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A. L. Gramajo, A. Jayaprakash Patil, A. Sharma, A. Neekhra, G. M. Seigel, B. D. Kuppermann, M. C. Kenney; Mechanisms of Nicotine Effect on Retinal and Vascular Cells in Culture. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3413.
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To evaluate the short-term in-vitro safety of nicotine in human retinal pigment epithelial (ARPE-19), rat neurosensory retinal (R28) and human microvascular endothelial cells (HMVEC) and the potential pathways of cell toxicity.
ARPE-19, R28 and HMVEC were treated with 10-2 and 10-4 M nicotine for 24 hours. In addition, R28 cells were pre-treated for one hour with 25 µM of ALLN (N-Acetyl-Leu-Leu-Nle-CHO, calpain inhibitor), 5 µM of ALLM (N-Acetyl-Leu-Leu-Met-CHO, calpain inhibitor); or 5 or 10µM of epicatechin, an antioxidant, before exposure to nicotine. Cell viability was measured using the trypan blue dye exclusion assay. Apoptosis was evaluated by measuring the caspase-3/7 activity and with the DNA laddering assay.
R28 and HMVEC cultures showed decreased cell viabilities with 10-2 M nicotine treatment compared with controls (47.7% ± 27.9 and 70.4% ± 9.5 versus 95.1% ± 4.5 and 86.9% ± 3.5 for R28 and HMVEC respectively, p<0.05). In the nicotine-treated R28 cultures, the decreased cell viability was partially reversed from 70.2% ± 0.9 to 82% ± 1.4 with 5µM of the antioxidant epicatechin and from 70.2% ± 0.9 to 86% ± 1.4 with 10µM of epicatechin. No reversal effects were seen with the ALLN and ALLM calpain inhibitors. Caspase-3/7 was not activated by nicotine treatment in any cell line. In the R28 cells, the DNA laddering assay showed a 200bp banding pattern consisting with apoptosis. HMVEC showed no bands by the DNA fragmentation assay. ARPE-19 cells were not affected by any of the nicotine concentrations used in this study.
Our findings suggest that nicotine, at the concentration of 10-2 M, is toxic in vitro to HMVEC and R28 cells but not to ARPE-19 cells. In R28 cells, the nicotine effects were in part through an antioxidant pathway that is non-caspase, non-calpain dependent. In HMVEC, the decrease in cell viability appears to be mediated via necrosis. This study show dissimilar responses to nicotine treatment in different cells line. Understanding the mechanisms of cell death involved may have therapeutic implications in the potential treatment of AMD.
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