Effects of Benzo(e)pyrene on reactive oxygen/nitrogen species and inflammatory cytokines induction in human RPE cells and attenuation by mitochondrial-involved mechanism

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 h 2 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. © 2016 journal of ophthalmic and vision research.