title: Effects of Benzo(e)pyrene on Reactive Oxygen/Nitrogen Species and Inflammatory Cytokines Induction in Human RPE Cells and Attenuation by Mitochondrial-involved Mechanism.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Ophthalmology, Gavin Herbert Eye Institute, University of California, Irvine, CA, USA; Clinica Dres Estrago, Corrientes, Argentina.
authors.forename: M Fernanda
authors.initials: MF
authors.lastname: Estrago-Franco
authors.affiliation: Department of Ophthalmology, Gavin Herbert Eye Institute, University of California, Irvine, CA, USA; Ophthalmology Department, Minia University, Egypt.
authors.forename: M Tarek
authors.initials: MT
authors.lastname: Moustafa
authors.affiliation: Department of Ophthalmology, Gavin Herbert Eye Institute, University of California, Irvine, CA, USA; Eye Research Center, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Riazi-Esfahani
authors.affiliation: Department of Ophthalmology, Gavin Herbert Eye Institute, University of California, Irvine, CA, USA.
authors.forename: Ashish U
authors.initials: AU
authors.lastname: Sapkal
authors.affiliation: Department of Ophthalmology, Gavin Herbert Eye Institute, University of California, Irvine, CA, USA.
authors.forename: Rhina
authors.initials: R
authors.lastname: Piche-Lopez
authors.affiliation: Department of Ophthalmology, Gavin Herbert Eye Institute, University of California, Irvine, CA, USA; Royal Lancaster Infirmary, University Hospitals of Morecambe Bay NHS Trust, Lancaster, UK.
authors.forename: A Jayaprakash
authors.initials: AJ
authors.lastname: Patil
authors.affiliation: Department of Ophthalmology, Gavin Herbert Eye Institute, University of California, Irvine, CA, USA; Lotus Eye Care Hospital, Coimbatore, Tamil Nadu, India.
authors.forename: Ashish
authors.initials: A
authors.lastname: Sharma
authors.affiliation: Department of Ophthalmology, Gavin Herbert Eye Institute, University of California, Irvine, CA, USA.
authors.forename: Payam
authors.initials: P
authors.lastname: Falatoonzadeh
authors.affiliation: Department of Ophthalmology, Gavin Herbert Eye Institute, University of California, Irvine, CA, USA.
authors.forename: Marilyn
authors.initials: M
authors.lastname: Chwa
authors.affiliation: Department of Pediatrics, University of California, Irvine, CA, USA.
authors.forename: Georgia
authors.initials: G
authors.lastname: Luczy-Bachman
authors.affiliation: Department of Ophthalmology, Gavin Herbert Eye Institute, University of California, Irvine, CA, USA.
authors.forename: Baruch D
authors.initials: BD
authors.lastname: Kuppermann
authors.affiliation: Department of Ophthalmology, Gavin Herbert Eye Institute, University of California, Irvine, CA, USA.
authors.forename: M Cristina
authors.initials: MC
authors.lastname: Kenney
keywords: Benzo(e)pyrene; Cytokines; Retinal Pigment Epithelium