ToxSci Advance Access originally published online on September 5, 2007
Toxicological Sciences 2007 100(2):433-444; doi:10.1093/toxsci/kfm236
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Glutathione Levels Modulate Domoic Acid–Induced Apoptosis in Mouse Cerebellar Granule Cells
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* Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington 98105
Department of Human Anatomy, Pharmacology, and Forensic Science, University of Parma Medical School, 43100 Parma, Italy
1 To whom correspondence should be addressed at Department of Environmental and Occupational Health Sciences, University of Washington, 4225 Roosevelt # 100, Seattle, WA 98105. Fax: (206) 685-4696. E-mail: lgcosta{at}u.washington.edu.
Received July 19, 2007; accepted August 28, 2007
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Abstract |
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Exposure of mouse cerebellar granule neurons (CGNs) to domoic acid induced cell death, either by apoptosis or by necrosis, depending on its concentration. Necrotic damage predominated in response to domoic acid above 0.1µM. In contrast, cell injury with apoptotic features (assessed by Hoechst staining and DNA laddering assay) was evident after exposure to lower concentrations of domoic acid (
0.1µM). The AMPA (
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)/kainate receptor antagonist 2,3-dihydroxy-6-nitro-sulfamoylbenzo [f] quinoxaline, but not the N-methyl-D-aspartate receptor antagonist MK-801, prevented domoic acid–induced apoptosis. To evaluate the role of oxidative stress in domoic acid–induced apoptosis, experiments were carried out in CGNs isolated from wild-type mice (Gclm (+/+)) and mice lacking the modifier subunit of glutamate-cysteine ligase, the first and rate-limiting step of glutathione (GSH) biosynthesis (Gclm (–/–)). CGNs from Gclm (–/–) mice have very low levels of GSH and were more sensitive to domoic acid–induced apoptosis and necrosis than Gclm (+/+) CGNs. The antioxidant melatonin (200µM) and the membrane-permeant GSH delivery agent GSH ethyl ester (2.5mM) prevented domoic acid–induced apoptosis. Domoic acid increased formation of reactive oxygen species but did not affect intracellular GSH levels. Domoic acid also increased cytosolic and mitochondrial calcium levels, increased oxidative stress in mitochondria, and altered mitochondrial membrane potential, which ultimately caused cytochrome c release, activation of caspase-3, and degradation of poly (ADP-ribose) polymerase. These results indicate that low concentrations of domoic acid cause apoptotic neuronal cell death mediated by oxidative stress.
Key Words: apoptosis; domoic acid; glutamate-cysteine ligase; glutamate receptors; glutathione; oxidative stress.