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.

National Institute of Health (NIH)
National Institute of Environmental Health Sciences (NIEHS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-ALTX-1 (01))
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Mcclure, Michael
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University of Florida
Schools of Veterinary Medicine
United States
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Gallagher, Evan P; Huisden, Christiaan M; Gardner, James L (2007) Transfection of HepG2 cells with hGSTA4 provides protection against 4-hydroxynonenal-mediated oxidative injury. Toxicol In Vitro 21:1365-72
Steiner, Claudia; Peters, Wilbert H M; Gallagher, Evan P et al. (2007) Genistein protects human mammary epithelial cells from benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide and 4-hydroxy-2-nonenal genotoxicity by modulating the glutathione/glutathione S-transferase system. Carcinogenesis 28:738-48
Gallagher, Evan P; Gardner, James L; Barber, David S (2006) Several glutathione S-transferase isozymes that protect against oxidative injury are expressed in human liver mitochondria. Biochem Pharmacol 71:1619-28
Moneypenny, Craig G; Gallagher, Evan P (2005) 4-Hydroxynonenal inhibits cell proliferation and alters differentiation pathways in human fetal liver hematopoietic stem cells. Biochem Pharmacol 69:105-12
Knoll, Nadine; Ruhe, Carola; Veeriah, Selvaraju et al. (2005) Genotoxicity of 4-hydroxy-2-nonenal in human colon tumor cells is associated with cellular levels of glutathione and the modulation of glutathione S-transferase A4 expression by butyrate. Toxicol Sci 86:27-35
Gardner, James L; Doi, Adriana M; Pham, Robert T et al. (2003) Ontogenic differences in human liver 4-hydroxynonenal detoxification are associated with in vitro injury to fetal hematopoietic stem cells. Toxicol Appl Pharmacol 191:95-106
Doi, Adriana M; Patterson, Paula E; Gallagher, Evan P (2002) Variability in aflatoxin B(1)-macromolecular binding and relationship to biotransformation enzyme expression in human prenatal and adult liver. Toxicol Appl Pharmacol 181:48-59
Gallagher, Evan P; Gardner, James L (2002) Comparative expression of two alpha class glutathione S-transferases in human adult and prenatal liver tissues. Biochem Pharmacol 63:2025-36
Gallagher, E P; Sheehy, K M (2001) Effects of phenytoin on glutathione status and oxidative stress biomarker gene mRNA levels in cultured precision human liver slices. Toxicol Sci 59:118-26
Gardner, J L; Gallagher, E P (2001) Development of a peptide antibody specific to human glutathione S-transferase alpha 4-4 (hGSTA4-4) reveals preferential localization in human liver mitochondria. Arch Biochem Biophys 390:19-27