Effects of Benzo(e)Pyrene, a Toxic Component of Cigarette Smoke, on Human Retinal Pigment Epithelial Cells In Vitro

• Published in: Investigative ophthalmology & visual science 2008-11, Vol.49 (11), p.5111-5117
• Main Authors: Sharma, Ashish, Neekhra, Aneesh, Gramajo, Ana L, Patil, Jayaprakash, Chwa, Marilyn, Kuppermann, Baruch D, Kenney, M. Cristina
• Format: Article
• Language: English
• Subjects: Apoptosis; Benzopyrenes - adverse effects; Biological and medical sciences; Caspases - metabolism; Cell Survival; Cells, Cultured; DNA - drug effects; DNA Fragmentation; Electrophoresis, Agar Gel; Eye and associated structures. Visual pathways and centers. Vision; Fundamental and applied biological sciences. Psychology; Humans; Macular Degeneration - etiology; Macular Degeneration - genetics; Macular Degeneration - pathology; Medical sciences; Ophthalmology; Pigment Epithelium of Eye - drug effects; Pigment Epithelium of Eye - pathology; Risk Factors; Tobacco Smoke Pollution - analysis; Vertebrates: nervous system and sense organs
• ISSN: 0146-0404; 1552-5783; 1552-5783
• DOI: 10.1167/iovs.08-2060

To better understand the cellular and molecular basis for the epidemiologic association between cigarette smoke and age-related macular degeneration (AMD), the authors examined the effects of Benzo(e)Pyrene (B(e)P), a toxic element in cigarette smoke, on human retinal pigment epithelial cells (ARPE-19). ARPE-19 cells were cultured in Dulbecco modified Eagle medium containing 10% fetal bovine serum. Cells were treated for 24 hours with 1000 microM, 400 microM, 200 microM, and 100 microM B(e)P. Cell viability was determined by a trypan blue dye-exclusion assay. Activities of caspase-3/7, caspase-8, caspase-9, and caspase-12 were measured by a fluorescence image scanner, and DNA laddering was evaluated by electrophoresis on 3% agarose gel. The mean percentage of cell viabilities of ARPE-19 cells was decreased in a dose-dependent manner after exposure to B(e)P at the higher concentrations of 1000 microM (20.0 +/- 0.4; P < 0.001), 400 microM (35.6 +/- 6.4; P < 0.001), and 200 microM (58.7 +/- 2.3; P < 0.001) but not at 100 microM (95.9 +/- 0.7; P > 0.05) compared with the equivalent dimethyl sulfoxide (DMSO)-treated control cultures. There were significant increases in caspase-3/7, -8, -9, and -12 activities compared with the DMSO-treated controls (P < 0.001). DNA laddering revealed bands at 200-bp intervals. These results show that B(e)P is a toxicant to human retinal pigment epithelial cells in vitro. It causes cell death and induces apoptosis by the involvement of multiple caspase pathways.