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 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.

[1]  Mariela C. Marazita,et al.  Oxidative stress-induced premature senescence dysregulates VEGF and CFH expression in retinal pigment epithelial cells: Implications for Age-related Macular Degeneration , 2015, Redox biology.

[2]  H. Bundgaard,et al.  Simvastatin effects on skeletal muscle: relation to decreased mitochondrial function and glucose intolerance. , 2013, Journal of the American College of Cardiology.

[3]  S. Jarrett,et al.  Consequences of oxidative stress in age-related macular degeneration. , 2012, Molecular aspects of medicine.

[4]  L. Folkersen,et al.  Retinal pigment epithelial cells upregulate expression of complement factors after co-culture with activated T cells. , 2011, Experimental eye research.

[5]  B. Kuppermann,et al.  Inhibition of apoptosis in human retinal pigment epithelial cells treated with benzo(e)pyrene, a toxic component of cigarette smoke. , 2010, Investigative ophthalmology & visual science.

[6]  S. Thanos,et al.  GM-CSF regulates the ERK1/2 pathways and protects injured retinal ganglion cells from induced death. , 2009, Experimental eye research.

[7]  G. Seigel,et al.  Differential effects of nicotine on retinal and vascular cells in vitro. , 2009, Toxicology.

[8]  J. Handa,et al.  Changes in Retinal Pigment Epithelium Related to Cigarette Smoke: Possible Relevance to Smoking as a Risk Factor for Age-Related Macular Degeneration , 2009, PloS one.

[9]  C. Barnstable,et al.  Bacterial endotoxin activates retinal pigment epithelial cells and induces their degeneration through IL-6 and IL-8 autocrine signaling. , 2009, Molecular immunology.

[10]  S. Kase,et al.  Regulated secretion of complement factor H by RPE and its role in RPE migration , 2009, Graefe's Archive for Clinical and Experimental Ophthalmology.

[11]  Joe G Hollyfield,et al.  Oxidative damage–induced inflammation initiates age-related macular degeneration , 2008, Nature Medicine.

[12]  C. Sommer,et al.  A Neuroprotective Function for the Hematopoietic Protein Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) , 2008, Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism.

[13]  S. D. de Córdoba,et al.  Translational Mini‐Review Series on Complement Factor H: Genetics and disease associations of human complement factor H , 2007, Clinical and experimental immunology.

[14]  W. Schäbitz,et al.  The hematopoietic factor GM-CSF (Granulocyte-macrophage colony-stimulating factor) promotes neuronal differentiation of adult neural stem cells in vitro , 2007, BMC Neuroscience.

[15]  T. Salt,et al.  Complement factor H deficiency in aged mice causes retinal abnormalities and visual dysfunction , 2007, Proceedings of the National Academy of Sciences.

[16]  Johanna M Seddon,et al.  Variation in complement factor 3 is associated with risk of age-related macular degeneration , 2007, Nature Genetics.

[17]  W. Park,et al.  An ROS generator, antimycin A, inhibits the growth of HeLa cells via apoptosis , 2007, Journal of cellular biochemistry.

[18]  J. Haines,et al.  Protective effect of complement factor B and complement component 2 variants in age-related macular degeneration. , 2007, Human molecular genetics.

[19]  I. Deary,et al.  Complement C3 variant and the risk of age-related macular degeneration. , 2007, The New England journal of medicine.

[20]  N. Sugihara,et al.  Effects of benzo(e)pyrene and benzo(a)pyrene on P-glycoprotein-mediated transport in Caco-2 cell monolayer: a comparative approach. , 2007, Toxicology in vitro : an international journal published in association with BIBRA.

[21]  H. Okano,et al.  Interleukin-6 receptor-mediated activation of signal transducer and activator of transcription-3 (STAT3) promotes choroidal neovascularization. , 2007, The American journal of pathology.

[22]  J. Forrester,et al.  Synthesis of complement factor H by retinal pigment epithelial cells is down-regulated by oxidized photoreceptor outer segments. , 2007, Experimental eye research.

[23]  S. Sivaprasad,et al.  Estimation of systemic complement C3 activity in age-related macular degeneration. , 2007, Archives of ophthalmology.

[24]  Tadashi Mabuchi,et al.  Genetic analysis of human complement factor H polymorphisms , 2007, Electrophoresis.

[25]  M. K. Sanyal,et al.  Differential metabolism of benzo[alpha]pyrene in vitro by human placental tissues exposed to active maternal cigarette smoke. , 2007, Birth defects research. Part B, Developmental and reproductive toxicology.

[26]  I. Giddings,et al.  Time- and concentration-dependent changes in gene expression induced by benzo(a)pyrene in two human cell lines, MCF-7 and HepG2 , 2006, BMC Genomics.

[27]  J. Haines,et al.  Cigarette smoking strongly modifies the association of LOC387715 and age-related macular degeneration. , 2006, American journal of human genetics.

[28]  R. T. Smith,et al.  Variation in factor B (BF) and complement component 2 (C2) genes is associated with age-related macular degeneration , 2006, Nature Genetics.

[29]  B. Rosner,et al.  Progression of age-related macular degeneration: prospective assessment of C-reactive protein, interleukin 6, and other cardiovascular biomarkers. , 2005, Archives of ophthalmology.

[30]  Dao-Yi Yu,et al.  Retinal degeneration and local oxygen metabolism. , 2005, Experimental eye research.

[31]  R. T. Smith,et al.  A common haplotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-related macular degeneration. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[32]  J. Gilbert,et al.  Complement Factor H Variant Increases the Risk of Age-Related Macular Degeneration , 2005, Science.

[33]  A. Edwards,et al.  Complement Factor H Polymorphism and Age-Related Macular Degeneration , 2005, Science.

[34]  J. Ott,et al.  Complement Factor H Polymorphism in Age-Related Macular Degeneration , 2005, Science.

[35]  Robert S. Balaban,et al.  Mitochondria, Oxidants, and Aging , 2005, Cell.

[36]  E. Tremoli,et al.  Apocynin prevents cyclooxygenase 2 expression in human monocytes through NADPH oxidase and glutathione redox-dependent mechanisms. , 2004, Free radical biology & medicine.

[37]  J. Keaney,et al.  Oxidative stress, antioxidants, and endothelial function. , 2004, Current medicinal chemistry.

[38]  L. Almasy,et al.  Genetic and environmental factors influencing the human factor H plasma levels , 2004, Immunogenetics.

[39]  N. Rifai,et al.  Association between C-reactive protein and age-related macular degeneration. , 2004, JAMA.

[40]  F. Murad,et al.  Novel effects of nitric oxide. , 2003, Annual review of pharmacology and toxicology.

[41]  Charles L. Hoppel,et al.  Production of Reactive Oxygen Species by Mitochondria , 2003, Journal of Biological Chemistry.

[42]  M. Brand,et al.  Topology of Superoxide Production from Different Sites in the Mitochondrial Electron Transport Chain* , 2002, The Journal of Biological Chemistry.

[43]  W. R. Biggs,et al.  Electronic spectral detection in liquid chromatography , 2002, Analytical and Bioanalytical Chemistry.

[44]  D. Archer,et al.  Retinal pigment epithelial cell DNA is damaged by exposure to benzo[a]pyrene, a constituent of cigarette smoke. , 2002, Experimental eye research.

[45]  P. Campochiaro,et al.  Nitric oxide is proangiogenic in the retina and choroid * , 2002, Journal of cellular physiology.

[46]  C. Wilcox.  Reactive oxygen species: Roles in blood pressure and kidney function , 2002, Current hypertension reports.

[47]  L. Scorrano,et al.  The Mitochondrial Permeability Transition, Release of Cytochrome c and Cell Death , 2001, The Journal of Biological Chemistry.

[48]  Y. Courtois,et al.  Inducible nitric oxide synthase mediates the change from retinal to vitreal neovascularization in ischemic retinopathy. , 2001, The Journal of clinical investigation.

[49]  B. Halliwell,et al.  Free Radicals and Antioxidants in the Year 2000: A Historical Look to the Future , 2000, Annals of the New York Academy of Sciences.

[50]  B. Halliwell,et al.  Nitric oxide and peroxynitrite. The ugly, the uglier and the not so good: a personal view of recent controversies. , 1999, Free radical research.

[51]  J. Hoek,et al.  Functional Consequences of the Sustained or Transient Activation by Bax of the Mitochondrial Permeability Transition Pore* , 1999, The Journal of Biological Chemistry.

[52]  M. Urashima,et al.  IL-6 triggers cell growth via the Ras-dependent mitogen-activated protein kinase cascade. , 1997, Journal of immunology.

[53]  J. Stolk,et al.  Characteristics of the inhibition of NADPH oxidase activation in neutrophils by apocynin, a methoxy-substituted catechol. , 1994, American journal of respiratory cell and molecular biology.

[54]  D. Schwartz.  The Beaver Dam Eye Study: the relation of age-related maculopathy to smoking. , 1994, Survey of ophthalmology.

[55]  R W Alexander,et al.  Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. , 1994, Circulation research.

[56]  D. Heistad,et al.  Does basal production of nitric oxide contribute to regulation of brain-fluid balance? , 1992, The American journal of physiology.

[57]  T. Hirano,et al.  Molecular cloning and expression of an IL-6 signal transducer, gp130 , 1990, Cell.

[58]  B. Freeman,et al.  Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[59]  B Chance,et al.  The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen. , 1973, The Biochemical journal.

[60]  I. Fridovich,et al.  Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). , 1969, The Journal of biological chemistry.

[61]  M. Lavail,et al.  Retinal degenerative diseases : laboratory and therapeutic investigations , 2010 .

[62]  J. H. Kim,et al.  Protective effect of clusterin from oxidative stress-induced apoptosis in human retinal pigment epithelial cells. , 2010, Investigative ophthalmology & visual science.

[63]  E. Souied,et al.  [Epidemiology of age related macular degeneration]. , 2009, Journal Francais d'Ophtalmologie.

[64]  G. Polunin,et al.  [Pathogenesis of age-related macular degeneration]. , 2006, Vestnik oftalmologii.

[65]  Benita J. O’Colmain,et al.  Prevalence of age-related macular degeneration in the United States. , 2004, Archives of ophthalmology.

[66]  Dean P. Jones,et al.  A randomized, placebo-controlled, clinical trial of high-dose supplementation with vitamins C and E and beta carotene for age-related cataract and vision loss: AREDS report no. 9. , 2001, Archives of ophthalmology.

[67]  G. Ciliberto,et al.  Impaired Stat3 activation following localized inflammatory stimulus in IL-6-deficient mice. , 1998, Cytokine.

[68]  M. S. Zedeck,et al.  Polycyclic aromatic hydrocarbons: a review. , 1980, Journal of environmental pathology and toxicology.

[69]  P. Shubik,et al.  Carcinogenicity of different polycyclic hydrocarbons in the respiratory tract of hamsters. , 1974, Journal of the National Cancer Institute.