April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Effects of Ethanol on ARPE-19 cells: Autophagy and Oxidative Stress
Author Affiliations & Notes
  • Luis Bonet
    Neurobiology and Neurophysiology, Catholic University of Valencia, Valencia, Spain
  • Miguel Flores-Bellver
    Neurobiology and Neurophysiology, Catholic University of Valencia, Valencia, Spain
  • Sara Saez-Atienzar
    Neurobiology and Neurophysiology, Catholic University of Valencia, Valencia, Spain
    Neuropsicopharmacology, Complejo Universitario Universidad de Albacete, Albacete, Spain
  • Natalia Martínez
    Neurobiology and Neurophysiology, Catholic University of Valencia, Valencia, Spain
  • Javier Sancho-Pelluz
    Neurobiology and Neurophysiology, Catholic University of Valencia, Valencia, Spain
  • Jorge M Barcia
    Neurobiology and Neurophysiology, Catholic University of Valencia, Valencia, Spain
  • Joaquin Jordan
    Ciencias Medicas, Universidad de Castilla la Mancha, Albacete, Spain
  • Maria F Galindo
    Neuropsicopharmacology, Complejo Universitario Universidad de Albacete, Albacete, Spain
  • Francisco J Romero
    Neurobiology and Neurophysiology, Catholic University of Valencia, Valencia, Spain
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 1743. doi:
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      Luis Bonet, Miguel Flores-Bellver, Sara Saez-Atienzar, Natalia Martínez, Javier Sancho-Pelluz, Jorge M Barcia, Joaquin Jordan, Maria F Galindo, Francisco J Romero, Autophagy in retinal damage; Effects of Ethanol on ARPE-19 cells: Autophagy and Oxidative Stress. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1743.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Despite the well-known pathological effects of ethanol (EtOH) consumption on the nervous system, how alcohol exposure affects retinal cells has yet to be clarified. Retinal pigment epithelium (RPE) plays a crucial role in the physiology of the retina due to its location and metabolism. Oxidative damage has been demonstrated as a pathogenic mechanism in several retinal diseases, and reactive oxygen species (ROS) are important by-products of ethanol metabolism. ROS production seems to promote autophagy, a mechanism designed to degrade damaged organelles and proteins. Recently, autophagy has been proposed as a cytoprotective mechanism against EtOH-induced toxicity in liver and brain cells. Thus, our goal is to study the effect of EtOH in ARPE-19 cells, and the role of autophagy

Methods: ARPE-19 cells were seeded and then exposed to different EtOH concentrations (80 mM, 200 mM, 400 mM, and 600 mM). Autophagy was measured by analyzing LC3 punctae with a GFP-LC3 plasmid and different protein expression (p62 and LC3-II) by western blot. Moreover, LC3 flux was analyzed by using chloroquine as a lysosome inhibitor and by using the RFP-GFP-LC3 plasmid to quantify autophagosome maturation. Mitochondrial morphology was analyzed using a pDsRed2-Mito plasmid and by electron microscopy. ROS levels were measured using 2’7’-dichlorodihydrofluorescein (DCFH). Lipid peroxidation products were analyzed by ELISA. 4-HNE aggregates were localized with immunocytochemistry.

Results: A significant reduction of MTT occurred first at 600 mM EtOH, after 24 h. EtOH exposure induced LC3 synthesis in a concentration dependent-manner (starting 80 mM). Moreover, EtOH also promoted a concentration-dependent increase in autophagosome-lysosome fusion and mitochondrial fragmentation. Obvious ultraestructural morphological changes were observed at every EtOH concentration used. Furthermore, EtOH increased DCFH fluorescence (ROS) levels in treated cells compared to non-treated. 4-HNE aggregates increased after EtOH exposure, forming large ball-shaped aggregates in the vicinity of the nucleus.

Conclusions: Alcohol exposure produces ROS and mitochondrial fragmentation. Furthermore, EtOH induces 4-HNE labelled protein aggregates. In order to eliminate damaged organelles, autophagy is activated. In summary, EtOH induces all neurodegenerative hallmarks in RPE cells such as mitochondrial damage, protein aggregation, and autophagy activation.

Keywords: 701 retinal pigment epithelium • 600 mitochondria • 634 oxidation/oxidative or free radical damage  
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