Effect of dietary inducer dimethylfumarate on glutathione in cultured human retinal pigment epithelial cells

PURPOSE. TO determine the effect of dimethylfumarate (DMF), an inducer of g lutathione (GSH)dependent detoxification, on intracellular GSH levels in cu ltured human retinal pigment epithelium (hRPE) cells, its mechanism of acti on, and its effect on hRPE cells subjected to oxidative injury. METHODS. Established hRPE cell lines were treated with DMF and assayed by h igh-pressure liquid chromatography for intracellular and extracellular GSH levels. Quantification of gamma-glutamylcysteine synthetase (GLCL) was dete rmined through northern and western blot analyses, and activity was measure d. Effects of pretreatment with DMF on GSH redox status of hRPE cells was d etermined. Sensitivity of hRPE cells to oxidative stress was determined usi ng tert-butylhydroperoxide as the oxidative agent. RESULTS. Dimethylfumarate caused a transient decrease followed by a signifi cant increase in intracellular GSH. Glutathione increased maximally at 24 h ours with 100 to 200 mu M DMF. The initial decrease could be accounted for by the formation of a DMF-GSH conjugate. Dimethylfumarate treatment increas ed the steady state mRNA expression of the regulatory subunit of GLCL, but no increase was seen for the catalytic subunit, However, protein levels wer e increased for both, and the catalytic activity of GLCL was also increased . Whereas the initial decrease in GSH made hRPE cells more susceptible to o xidative damage, pretreatment with DMF under conditions that increased intr acellular GSH protected hRPE cells against oxidative damage. CONCLUSIONS. These results suggest a means by which the antioxidant capabil ity of hRPE may be augmented without direct antioxidant supplementation. Sp ecifically, a dietary compound that conjugates with GSH can induce GSH synt hesis, 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 d ietary inducers.