The overall goal of this proposal is to define the function of glutathione (GSH) in mammals. Mice deficient in the heavy (catalytic) subunit of gamma-glutamyl cysteine synthetase (gamma-GCS-HSU), the rate limiting enzyme in GSH synthesis, diet at post coital day 7.5 and completely lack GSH; however, cell lines derived from gamma-GCS- HSU-deficient embryos survive if GSH is provided in the medium. These cells contain only 50-60 mum GSH (1-2% of wild type levels). Preliminary data indicate that removal of GSH from the medium produces a fall in GSH to undetectable levels, increased hydroxyl radical formation, activation of JNK/SAPK and cell death. These data suggest that very low GSH levels (,1% of wild type cells) whether """"""""spontaneous"""""""" or induced by chemicals/radiation may be a key trigger that initiates the oxidative stress response and apoptosis. The gamma-GCS-HSU-deficient mice and cells we have developed and additional knock-out mice and cells we plan to construct provide a unique opportunity to test four hypotheses about GSH function: 1. Only low levels of GSH are required for survival of unstressed cells, but high levels are necessary to protect cells from chemical and radiation injury, and this protection is directly proportional to GSH level. 2. Very low/absent levels of cellular GSH activate a cellular stress response program that includes JNK/SAPK, p38 MAPK and NFkappaB, and activation of these pathways by chemicals radiation depends upon their ability to lower GSH levels. 3.Very low-absent levels of cellular GSH potentiate apoptotic cell death, and chemicals/radiation that induce apoptosis do so by lowering cellular GSH levels. The process involves suppression of anti-apoptotic factors and activation of pro-apoptotic factors. 4. Only low levels of GSH are needed for development and survival of unstressed mice. These low levels of GSH render mice hypersensitive to chemical injury.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES008668-07
Application #
6624932
Study Section
Chemical Pathology Study Section (CPA)
Program Officer
Thompson, Claudia L
Project Start
1996-12-01
Project End
2004-11-30
Budget Start
2002-12-01
Budget End
2003-11-30
Support Year
7
Fiscal Year
2003
Total Cost
$231,247
Indirect Cost
Name
Baylor College of Medicine
Department
Pathology
Type
Schools of Medicine
DUNS #
051113330
City
Houston
State
TX
Country
United States
Zip Code
77030
Habib, Geetha M (2010) Arsenite causes down-regulation of Akt and c-Fos, cell cycle dysfunction and apoptosis in glutathione-deficient cells. J Cell Biochem 110:363-71
Habib, Geetha M (2009) p53 regulates Hsp90beta during arsenite-induced cytotoxicity in glutathione-deficient cells. Arch Biochem Biophys 481:101-9
Habib, Geetha M; Shi, Zheng-Zheng; Lieberman, Michael W (2007) Glutathione protects cells against arsenite-induced toxicity. Free Radic Biol Med 42:191-201
Valverde, Mahara; Rojas, Emilio; Kala, Subbarao V et al. (2006) Survival and cell death in cells constitutively unable to synthesize glutathione. Mutat Res 594:172-80
Rojas, E; Shi, Z-Z; Valverde, M et al. (2003) Cell survival and changes in gene expression in cells unable to synthesize glutathione. Biofactors 17:13-9
Shi, Z Z; Osei-Frimpong, J; Kala, G et al. (2000) Glutathione synthesis is essential for mouse development but not for cell growth in culture. Proc Natl Acad Sci U S A 97:5101-6
Habib, G M; Lieberman, M W (1999) Cleavage of leukotriene D4 in mice with targeted disruption of a membrane-bound dipeptidase gene. Adv Exp Med Biol 469:295-300
Habib, G M; Cuevas, A A; Barrios, R et al. (1999) Mouse leukotriene A4 hydrolase is expressed at high levels in intestinal crypt cells and splenic lymphocytes. Gene 234:249-55
Carter, B Z; Shi, Z Z; Barrios, R et al. (1998) gamma-glutamyl leukotrienase, a gamma-glutamyl transpeptidase gene family member, is expressed primarily in spleen. J Biol Chem 273:28277-85
Habib, G M; Shi, Z Z; Cuevas, A A et al. (1998) Leukotriene D4 and cystinyl-bis-glycine metabolism in membrane-bound dipeptidase-deficient mice. Proc Natl Acad Sci U S A 95:4859-63