Conjugation of potentially toxic electrophilic compounds by glutathione (GSH) is a major function of the mammalian liver and a significant mechanism of cellular defense against many hepatotoxic environmental contaminants. Acute depletion of GSH presents a clear threat to the organism by alteration of the GSH/GSSG ratio, the NAD(P)+/NAD(P)H ratio and the cytotoxic events associated with oxidative challenge. Chronic depletion of GSH due to protracted exposure to GSH depleting agents through air and water contamination presents a different set of toxin related challenges induced by the continuous depletion of GSH and the presence of elevated intracellular levels of the GSH conjugate. Resynthesis of GSH is limited by cysteine concentration. The mechanism by which cysteine is supplied varies between cell types; some dependent on dietary absorbtion, some synthesizing cysteine from methionine, while others are proposed to depend on recovery of cysteine by degradation of GSH by Gamma-glutamyl transpeptidase. The proposed research will examine the effect of GSH depletion by diethylmaleate, ethacrynic acid, t-butylhydroperoxide, and 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD), chemicals representative of the major class of xenobiotic GSH depleting agents, or the thiol status of selected cell types. The cells to be examined are primary cultures of rat and human hepatocytes, hepatocarcinoma HR-35 cells, the nontumorigenic epithelial adult rat liver ARL-15C1 and the tumorigenic line ARL-16T2. This collection of cells presents a developmental and transformational gradient from normal to transformed lines with intermediate stages also represented. More importantly, an apparent range of cysteine recovery mechanisms is also represented, from primarily transsulfuration to a strict cysteine requirement for growth. The specific goals to be accomplished are: 1) Evaluation of the thiol status in ARL-15C1 and 16T2 cell lines. 2) Characterization of the mitochondrial pool of GSH in ARL-15C1 and 16T2 and the effect of pool depletion on toxicity. 3) Characterize the thiol status of HR-35 hepatocarcinoma cells. 4) Determine the effect of chronic GSH depletion on chemical toxicity in Gamma-glutamyl transpeptidase containing cells. 5) By cell fusion introduce Gamma-glutamyl transpeptidase into nontumorigenic cells to evaluate alterations in the thiol status. 6) Elucidate mechanism of acute toxicity of TCDD in ARL-15C1. 7) Investigate the regulation of Gamma-cystathionase activity in rat hepatocytes before and after GSH depletion. 8) Examine the thiol status of human hepatocytes and evaluate the importance of the mitochondrial GSH pool.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES003272-06
Application #
3250453
Study Section
Toxicology Subcommittee 2 (TOX)
Project Start
1984-07-01
Project End
1992-12-31
Budget Start
1991-01-01
Budget End
1991-12-31
Support Year
6
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Oregon Health and Science University
Department
Type
Schools of Medicine
DUNS #
009584210
City
Portland
State
OR
Country
United States
Zip Code
97239
Sierra-Rivera, E; Meredith, M J; Voorhees, G J et al. (1994) Synthesis of heat shock proteins following oxidative challenge: role of glutathione. Int J Hyperthermia 10:573-86
Malinow, M R; Axthelm, M K; Meredith, M J et al. (1994) Synthesis and transsulfuration of homocysteine in blood. J Lab Clin Med 123:421-9
Freeman, M L; Sierra-Rivera, E; Voorhees, G J et al. (1993) Synthesis of hsp-70 is enhanced in glutathione-depleted Hep G2 cells. Radiat Res 135:387-93
Saunders, E L; Maines, M D; Meredith, M J et al. (1991) Enhancement of heme oxygenase-1 synthesis by glutathione depletion in Chinese hamster ovary cells. Arch Biochem Biophys 288:368-73
Meredith, M J (1991) Variations in gamma-glutamyl transpeptidase glycosylation and kinetic parameters in cultured liver cells. Biochem Int 25:321-30
Meredith, M J (1991) Glutathione and glutathione conjugate efflux from cultured liver cells. Cell Biol Toxicol 7:15-33
Saunders, E L; Meredith, M J; Eisert, D R et al. (1991) Depletion of glutathione after gamma irradiation modifies survival. Radiat Res 125:267-76
Freeman, M L; Meredith, M J; Eisert, D R (1990) Failure of chronic glutathione elevation to reduce cytotoxicity produced by exposure to cis-diamminedichloroplatinum(II), ionizing radiation, or hyperthermia. Cancer Res 50:5296-300
Gale, G R; Smith, A B; Atkins, L M et al. (1990) Glutathione status, glutathione monoisopropyl ester, and cadmium metabolism in mice. Res Commun Chem Pathol Pharmacol 70:71-91
Freeman, M L; Spitz, D R; Meredith, M J (1990) Does heat shock enhance oxidative stress? Studies with ferrous and ferric iron. Radiat Res 124:288-93

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