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

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.
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).
The ARPE‑19 cultures treated with B(e) p showed significantly increased ROS/RNS levels (P 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.