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 H2 DCFDA 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.
Highlights
Age‐related macular degeneration (AMD) is the leading cause of irreversible blindness and low vision in individuals over 55 years of age in the industrialized world.[1]
B(e)p Creates a Pro‐inflammatory Environment; Estrago‐Franco et al cultures demonstrated increased levels of IL‐6 (33%; P = 0.016) and GM‐CSF (29%; P = 0.0001) compared to dimethyl sulfoxide (DMSO)‐equivalent controls, while the expression levels for components of the complement pathway (C3, Complement Factor H (CFH), CD59 and CD55/DAF) were not changed
Supernatants of the ARPE‐19 cell cultures treated with 50, 100 and 200 μM B(e)p or DMSO‐equivalent controls were analyzed with the multiplex bead array, which scanned for IL‐6, IL‐8, GM‐CSF, TGF‐α and VEGF proteins
Summary
Age‐related macular degeneration (AMD) is the leading cause of irreversible blindness and low vision in individuals over 55 years of age in the industrialized world.[1] AMD can be classified into two types: wet or neovascular and dry or atrophic. In wet AMD, choroidal neovascular membrane (CNV) develops, which causes hemorrhage, swelling, and macular scarring resulting in severe visual loss. The combined prevalence of neovascular AMD and geographic atrophy has been estimated to be 1.4% in the United States, affecting 1.75 million individuals presently with a 50% expected increase by the year 2020.[2] the clinical features of AMD have been well documented for more than a century, the detailed pathogenesis of AMD is not fully understood.[3] cigarette smoking is one of the most consistent risk factors for AMD in several worldwide population based studies. Epidemiologic studies have confirmed that smoking is a risk factor in both forms of AMD.[5]
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