PURPOSE: Cytochrome p450 2E1 (CYP2E1) is a detoxifying enzyme with particular affinity for ethanol (EtOH) expressed in several tissues. Although CYP2E1 has been identified in human RPE, nothing is known about its metabolic activity. Expression of CYP2E1 and activity after EtOH exposure have been studied in human RPE and ARPE-19 cells. METHODS: Ethanol-induced CYP2E1 mRNA expression was analyzed by RT-PCR and quantitative PCR (qPCR) from human donor RPE as well as from ARPE-19 cells. Expression of CYP2E1 protein was determined by Western blot. Cytoplasmic CYP2E1 location also was demonstrated by immunocytochemistry. Cell viability was studied by the colorimetric assay XTT after EtOH treatment. Diallyl sulfide (DAS) was used to inhibit CYP2E1 activity. The microsomal CYP2E1 activity assay was determined by quantification of 4-nitrocatechol (4NC) formation through HPLC. RESULTS: Relevant CYP2E1 mRNA levels are present in human RPE. Ethanol augmented the formation of reactive oxygen species (ROS) in ARPE-19 cells. Expression of CYP2E1 mRNA, CYP2E1 protein activity, and ROS production were induced by ethanol in a concentration-dependent manner. Interestingly, the treatment with DAS reduced all the aforementioned increased values. The presence of CYP2E1 in both hRPE and ARPE-19 cells reinforces the protective role of the RPE and strongly suggests additional roles for CYP2E1 related to vision.
PURPOSE:Cytochrome p450 2E1 (CYP2E1) is a detoxifying enzyme with particular affinity for ethanol (EtOH) expressed in several tissues. Although CYP2E1 has been identified in humanRPE, nothing is known about its metabolic activity. Expression of CYP2E1 and activity after EtOH exposure have been studied in humanRPE and ARPE-19 cells. METHODS:Ethanol-induced CYP2E1 mRNA expression was analyzed by RT-PCR and quantitative PCR (qPCR) from humandonorRPE as well as from ARPE-19 cells. Expression of CYP2E1 protein was determined by Western blot. Cytoplasmic CYP2E1 location also was demonstrated by immunocytochemistry. Cell viability was studied by the colorimetric assay XTT after EtOH treatment. Diallyl sulfide (DAS) was used to inhibit CYP2E1 activity. The microsomal CYP2E1 activity assay was determined by quantification of 4-nitrocatechol (4NC) formation through HPLC. RESULTS: Relevant CYP2E1 mRNA levels are present in humanRPE. Ethanol augmented the formation of reactive oxygen species (ROS) in ARPE-19 cells. Expression of CYP2E1 mRNA, CYP2E1 protein activity, and ROS production were induced by ethanol in a concentration-dependent manner. Interestingly, the treatment with DAS reduced all the aforementioned increased values. The presence of CYP2E1 in both hRPE and ARPE-19 cells reinforces the protective role of the RPE and strongly suggests additional roles for CYP2E1 related to vision.
Authors: W A García-Suástegui; L A Ramos-Chávez; M Rubio-Osornio; M Calvillo-Velasco; J A Atzin-Méndez; J Guevara; D Silva-Adaya Journal: Oxid Med Cell Longev Date: 2017-01-10 Impact factor: 6.543
Authors: Jorge M Barcia; Sandra Portolés; Laura Portolés; Alba C Urdaneta; Verónica Ausina; Gema M A Pérez-Pastor; Francisco J Romero; Vincent M Villar Journal: Front Physiol Date: 2017-01-25 Impact factor: 4.566
Authors: Sandra Atienzar-Aroca; Gemma Serrano-Heras; Aida Freire Valls; Carmen Ruiz de Almodovar; Maria Muriach; Jorge M Barcia; Jose M Garcia-Verdugo; Francisco J Romero; Javier Sancho-Pelluz Journal: J Cell Mol Med Date: 2018-08-21 Impact factor: 5.310