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Degree

PhD, University of Cincinnati, Medicine : Toxicology (Environmental Health), 2007.

Abstract

Glutathione (GSH) is the most abundant cellular thiol antioxidant, disturbance in GSH homeostasis has been associated with several oxidative stress-related diseases and aging. The rate-limiting enzyme in GSH biosynthesis, glutamate-cysteine ligase (GCL), is composed of a catalytic subunit (GCLC) and a modifier subunit (GCLM). Using mouse recombinant proteins, we demonstrate that: (a) binding of GCLM to GCLC is efficient in vitroto form the holoenyzme; (b) compared with GCLC, the holoenzyme has a higher Vmax, lower Kms for glutamate and ATP, and a higher Ki for GSH feedback inhibition; (c) GCLM may limit the levels of GSH biosynthesis in most of the tissues. In agreement with the in vitroresults, Gclm(-/-)mice have a 60-90% depletion of tissue GSH, whereas showing no overt phenotype; Gclm(-/-)mouse fetal fibroblasts (MFFs), however, are highly sensitive to oxidative insults. We therefore used Gclm(-/-)MFFs to study the role of GSH in cellular aging. Gclm(-/-)MFFs, having 25% of normal intracellular GSH, undergo premature senescence in culture. This phenotype is accompanied by increased cellular ROS and DNA damage, and induction of p53 and p21 proteins. N-acetylcysteine (NAC) supplementation restores intracellular GSH to control levels and prevents premature senescence in Gclm(-/-)MFFs. We conclude that GSH homeostasis is an important determinant in cellular senescence. As GSH depletion has been associated with numerous liver diseases, the specific role of GSH deficiency in liver pathophysiology has been investigated using the hepatocyte-specific GCLC knockout [ Gclc(h/h)] mice. Having progressive depletion of hepatic GSH to 4.5% of control levels, Gclc(h/h)mice develop severe steatosis and die of liver failure within a month. Hepatic mitochondria have concomitant depletion of GSH to 15% and appear to be the major affected organelle, showing atypical morphology and dysfunction. NAC supplementation in the drinking water prevents mortality in the Gclc(h/h)mice, by preserving mitochondrial GSH up to 65% of control levels and partially restoring their function. These NAC-rescued Gclc(h/h)mice, however, still reveal persistent oxidative stress in the liver and develop liver cirrhosis with age. These data indicate the essential role of GSH homeostasis in maintaining normal liver function.

Subject Headings

Health Sciences, Toxicology

Keywords

glutathione; oxidative stress; knockout; senescence; steatosis

Advisor

Dr. Daniel W. Nebert

Pages

181p.

Document number: ucin1172086646
Permalink: http://rave.ohiolink.edu/etdc/view?acc_num=ucin1172086646

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