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M. F. Estrago-Franco, A. Jayaprakash Patil, A. Sharma, L. C. Zacharias, M. Chwa, G. Luczy-Bachman, B. D. Kuppermann, M. C. Kenney; Effect of Benzo(e)Pyrene on Human Retinal Pigment Epithelial Cells in vitro. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2332.
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© ARVO (1962-2015); The Authors (2016-present)
To study the biological effects of Benzo(e)Pyrene (B(e)P), a toxicant in cigarette smoke, on human retinal pigment epithelial (ARPE-19) cells in vitro.
ARPE-19 cells were treated for 24 hours with B(e)P (200 µM/ml, 100 µM/ml or 50 µM/ml) and with equivalent concentrations of DMSO. Cell viability was determined by a dye-exclusion assay. Reactive Oxygen Species (ROS) production was measured with the fluorescent H2DCFDA assay, which detects hydrogen peroxide, peroxyl radicals, and peroxynitrites anions. Western blotting was performed using the goat polyclonal to Complement Factor H (CFH). The blots were developed with Immuno-Star chemiluminescent substrate buffer (Bio-Rad). Supernatants of cell cultures treated with B(e)P were analyzed for cytokine synthesis using multiplex bead arrays.
ARPE-19 cells showed a concentration-dependent decrease in cell viability after exposure to B(e)P. Untreated and DMSO treated equivalent cultures of 200 µM, 100 µM and 50 µM showed cell viabilities of 94.45% ± 4.2, 93.37% ± 4.2, 90.62% ± 5.3 and 93.05% ±4.5 respectively. Cell viabilities of B(e)P treated cells were 83.4% ±3.6 (P<0.05), 73.85% ±3.7 (P<0.001) and 57.9% ±9 (P<0.001) at doses of 50 µM,100 µM and 200 µM of B(e)P, respectively.ROS/RSN production was not changed after exposure to any concentration of B(e)P. Western blot analysis of Complement factor H showed a decreased staining in the bands of 28.57% and 23.80% with 200 and 100 µM of B(e)P respectively.ARPE-19 cells treated with B(e)P (50 µM,100 µM and 200 µM) presented an increase of IL-6 of 48%, 27% and 22% respectively. Other tested cytokines showed less significant upregulation.
B(e)P treatment of ARPE-19 cells resulted in decreased cell viability and decreased levels of Complement Factor H, a protein which is part of the alternative complement pathway and that inhibits inflammation.The proinflammatory cytokine IL-6 exhibited increased levels after B(e)P exposure. These data show a relationship between B(e)P and protein levels of Complement Factor H. This may represent a mechanism by which smoking influences AMD and other retinal diseases.
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