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Khoa Pham, Claudio Ramirez, Lokesh Tulasiram, Baruch D. Kuppermann, M. C. Kenney; The Autophagy Inhibitor 3-Methyladenine Reduces Hydroquinone- Induced Reactive Oxygen Species Formation in ARPE-19 Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2346.
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Cigarette smoking has been linked to the development of age-related macular degeneration. Hydroquinone (HQ), a major component of cigarette smoke, has been hypothesized to be toxic to retinal pigment epithelial cells through the production of reactive oxygen/nitrogen species (ROS/RNS). The exact mechanisms of HQ toxicity, however, are unknown. Here we show that the increase in reactive oxygen species in HQ exposed ARPE-19 cells, a human retinal pigment epithelial cell line, can be reversed by the autophagy inhibitor 3-methyladenine (3-MA).
ARPE-19 cells were exposed to 200 µM HQ for 12 hours with and without pretreatment with 5mM 3-MA (Acros Organics, New Jersey, USA) for 4 hours. Levels of ROS/RNS were then measured using ',7'-dichlorodihydrofluorescein diacetate (H2DCFDA; Invitrogen-Molecular Probes, Eugene, OR), a fluorescent dye which detects hydrogen peroxide, peroxyl radicals, and peroxynitrite anions. As controls, ARPE-19 cells were treated with 4 µM rapamycin (LCLabs, Woburn, MA), a known inducer of autophagy with and without pretreatment with 3-MA. Relative fluorescence measurements were made using a fluorescence imager (FMBIO III, Hitachi, Yokohama, Japan).
Exposure of ARPE-19 cells to 200 µM HQ resulted in a significant increase in ROS/RNS levels (14,450 ± 1,351) when compared to a DMSO-equivalent control (4,479 ± 417.6, p<0.005). Pretreatment of ARPE-19 cells with 3-MA reversed the increase in ROS/RNS levels (6,491 ± 1,128, p <0.005). Rapamycin exposure resulted in elevated levels of ROS/RNS (19,580 ± 2,604, p<0.005) that was reversed with pretreatment with 3-MA (4,293 ± 751.7, p<0.005).
ARPE-19 cells exposed to HQ undergo autophagy, a catabolic process by which a cell degrades its own structures. Inhibition of autophagy reversed the increase in ROS/RNS caused by HQ exposure. As smoking has been linked to the development of macular degeneration, the process of autophagy may represent a therapeutic target.
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