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R. M. Piche Lopez, N. Gupta, C. A. Ramirez, S. Mansoor, A. G. Limb, B. D. Kuppermann, M. C. Kenney; Effects of Benzo(e)Pyrene, a Toxic Component of Cigarette Smoke, on Müller Cells (MIO-M1) in vitro. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4108.
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To understand the cellular and molecular basis of the association between cigarette smoking and age-related macular degeneration (AMD), we examined the effect of Benzo(e)Pyrene (B(e)P), a toxic element in cigarette smoke, on a Müller cell line (MIO-M1).
MIO-M1cells were treated for 24 hours with 200µM, 100µM, 50µM and 25µM of B(e)P. The cytotoxicity was measured by: Cell viability trypan blue dye-exclusion assay, reactive oxygen species/reactive nitrogen species (ROS/RNS) assay, mitochondrial membrane potential (ΔΨ[[Unsupported Character - М]]), caspase-3/7 activity, and DNA fragmentation assay
All concentrations of B(e)P decreased the cell viability of the MIO-M1 cells:200µM (75.13±1.52, P<0.001), 100µM (84.3±0.16,P<0.001), 50µM (87.18±0.69,P<0.05), and 25µM (89.25±1.47,P<0.05) compared to DMSO equivalent controls 86.9±0.48, 90.3±0.55, 90.8±0.27, 91.3±0.30 respectively. The increase in ROS/RNS production was 2835 ± 190.0, P<0.001 (200µM); 2704 ± 111.7, P<0.001 (100µM); 2339 ± 76.0, P<0.05 (50µM) and 2274 ± 67.6, P<0.05 (25µM), compared to with equivalent DMSO controls 1199±36.1, 1193±49.3, 1369±28.9, 1281±26.4 respectively. The ΔΨ[[Unsupported Character - М]] was significantly decreased after treatment with B(e)P at 200µM (3.25±0.14, P<0.001), 100µM (4.0±0.14, P<0.001), and 50µM (5.16±0.19, P<0.001),compared to with equivalent DMSO controls 6.64±0.14, 6.44±0.22, 6.78±0.14 and 6.81±0.15 respectively. Caspase-3/7 activity was increased for cells treated with 200µM (9804 ± 150.4, P<0.001), 100µM (8999 ± 146.5, P<0.001), 50µM (6864 ± 283.7, P<0.001) and 25µM (4592 ± 193.4, P<0.05), compared to DMSO controls 4113±80.5, 2619±232.3, 2559±211.9 and 2535±254.2 respectively. There was marked DNA fragmentation in cells exposed to B(e)P at 200 µM only.
B(e)P, an important toxin of cigarette smoke, causes oxidative damage and apoptosis of MIO-M1 cells. Disruption of these important functions by B(e)P may contribute to the significant risk that smoking has for cells of AMD.
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