CYP2E1 in the Human Retinal Pigment Epithelium: Expression, Activity, and Induction by Ethanol
| Autor: | Alba C. Urdaneta, D. López Malo, Javier Sancho-Pelluz, Sandra Atienzar-Aroca, Miguel Flores-Bellver, Francisco J. Romero, Luis Bonet-Ponce, Natalia Martínez-Gil, Jorge M. Barcia, Cristina Peris-Martínez |
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| Rok vydání: | 2015 |
| Předmět: |
Cell Survival
Blotting Western Immunocytochemistry Retinal Pigment Epithelium Polymerase Chain Reaction Cell Line Enzyme activator Western blot medicine Humans RNA Messenger Viability assay chemistry.chemical_classification Analysis of Variance Reactive oxygen species Ethanol medicine.diagnostic_test Chemistry Cytochrome P-450 CYP2E1 CYP2E1 Immunohistochemistry Molecular biology Enzyme Activation Blot Oxidative Stress Cell culture Reactive Oxygen Species |
| Zdroj: | Investigative Opthalmology & Visual Science. 56:6855 |
| ISSN: | 1552-5783 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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