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.
