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A. U. Sapkal, V. R. Sharma, C. R. Ramirez, R. Z. Migon, N. Gupta, M. Chwa, B. D. Kuppermann, M. C. Kenney; Protective Effect of 17Beta Estradiol on Benzo(e)pyrene Induced Toxicity in ARPE-19 Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1419.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the in vitro interaction of 17Beta Estradiol (βE2, a hormonal antioxidant) and Benzo(e)pyrene (B(e)P, a major component of cigarette smoke) on a human retinal pigment epithelial cell line (ARPE-19).
ARPE-19 cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% bovine growth serum. Cells were pretreated with 0 nM (no protective pretreatment) or 20 nM or 40 nM βE2 for 6 hrs and then 300 µM B(e)P was added to all cells for an additional 24 hrs. Because B(e)P requires dimethyl sulfoxide (DMSO) for dissolution, DMSO control was also performed on cells without βE2 pretreatment. Cell viability was measured using a trypan blue dye-exclusion assay. JC-1 assay was performed to measure mitochondrial membrane potential (ΔΨm) and caspase-3/7 activity was assayed to measure apoptosis.
The mean cell viability percentage of ARPE-19 cells treated with 300 µM B(e)P alone decreased to 71.6 ± 1.3 and it increased to 82.4 ± 0.8 with 20 nM βE2 pretreatment (P<0.05). DMSO equivalent control for B(e)P was 98.40 ± 0.30. ΔΨm after B(e)P treatment alone decreased to 3.14 ± 0.03 and after 20nM βE2 pretreatment increased to 6.007 ± 0.08 (P < 0.001) and to 5.423 ± 0.75 in cells pretreated with 40 nM βE2 (P < 0.05). DMSO equivalent control for B(e)P was 6.322 ± 0.7991. Caspase-3/7 activity in ARPE-19 cells treated with 300 µM B(e)P alone increased to 17260 ± 552.5, and decreased to 6386 ± 132.3 with 20 nM βE2 pretreatment (P<0.001) and to 12060 ± 611.2 with 40 nM βE2 pretreatment (P<0.01). DMSO equivalent control for B(e)P was 2817 ± 395.8.
B(e)P is a major component of cigarette smoke known to induce apoptotic cell death. Estrogen has been previously shown to protect ARPE-19 cells from oxidative stress. Smoking increases the chances of Age-Related Macular Degeneration (AMD) while Estrogen may protect against AMD. Our study demonstrates that βE2 plays a protective role in ARPE-19 cells against B(e)P induced toxicity by reducing apoptosis, and increasing both cell viability and mitochondrial membrane potential.
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