Reduced glutathione (GSH) is one of the cell's major defenses against oxidative stress. Lowered GSH levels have been implicated in susceptibility to numerous complex diseases (including neurodegenerative disorders, cancer, diabetes mellitus, cataracts, and AIDS) plus toxicity to environmental chemicals and heavy metal ions such as cadmium. Levels of GSH vary 10-fold between different cell types. The rate-limiting step in GSH biosynthesis is glutamate-cysteine ligase (GCL). GCL activity exists as the GCL catalytic subunit (GCLC) or as the GCL holoenzyme, a heterodimer composed of GCLC and a modifier subunit GCLM. Using gene targeting, we have generated conventional Gclm(-/-) and Cre-inducible Gclc(-/-) knockout mouse lines. GSH levels in Gclm(-/-) mice are only approximately 10% of that in Gclm(+/+) littermates in all tissues surveyed, yet, surprisingly, they are viable and fertile. Liver-specific Gclc(-/-) mice die by age 4 weeks, but can be rescued by N-acetylcysteine in the drinking water. With these mice, we are in a unique position to address the hypothesis that Gclm(-/-) mice will be susceptible to both environmental and endogenous toxicants because although the amount of GCLC controls the potential maximum level of cellular GSH, the GCLC/GCLM ratio determines the actual level. Thus, we will: [a] Evaluate endogenous, genotoxicity and oxidative stress in untreated Gclm(+/+) and Gclm(-/-) mice; [b] Assess cadmium-induced liver and kidney toxicity in Gclm(+/+) and Gclm(-/-) mice; and [c] Dissect the role of GCLC and GCLM in controlling GSH levels through inducible expression of GCLC and GCLM in double-knockout Gclc/Gclm(-/-) immortalized hepatocytes. By way of these studies, we will define further the role of GCLM and GSH during both heavy metal-induced as well as endogenously-induced oxidative stress, while evaluating the Gclm(-/-) mouse as a model for a compromised oxidative stress response. Further, we will delineate the roles of GCLC and GCLM in controlling GSH levels. These studies will provide valuable insight into understanding the etiology, preventive medicine, and the possible development of therapeutic intervention in the above-mentioned diseases and toxicities. For example, studies suggest that human genetic differences exist in toxicity to cadmium and other environmental toxicants that cause oxidative stress; moreover, the GCLC and GCLM genes are highly polymorphic. Our proposed research should therefore help focus future genotype-phenotype association studies between the appropriate DNA variant sites in the GCLC and GCLM genes and disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES012463-04
Application #
7072779
Study Section
Alcohol and Toxicology Subcommittee 4 (ALTX)
Program Officer
Maull, Elizabeth A
Project Start
2003-09-01
Project End
2008-05-31
Budget Start
2006-06-01
Budget End
2007-05-31
Support Year
4
Fiscal Year
2006
Total Cost
$316,042
Indirect Cost
Name
University of Cincinnati
Department
Public Health & Prev Medicine
Type
Schools of Medicine
DUNS #
041064767
City
Cincinnati
State
OH
Country
United States
Zip Code
45221
Chen, Ying; Curran, Christine P; Nebert, Daniel W et al. (2012) Effect of chronic glutathione deficiency on the behavioral phenotype of Gclm-/- knockout mice. Neurotoxicol Teratol 34:450-7
Chen, Ying; Johansson, Elisabet; Yang, Yi et al. (2010) Oral N-acetylcysteine rescues lethality of hepatocyte-specific Gclc-knockout mice, providing a model for hepatic cirrhosis. J Hepatol 53:1085-94
Johansson, Elisabet; Wesselkamper, Scott C; Shertzer, Howard G et al. (2010) Glutathione deficient C57BL/6J mice are not sensitized to ozone-induced lung injury. Biochem Biophys Res Commun 396:407-12
Chen, Ying; Johansson, Elisabet; Fan, Yunxia et al. (2009) Early onset senescence occurs when fibroblasts lack the glutamate-cysteine ligase modifier subunit. Free Radic Biol Med 47:410-8
Shertzer, Howard G; Schneider, Scott N; Kendig, Eric L et al. (2009) Tetrahydroindenoindole inhibits the progression of diabetes in mice. Chem Biol Interact 177:71-80
Gao, Ling; Wang, Jiakun; Sekhar, Konjeti R et al. (2007) Novel n-3 fatty acid oxidation products activate Nrf2 by destabilizing the association between Keap1 and Cullin3. J Biol Chem 282:2529-37
Chen, Ying; Yang, Yi; Miller, Marian L et al. (2007) Hepatocyte-specific Gclc deletion leads to rapid onset of steatosis with mitochondrial injury and liver failure. Hepatology 45:1118-28
Shertzer, Howard G; Genter, Mary B; Talaska, Glenn et al. (2007) 7H-dibenzo[c,g]carbazole metabolism by the mouse and human CYP1 family of enzymes. Carcinogenesis 28:1371-8
Yang, Yi; Chen, Ying; Johansson, Elisabet et al. (2007) Interaction between the catalytic and modifier subunits of glutamate-cysteine ligase. Biochem Pharmacol 74:372-81
Genter, Mary Beth; Clay, Corey D; Dalton, Timothy P et al. (2006) Comparison of mouse hepatic mitochondrial versus microsomal cytochromes P450 following TCDD treatment. Biochem Biophys Res Commun 342:1375-81

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