Effect of Benzo(e)pyrene, a Toxicant in Cigarette Smoke, on Human Retinal Pigment Epithelial Cells in vitro

Abstract

Purpose: : To better understand the cellular and molecular basis for the epidemiologic association between cigarette smoke and age-related macular degeneration (AMD), we examined the effects of Benzo(e)Pyrene (B(e)P), a toxic element in cigarette smoke, on human retinal pigment epithelial cells (ARPE-19).

Methods: : ARPE-19 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum. Cells were treated for 24 hours with 1000µM, 400µM, 200µM and 100µM B(e)P constituted in DMSO. 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 fluorescence caspase kits and DNA laddering was evaluated by electrophoresis on 3% agarose gel.

Results: : All the concentrations of B(e)P except 100µM were found to be toxic in comparision to its DMSO equivalent. The mean cell viabilities of ARPE-19 after 24 hours exposure to B(e)P 1000µM, 400µM, 200µM and 100µM were 20.0 ± 0.4 (P < 0.001); 35.6 ± 6.4 (P < 0.001); 58.7 ± 2.3 (P < 0.001) and 95.9 ± 0.7 (P > 0.05), respectively. The mean cell viabilities of ARPE-19 with 1000µM, 400µM, 200µM and 100µM DMSO control were 91.1 ± 3.6; 97.3 ± 1.5; 98.3 ± 1.7 and 98.4 ± 0.6 respectively. There was a significant increase in caspase-3/7, -8, -9 and -12 activities compared to the DMSO-treated controls (P < 0.001). DNA laddering revealed bands at 200bp intervals.

Conclusions: : B(e)P is a major Polycyclic Aromatic Hydrocarbon (PAH) component of cigarette smoke. Association between AMD and cigarette smoke has been proven by epidemiological studies. Evidences for the molecular mechanism responsible for AMD is still lacking. In the present study with human ARPE-19 cells, our findings demonstrate that B(e)P, an element associated with smoking, causes caspase dependent apoptosis which may be a major cause in promoting the onset and progression of AMD.

Keywords: age-related macular degeneration • apoptosis/cell death