HUMAN FETAL LIVER GST--HYDROXYNONENAL CONJUGATION

人胎肝 GST--羟基壬醛结合

基本信息

批准号：
6917762
负责人：
EVAN P GALLAGHER
金额：
$ 20.01万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-05-01 至 2005-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6917762
关键词：
aldehydes cell line cytotoxicity embryo /fetus cell /tissue gene expression glutathione transferase isozymes liver messenger RNA metabolism placenta polymerase chain reaction protein purification protein sequence tissue /cell culture transfection western blottings

项目摘要

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.

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，也是迄今为止测试的最具细胞毒性和致突变性的醛之一。 4HNE 被认为是许多环境相关疾病的致病因素，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 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捐献者没有可检测到的肝脏 GST-4HNE 活性。此外，中期妊娠胎儿不能通过 ADH 有效解毒 4HNE，ADH 是 4HNE 去除的竞争途径。因此，未出生的孩子可能是对子宫内促氧化剂敏感的亚群。该资助的具体目标和方法是 1) 使用体外部分和人类肝脏切片培养物来表征和比较成人和胎儿肝脏中的 4HNE 代谢，2) 全面表征肝脏 GST-4HNE 活性和 hGSTA4- 的变化使用定量 PCR 和定量蛋白质印迹法检测成人和胎儿肝脏以及胎盘中的 4 同工酶表达，3) 鉴定胎儿肝脏和胎盘中代谢的潜在新型 GST 亚型通过 GST 蛋白纯化、测序和免疫印迹分析 4HNE，4) 使用转染的细胞系和精密肝切片的动态培养来确定较差的 4HNE 接合器和胎儿是否面临 4HNE 相关细胞毒性的风险增加，以及 5) 确定 hGSTA4 mRNA 是否表达在成人和胎儿肝脏中是可诱导的，如果是这样，则诱导性是否具有原位保护性。该项目的结果将加深我们对人类因暴露于与多种疾病状态相关的环境化学物质而产生的高毒性代谢物的风险程度和变化的理解。此外，该项目对于孕妇接触的促氧化药物或化学物质的子宫内影响具有重要意义。