Effects of Benzo(e)Pyrene, a toxic component of cigarette smoke, on human retinal pigment epithelial cells in vitro.
OBJECTIVE
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).


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Sharma
Ashish
A
Department of Ophthalmology, School of Medicine, University of California, Irvine, California 92868, USA.
Neekhra
Aneesh
A
Gramajo
Ana L
AL
Patil
Jayaprakash
J
Chwa
Marilyn
M
Kuppermann
Baruch D
BD
Kenney
M Cristina
MC
D017209:Apoptosis; D001580:Benzopyrenes / Q000009:adverse effects*; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D004247:DNA / Q000187:drug effects; D053938:DNA Fragmentation; D004587:Electrophoresis, Agar Gel; D006801:Humans; D008268:Macular Degeneration / Q000209:etiology / Q000235:genetics / Q000473:pathology; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000473:pathology; D012307:Risk Factors; D014028:Tobacco Smoke Pollution / Q000032:analysis*
D001580:Benzopyrenes; D014028:Tobacco Smoke Pollution; C026487:benzo(e)pyrene; D004247:DNA; D020169:Caspases