Martinez-Gil Natalia, Flores-Bellver Miguel, Atienzar-Aroca Sandra, Lopez-Malo Daniel, Urdaneta Alba C, Sancho-Pelluz Javier, Peris-Martínez Cristina, Bonet-Ponce Luis, Romero Francisco J, Barcia Jorge M
Invest Ophthalmol Vis Sci. 2015 Oct;56(11):6855-63. doi: 10.1167/iovs.14-16291.
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.
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.
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.
细胞色素P450 2E1（CYP2E1）是一种解毒酶，对多种组织中表达的乙醇（EtOH）具有特殊亲和力。尽管已在人视网膜色素上皮（RPE）中鉴定出CYP2E1，但其代谢活性尚不清楚。已对人RPE和ARPE - 19细胞中EtOH暴露后的CYP2E1表达及活性进行了研究。
通过逆转录聚合酶链反应（RT - PCR）和定量聚合酶链反应（qPCR）分析来自人供体RPE以及ARPE - 19细胞的乙醇诱导的CYP2E1 mRNA表达。通过蛋白质免疫印迹法测定CYP2E1蛋白的表达。免疫细胞化学也证实了细胞质中CYP2E1的定位。乙醇处理后，通过比色法XTT研究细胞活力。使用二烯丙基硫醚（DAS）抑制CYP2E1活性。通过高效液相色谱法（HPLC）定量4 - 硝基邻苯二酚（4NC）形成来测定微粒体CYP2E1活性。
人RPE中存在相关的CYP2E1 mRNA水平。乙醇增加了ARPE - 19细胞中活性氧（ROS）的形成。乙醇以浓度依赖性方式诱导CYP2E1 mRNA表达、CYP2E1蛋白活性和ROS产生。有趣的是，DAS处理降低了所有上述升高的值。hRPE和ARPE - 19细胞中CYP2E1的存在强化了RPE的保护作用，并强烈提示CYP2E1与视觉相关的其他作用。
