Author/Authors :
Estrago-Franco, M Fernanda 1Department of Ophthalmology, Gavin Herbert Eye Institute, University of California, Irvine, CA, USA , Moustafa, M Tarek 1Department of Ophthalmology, Gavin Herbert Eye Institute, University of California, Irvine, CA, USA , Riazi-Esfahani, Mohammad 1Department of Ophthalmology, Gavin Herbert Eye Institute, University of California, Irvine, CA, USA , Sapkal, Ashish U 1Department of Ophthalmology, Gavin Herbert Eye Institute, University of California, Irvine, CA, USA , Piche-Lopez, Rhina 1Department of Ophthalmology, Gavin Herbert Eye Institute, University of California, Irvine, CA, USA , Jayaprakash Patil, A 1Department of Ophthalmology, Gavin Herbert Eye Institute, University of California, Irvine, CA, USA , Sharma, Ashish 1Department of Ophthalmology, Gavin Herbert Eye Institute, University of California, Irvine, CA, USA , Falatoonzadeh, Payam 1Department of Ophthalmology, Gavin Herbert Eye Institute, University of California, Irvine, CA, USA , Chwa, Marilyn 1Department of Ophthalmology, Gavin Herbert Eye Institute, University of California, Irvine, CA, USA , Luczy-Bachman, Georgia Department of Pediatrics - University of California, Irvine, CA, USA , Kuppermann, Baruch D 1Department of Ophthalmology, Gavin Herbert Eye Institute, University of California, Irvine, CA, USA , Kenney, M Cristina Department of Ophthalmology - Gavin Herbert Eye Institute - University of California, Irvine, CA, USA
Abstract :
Purpose: To identify inhibitors that could effectively lower reactive oxygen/nitrogen species (ROS/RNS),
complement and inflammatory cytokine levels induced by Benzo(e)pyrene [B(e)p], an element of cigarette
smoke, in human retinal pigment epithelial cells (ARPE‑19) in vitro.
Methods: ARPE‑19 cells were treated for 24 hours with 200 µM, 100 µM, and 50 µM B(e)p or DMSO (dimethyl
sulfoxide)‑equivalent concentrations. Some cultures were pre‑treated with ROS/RNS inhibitors (NG
nitro‑L‑arginine, inhibits nitric oxide synthase; Apocynin, inhibits NADPH oxidase; Rotenone, inhibits
mitochondrial complex I; Antimycin A, inhibits mitochondria complex III) and ROS/RNS levels were measured
with a fluorescent H2DCFDA assay. Multiplex bead arrays were used to measure levels of Interleukin‑6 (IL‑6),
Interleukin‑8 (IL‑8), Granulocyte‑Macrophage Colony Stimulating Factor (GM‑CSF), Transforming Growth
Factor alpha (TGF‑α) and Vascular Endothelial Growth Factor (VEGF). IL‑6 levels were also measured by
an enzyme‑linked immunosorbent assay. Real‑time qPCR analyses were performed with primers for C3
(component 3), CFH (inhibits complement activation),
CD59 (inhibitor of the complement membrane attack
complex (MAC)) and CD55/DAF (accelerates decay
of target complement target proteins).
Results: The ARPE‑19 cultures treated with B(e)
p showed significantly increased ROS/RNS levels
(P < 0.001), which were then partially reversed by
6 µM Antimycin A (19%, P = 0.03), but not affected
by the other ROS/RNS inhibitors. The B(e)p treated cultures demonstrated increased levels of IL‑6 (33%; P = 0.016) and GM‑CSF (29%; P = 0.0001) compared to
DMSO‑equivalent controls, while the expression levels for components of the complement pathway (C3, CFH,
CD59 and CD55/DAF) were not changed.
Conclusion: The cytotoxic effects of B(e)p include elevated ROS/RNS levels along with pro‑inflammatory
IL‑6 and GM‑CSF proteins. Blocking the Qi site of cytochrome c reductase (complex III) with Antimycin A
led to partial reduction in B(e)p induced ROS production. Our findings suggest that inhibitors for multiple
pathways would be necessary to protect the retinal cells from B(e)p induced toxicity.
