儿肝脏和胎盘中肝脏GST-4HNE活性和hGSTA4-4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出在胎儿肝脏和胎盘中代谢4HNE的潜在的新型GST亚型；4)利用转染细胞系和精密肝脏切片的动态培养来确定不良的4HNE共轭子和胎儿发生4HNE相关细胞毒性的风险是否增加；5)确定hGSTA4 mRNA在成人和胎儿肝脏中是否可诱导表达，如果可诱导，是否具有原位保护性。该项目的结果将扩大我们对接触环境化学品所产生的一种剧毒代谢物的人类风险的程度和变化的理解，这种代谢物与若干疾病状态有关。此外，该项目对孕妇接触的促氧化药物或化学物质在子宫内的影响具有重要意义。

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.