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1 From the Departments of Biochemistry and 2 Ophthalmology, Program in Nutritional Health Sciences, School of Medicine, Emory University, Atlanta, Georgia; and the 3 Department of Environmental Health, University of Washington Seattle.
Abstract
PURPOSE. To determine the effect of dimethylfumarate (DMF), an inducer of glutathione (GSH)-dependent detoxification, on intracellular GSH levels in cultured human retinal pigment epithelium (hRPE) cells, its mechanism of action, and its effect on hRPE cells subjected to oxidative injury.
METHODS. Established hRPE cell lines were treated with DMF and assayed by
high-pressure liquid chromatography for intracellular and extracellular
GSH levels. Quantification of 
-glutamylcysteine synthetase (GLCL)
was determined through northern and western blot analyses, and activity
was measured. Effects of pretreatment with DMF on GSH redox status of
hRPE cells was determined. Sensitivity of hRPE cells to oxidative
stress was determined using tert-butylhydroperoxide as the
oxidative agent.
RESULTS. Dimethylfumarate caused a transient decrease followed by a significant increase in intracellular GSH. Glutathione increased maximally at 24 hours with 100 to 200 µM DMF. The initial decrease could be accounted for by the formation of a DMF-GSH conjugate. Dimethylfumarate treatment increased the steady state mRNA expression of the regulatory subunit of GLCL, but no increase was seen for the catalytic subunit. However, protein levels were increased for both, and the catalytic activity of GLCL was also increased. Whereas the initial decrease in GSH made hRPE cells more susceptible to oxidative damage, pretreatment with DMF under conditions that increased intracellular GSH protected hRPE cells against oxidative damage.
CONCLUSIONS. These results suggest a means by which the antioxidant capability of hRPE may be augmented without direct antioxidant supplementation. Specifically, a dietary compound that conjugates with GSH can induce GSH synthesis, increase GSH concentration, and improve protection by GSH-dependent detoxification pathways in hRPE. However, the early depletion of GSH before stimulated synthesis necessitates caution in prevention strategies using dietary inducers.
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