Prior studies in our laboratory and others have documented ethanol- related oxidative stress in the developing brain and there is evidence suggesting that it may cause at least some of the neurotoxic effects of ethanol (E). The mechanism(s) by which E causes this oxidative stress remain to be determined, but it is likely related to relatively low anti- oxidant status. There is a compelling literature illustrating that astrocytes play a vital neuroprotective role by producing and exporting reduced glutathione (GSH) and we include, as preliminary data, evidence that astrocyte GSH homeostasis may be perturbed by E in the developing brain. Hypothesis. We hypothesize that one mechanism underlying the E- mediated oxidative stress and subsequent damage to the developing brain is an impairment of the ability of astrocytes to generate and/or export GSH. Hypothesis: We hypothesize that one mechanisms underlying the E- mediated oxidative stress and subsequent damage to the developing brain is an impairment of the ability of astrocytes to generate and/or export GSH.
Specific Aims. The following specific aims are extensions of our preliminary studies and those of others which document an E-mediated reduction of GSH in cultured neonatal cortical astrocytes. They will determine the means by which E causes this effect. They focus on E effects on well established control points in GSH synthesis and on its efflux. Additionally, experiments will determine the effect of E on viability of astrocytes with respect to mitochondrial functions key to survival and generation of oxygen species which may deplete astrocyte GSH.
Specific Aim One will determine the effect(s) of E on astrocyte uptake of precursors required for the synthesis of GSH. We will focus on the effects of E on inward directed transport of cystine and cysteine, which are known determinants of GSH synthesis, but we will also determine effects of E on cellular content of other relevant amino acids e.g. glutamate glycine. These studies will also determine effects of E on GSH uptake and efflux by astrocytes.
Specific Aim Two will determine effects of E on the enzymatic synthesis of GSH by astrocytes with a focus on the impact on activity of gamma- glutamylcysteine synthetase, the rate-limiting enzyme in GSH synthesis. Additionally, effects of E on astrocyte-bound gamma-glutamyl transpeptidas4e as a source on CysGly for neuronal GSH synthesis. will be determined.
Specific Aim Three will determine the effects of E on astrocyte variability at the mitochondrial . The emphasis on the mitochondrion is due to it being a target for E toxicity, the source of ATP for GSH synthesis, and a primary source of oxidative stress. These experiments will determine E-related depletion of mitochondrial GSH, production of oxidative products which deplete mitochondria of GSH, and induction of mitochondrially-mediated cell death which we document in the appendix.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21AA013431-02
Application #
6620857
Study Section
Health Services Research Review Subcommittee (AA)
Program Officer
Foudin, Laurie L
Project Start
2002-03-01
Project End
2005-02-28
Budget Start
2003-03-01
Budget End
2004-02-29
Support Year
2
Fiscal Year
2003
Total Cost
$146,000
Indirect Cost
Name
University of Texas Health Science Center San Antonio
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
800772162
City
San Antonio
State
TX
Country
United States
Zip Code
78229
Green, C R; Watts, L T; Kobus, S M et al. (2006) Effects of chronic prenatal ethanol exposure on mitochondrial glutathione and 8-iso-prostaglandin F2alpha concentrations in the hippocampus of the perinatal guinea pig. Reprod Fertil Dev 18:517-24
Rathinam, Mary Latha; Watts, Lora Talley; Stark, Avishay A et al. (2006) Astrocyte control of fetal cortical neuron glutathione homeostasis: up-regulation by ethanol. J Neurochem 96:1289-300
Watts, Lora Talley; Rathinam, Mary Latha; Schenker, Steven et al. (2005) Astrocytes protect neurons from ethanol-induced oxidative stress and apoptotic death. J Neurosci Res 80:655-66