HUMAN FETAL LIVER GST--HYDROXYNONENAL CONJUGATION

4-羟基壬烯醛（4 HNE，C9 H16 O2）是脂质过氧化过程中形成的主要α，β-不饱和羰基，是迄今为止测试的最具细胞毒性和致突变性的醛之一。 4 HNE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。 4 HNE在人体组织中通过乙醇脱氢酶（ADH）、乙醛脱氢酶（ALDH）和谷胱甘肽S-转移酶（GST）解毒。 在这些酶中，人GST同工酶A4（hGSTA 4 -4）对4 HNE具有独特且特别高的活性。 我们已经发现，一些个人和中期妊娠胎儿捐赠者没有检测到肝脏GST-4 HNE活性。 此外，孕中期胎儿不能通过ADH有效地使4 HNE解毒，ADH是4 HNE去除的竞争途径。 因此，未出生的孩子可能是一个敏感的亚群在子宫内促氧化剂。 本基金的具体目标和方法是：1）使用人肝切片的体外组分和培养物表征和比较人成人和胎儿肝脏中的4 HNE代谢，2）使用定量PCR和定量蛋白质印迹法充分表征成人和胎儿肝脏以及胎盘中肝脏GST-4 HNE活性和hGSTA 4 -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胎儿肝脏和胎盘中代谢4 HNE的潜在的新GST同种型，4）使用转染的细胞系和精确肝脏切片的动态培养来确定不良4 HNE缀合物和胎儿是否处于4 HNE相关细胞毒性的增加的风险中，和5）确定hGSTA 4 mRNA表达在人成人和胎儿肝脏中是否是可诱导的，如果是，诱导是否在原位具有保护性。 该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，这种代谢物与一些疾病状态有关。 此外，该项目对孕妇接触的促氧化剂药物或化学品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.