CYP2E1 activity on ARPE-19 cells: New approach to RPE ethanol metabolism

Abstract

Purpose: Cytochrome p450 2E1 (CYP2E1) is a detoxifying enzyme found in several tissues with particular affinity for ethanol as part of the microsomal ethanol oxidizing system. Ethanol is mainly catabolized in the liver by CYP2E1 among others detoxifying enzymes. The presence of CYP2E1 in other tissues strongly suggests an extra-hepatic ethanol metabolism, and this fact may lead to specific and local ethanol-related damage. Previous data from our laboratory shows that chronic ethanol consumption negatively affects rat retina and these negative alterations can be reversed by antioxidants. The finding of CYP2E1 in the human retinal pigment epithelium cell line ARPE-19 gives support to the aforementioned idea of an extra-hepatic ethanol metabolism. Results herein show for the first time the microsomal activity of CYP2E1 in ARPE 19 cells, mRNA and protein over expression after ethanol exposure.

Methods: CYP2E1 activity assay was determined by quantification of 4-nitrocatechol (4NC) formation. Preliminary experiments were conducted to determine linear metabolite formation kinetics with respect to time and microsomal protein concentration. Metabolite formation rate was calculated using known concentrations of 4NC as calibration standards (0 - 1000 nM) and dividing the amount of the metabolite formed by the incubation time and microsomal protein content (nmol/min.mg). The quantification of the metabolite formed was assayed by HPLC. RT PCR, q PCR and western blot were also used.

Results: We show for the first time the activity of microsomal CYP2E1 in ARPE-19 cells. Its activity was induced by ethanol and inhibited by diallyl sulphide (DAS). The same pattern was found for CYP2E1 mRNA and protein expression.

Conclusions: CYP2E1 is present in retinal pigment epithelium cells, metabolizes ethanol and it is inducible. DAS is able to inhibit its activity and expression