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Praveena Gupta, Layla Ghergherehchi, Gabriela A. Kulp, Nima Tirgan, Bernard F. Godley; Effect Of Tobacco Specific N-nitrosamines, NNN And NNK On Cultured Retinal Pigment Epithelial Cells: Implications To Age Related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4428. doi: https://doi.org/.
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To investigate the in vitro effects of tobacco-specific n- nitrosamines (TSNA); (N-Nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) which are the major components of cigarette smoke on human retinal pigment epithelial cells.
Human ARPE-19 cultures were exposed to varying concentrations of NNN and NNK (1mM, 100µM, 10µM, 1 µM) in DMEM/F12 medium for 24 and 48 hours. MTT cell proliferation assays were performed to find a non toxic dose that would not kill the cells within the 24-48 hour period. For both NNN and NNK a 10µM dose of 24 hours was chosen to carry out the rest of the experiments. Total cell protein extracts were analyzed for the presence of OGG1, a DNA repair enzyme and VEGF. Live cells were stained with Hoechst and Mitotracker Green to visualize the nucleus and mitochondrial mass respectively using a confocal microscope. Cells were also labeled for anti- VEGFR2 and imaged using a confocal microscope. Comet assay was performed on whole cells to detect DNA damage.
Our studies show that both NNN and NNK behave similarly and caused significant DNA damage and compensatory upregulation of the DNA repair enzyme "OGG1" in RPE cells by about 7 %. Also, a 13% decrease in the mitochondrial mass is noted in the NNN and NNK treated groups when compared to the controls. There is an increasing trend of VEGF and VEGFR2 expression in both the treatment groups when compared with the controls.
Both NNN and NNK are toxic components of cigarette smoke. Both compounds cause DNA damage, loss of mitochondrial mass and up regulation of VEGF in cultures of RPE. Since cigarette smoking has been linked to increased risk of AMD, therefore components of cigarette smoke like, NNN and NNK can be important causative mediators of RPE damage in the eye.
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