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Lokesh Tulasi Ram, Khoa Pham, Claudio A. Ramirez, Baruch Kuppermann, M C. Kenney; Reversal Of Hydroquinone Induced Increase In Lactate Dehydrogenase By 3-methyl-adenine In Arpe-19 Cells In Vitro. Invest. Ophthalmol. Vis. Sci. 2011;52(14):880.
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Cigarette smoking is one of the important risk factors associated with Age-related Macular Degeneration (AMD). Hydroquinone (HQ) is a major component of cigarette smoke and can be cytotoxic in vitro and in vivo. An early event in AMD is loss of the retinal pigment epithelial cells (RPE) so it is important to understand the mechanisms by which the cell death occurs. The present study was designed to test whether HQ induced autophagy in the human RPE cells.
ARPE-19 cells with and without pretreatment with 5mM 3-methyl-adenine (3-MA. Acros Organics, New Jersey, USA) were exposed to 200µM HQ for 12 hours. Lactate dehydrogenase (LDH) levels were then measured with LDH Cytotoxicity Assay Kit II which is based upon a WST reagent that detects LDH released from the damaged cells by a coupling reaction, wherein lactic acid is oxidized to generate NADH which in turn gives a yellow color. LDH activity was quantified by spectrophotometry at a wave length of 450nM. Rapamycin 4µM was used as a positive control to induce autophagy in ARPE-19 cells with and without pretreatment with 3-MA.
Exposure of ARPE-19 cells to 200µM HQ resulted in a significant increase in LDH levels (0.666±0.005) when compared to DMSO-equivalent cultures (0.426±0.004, p<0.0001). LDH release was reduced significantly by pretreatment with 5mM 3-MA (0.431±0.004, p<0.0001) compared to the 200µM HQ-treated cultures. Rapamycin exposure resulted in elevated levels of LDH (0.680±0.004) that was reversed by pretreatment with 5mM 3-MA (0.596±0.002, p<0.0001).
Autophagy is a mechanism of cell death in ARPE-19 cells treated with HQ and this can be reversed by 3-MA, a known inhibitor of autophagy.
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