Abstract

This project will investigate the mechanism of paraquat cytotoxicity. Despite a consensus that oxygen and the redox properties of paraquat are involved, the ultimate cytotoxic event and the protective role of individual anti-oxidant enzymes have not been identified. This project will investigate these events by a novel approach. The ability of anti- oxidant enzymes to protect against paraquat cytotoxicity will be studied on an individual basis. This will be accomplished by coupling the cDNA of these enzymes to suitable expression vectors. Anti-oxidant enzymes to be studied include copper-zinc superoxide dismutase, manganese superoxide dismutase, glutathione peroxidase, catalase, metallothionein, and glutathione transferases. Two different, selectable, expression vectors will be constructed for each enzyme. One vector will use the noninducible SV promoter, and the other will use the inducible mouse metallothionein (mMT). Preliminary results with a copper-zinc superoxide dismutase expression vector [SV-CuZnSOD-SVneo] indicate the enzyme is produced in large quantities and paraquat resistance develops. The increase in resistance does not correlate to superoxide dismutase activity, but to increase activity of each enzyme against paraquat will be compared to others to determine which enzyme is most effective. The induction of the anti-oxidant enzymes copper-zinc superoxide dismutase, manganese superoxide dismutase, and catalase by paraquat will be studied to determine if the increase occurs by gene amplification, alterations in mRNA, or protein synthesis. In contrast to these anti-oxidant enzymes, glutathione peroxidase appears to be selectively inhibited by paraquat. The mechanism of cell line. This cell line appears to be resistant to paraquat on the basis of increased glutathione peroxidase activity, and not other anti-oxidant enzymes (such as superoxide dismutase), again suggesting that glutathione peroxidase plays an important role in detoxifying paraquat. The mechanism and cellular level (DNA, RNA, protein synthesis) by which this cell continues to express glutathione peroxidase, even when exposed to paraquat, will be determined.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES004989-04
Application #
3253191
Study Section
Respiratory and Applied Physiology Study Section (RAP)
Project Start
1990-07-01
Project End
1995-06-30
Budget Start
1993-07-01
Budget End
1994-06-30
Support Year
4
Fiscal Year
1993
Total Cost
Indirect Cost

  Institution

Name
University of California San Diego
Department
Type
Schools of Medicine
DUNS #
077758407
City
La Jolla
State
CA
Country
United States
Zip Code
92093

  Publications

Estonius, M; Forsberg, L; Danielsson, O et al. (1999) Distribution of microsomal glutathione transferase 1 in mammalian tissues. A predominant alternate first exon in human tissues. Eur J Biochem 260:409-13
de Haan, J B; Bladier, C; Griffiths, P et al. (1998) Mice with a homozygous null mutation for the most abundant glutathione peroxidase, Gpx1, show increased susceptibility to the oxidative stress-inducing agents paraquat and hydrogen peroxide. J Biol Chem 273:22528-36
de Haan, J B; Wolvetang, E J; Cristiano, F et al. (1997) Reactive oxygen species and their contribution to pathology in Down syndrome. Adv Pharmacol 38:379-402
Kelner, M J; Estes, L; Rutherford, M et al. (1997) Heterologous expression of carbonyl reductase: demonstration of prostaglandin 9-ketoreductase activity and paraquat resistance. Life Sci 61:2317-22
de Haan, J B; Cristiano, F; Iannello, R et al. (1996) Elevation in the ratio of Cu/Zn-superoxide dismutase to glutathione peroxidase activity induces features of cellular senescence and this effect is mediated by hydrogen peroxide. Hum Mol Genet 5:283-92
Kelner, M J; Stokely, M N; Stovall, N E et al. (1996) Structural organization of the human microsomal glutathione S-transferase gene (GST12). Genomics 36:100-3
Kelner, M J; Uglik, S F (1995) Superoxide dismutase abolishes the platelet-derived growth factor-induced release of prostaglandin E2 by blocking induction of nitric oxide synthase: role of superoxide. Arch Biochem Biophys 322:31-8
Kelner, M J; Bagnell, R D; Uglik, S F et al. (1995) Heterologous expression of selenium-dependent glutathione peroxidase affords cellular resistance to paraquat. Arch Biochem Biophys 323:40-6
Kelner, M J; Bagnell, R; Montoya, M et al. (1995) Transfection with human copper-zinc superoxide dismutase induces bidirectional alterations in other antioxidant enzymes, proteins, growth factor response, and paraquat resistance. Free Radic Biol Med 18:497-506
Kelner, M J; Uglik, S F (1994) Mechanism of prostaglandin E2 release and increase in PGH2/PGE2 isomerase activity by PDGF: involvement of nitric oxide. Arch Biochem Biophys 312:240-3

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