Elevation of brain glutathione by γ-glutamylcysteine ethyl ester protects against peroxynitrite-induced oxidative stress

Elevation of glutathione (GSH) has been recognized as an important method for modulating levels of reactive oxygen species (ROS) in the brain. We investigated the antioxidant properties of γ-glu-cys-ethyl ester (GCEE) in vitro and its ability to increase GSH levels upon in vivo i.p. injection. GCEE displays antioxidant activity similar to GSH as assessed by various in vitro indices such as hydroxyl radical scavenging, dichlorofluorescein fluorescence (DCF), protein specific spin labeling, glutamine synthetase (GS) activity, and protein carbonyls. Intraperitoneal injection of GCEE to gerbils resulted in a 41% increase in brain total GSH levels in vivo as determined by the DTNB-GSH reductase recycling method. Gerbils injected with buthionine sulfoximine (BSO), an inhibitor of γ-glutamylcysteine synthetase, had 40% less total brain glutathione. Gerbils injected with BSO followed by a GCEE injection had GSH levels similar to vehicle-injected controls, suggesting that GCEE upregulates GSH biosynthesis by providing γ-glutamylcysteine and not cysteine. Cortical synaptosomes from GCEE-injected animals were less susceptible to peroxynitrite-induced oxidative damage as assessed by DCF fluorescence, protein-specific spin labeling, and GS activity. These experiments suggest that GCEE is effective in increasing brain GSH levels and may potentially play an important therapeutic role in attenuating oxidative stress in neurodegenerative diseases associated with oxidative stress such as Alzheimer disease.