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Natalia Martínez, Sandra Atienzar, Miguel Flores-Bellver, Luis Bonet, Javier Sancho-Pelluz, Jorge M Barcia, Francisco J Romero; CYP2E1 mediated ethanol metabolism in ARPE-19 cells.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6322.
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Alcohol consumption has a high prevalence in most countries and it is the drug with the highest use and abuse worldwide. Chronic ethanol consumption produces an increase in lipid peroxidation products (MDA) and a decrease in antioxidant factors, (GSH) and its related enzymes, which can eventually induce apoptosis-mediated cell death on central nervous system. Retina is particularly sensitive to free radicals (ROS) and lipid peroxidation. Pigmented epithelial (RPE) cells play a major role in retinal homeostasis. There are few studies on ethanol-induced RPE injury related to oxidative stress. We used ARPE-19 cells to study if there is ethanol metabolism via CYP2E1 (P450) in retinal pigment epithelium.
The presence of P450 in ARPE-19 cells was studied by immunocytochemistry and Western Blot. Cell viability and apoptosis were studied by MTT and Western Blot. Regulation of CYP2E1 expression by ethanol-induced oxidative stress was studied by Western Blot, immunocytochemistry and RT-PCR, using a CYP2E1 selective inhibitor, Diallyl Sulfide (DAS). CYP2E1 activity was measured by colorimetric assay. The involvement of CYP2E1 in oxidative stress-mediated apoptosis and/or autophagy was analyzed by Western Blot, ELISA, and a proteome profiler cell stress assay.
We show for the first time the presence of CYP2E1 in RPE. Its expression was induced by ethanol and was concentration dependent. DAS treatment decreased the expression of the CYP2E1 and the amount of mRNA, reducing the formation of ROS in cultured cells.
CYP2E1 is present in retinal pigment epithelium and metabolizes ethanol; it is inducible on a concentration-dependent manner and DAS is able to inhibit its expression, reducing the formation of ROS.
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