Coordinate regulation of glutathione metabolism in astrocytes by Nrf2 Xiaojian Sun a , Heidi Erb a , Timothy H. Murphy a,b, * a Department of Psychiatry, Kinsmen Laboratory of Neurological Research and Brain Research Center, 4835-2255 Wesbrook Mall, University of British Columbia, Vancouver, BC, Canada V6T 1Z3 b Department of Physiology, Kinsmen Laboratory of Neurological Research and Brain Research Center, 4835-2255 Wesbrook Mall, University of British Columbia, Vancouver, BC, Canada V6T 1Z3 Received 8 November 2004 Available online 18 November 2004 Abstract The tripeptide glutathione (GSH) represents the major brain thiol and is essential for prevention of oxidative stress. Using mono- chlorobimane to label intracellular GSH in a glutathione S-transferase catalyzed reaction we have examined the kinetics of GSH metabolism including its rate of conjugation, total GSH content, synthesis, and efflux in astrocyte cultures under basal conditions and after induction of antioxidant response element (ARE)-mediated gene expression by the transcription factor Nrf2. In the pres- ence of a cerebral spinal fluid-like salt solution astrocytes could not synthesize detectable levels of GSH. Addition of GSH precur- sors, cystine, glutamate, and glycine, rapidly restored GSH synthesis. Astrocytes were able to use either glutamate or glutamine as precursors equally for GSH synthesis. Using the small molecule chemical inducer tert-butylhydroqunione (tBHQ) we report that induction of ARE-mediated gene expression is associated with a coordinated increase in GSH content and synthesis rate with little effect on the rate of GSH conjugation or efflux. Consistent with the effect of the inducer, adenovirus-mediated overexpression of the transcription factor Nrf2 that mediates tBHQÕs effects also increased GSH content, confirming that GSH metabolism can be regu- lated by the Nrf2 pathway. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Glutathione; Monochlorobimane; Phase II detoxification enzymes; Nrf2; tert-Butylhydroquinone; Antioxidant response element; Oxidative stress; Neuroprotection The tripeptide glutathione (GSH; c-L-glutamyl-L-cys- teinyl-glycine) is the most abundant thiol in mammalian cells with concentrations of up to 12 mM [9]. It is a cen- tral component of the antioxidant defences of cells, act- ing both to directly detoxify reactive oxygen species (ROS) and as a cofactor for several peroxidases [11]. It can also detoxify xenobiotics by forming S-conjugates, catalyzed by glutathione S-transferase (GST). A com- promised GSH system in the brain has been connected with oxidative stress occurring in neurological diseases, such as ParkinsonÕs disease (PD), AlzheimerÕs disease, HuntingtonÕs disease, and stroke [14]. Examining the mechanism of glutathione metabolism will provide in- sight into the understanding of how brain copes with oxidative stress and may provide therapeutic insight. It is well established that, in the central nervous sys- tem, there is a compartmentalization of GSH between neurons and glia, with glia, especially astrocytes, in gen- eral exhibiting higher levels of GSH [29,30]. The func- tion of this astrocytic GSH is unclear. Some studies suggest that it may be exported to neighboring cells [36] to provide antioxidant substrates. Cell culture is an attractive model for examining the function of astro- cytic GSH since relatively pure populations of astrocytes can be cultivated [34]. Since GSH plays a pivotal role in protecting cells, there has been a considerable interest in the develop- ment of methods to quantify the cellular GSH content. 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.11.031 * Corresponding author. Fax: +1 604 822 7981. E-mail address: thmurphy@interchange.ubc.ca (T.H. Murphy). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 326 (2005) 371–377 BBRC