May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Effects of Benzo(e)Pyrene on Rat Retinal Neurosensory Cells and Human Microvascular Endothelial Cells in vitro
Author Affiliations & Notes
  • A. Jayaprakash Patil
    Ophthalmology, Univ of California Irvine, Irvine, California
  • A. L. Gramajo
    Ophthalmology, Univ of California Irvine, Irvine, California
    Departamento de Oftalmologia, Fundacion VER, Cordoba, Argentina
  • A. Sharma
    Ophthalmology, Univ of California Irvine, Irvine, California
  • M. F. Estrago Franco
    Ophthalmology, Univ of California Irvine, Irvine, California
  • M. Chwa
    Ophthalmology, Univ of California Irvine, Irvine, California
  • G. M. Seigel
    Ophthalmology, Physiology and Biophysics, Ross Eye Institute, University at Buffalo, The State University of New York, Buffalo, New York
  • B. D. Kuppermann
    Ophthalmology, Univ of California Irvine, Irvine, California
  • M. C. Kenney
    Ophthalmology, Univ of California Irvine, Irvine, California
  • Footnotes
    Commercial Relationships  A. Jayaprakash Patil, None; A.L. Gramajo, None; A. Sharma, None; M.F. Estrago Franco, None; M. Chwa, None; G.M. Seigel, None; B.D. Kuppermann, None; M.C. Kenney, None.
  • Footnotes
    Support  Discovery Eye, Henry L. Guenther, Iris and B. Gerald Cantor, RPB, and Gilbert Foundations, Skirball Molecular Ophthalmology Program, PAAO David&Julianna Pyott Retina Fellowhsip
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 3968. doi:https://doi.org/
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      A. Jayaprakash Patil, A. L. Gramajo, A. Sharma, M. F. Estrago Franco, M. Chwa, G. M. Seigel, B. D. Kuppermann, M. C. Kenney; Effects of Benzo(e)Pyrene on Rat Retinal Neurosensory Cells and Human Microvascular Endothelial Cells in vitro. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3968. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : In order to study the effects of different components of cigarette smoke on the retina and its correlation with the Age-related Macular Degeneration (AMD), we examined the effects of Benzo(e)Pyrene (B(e)P), a cigarette smoke toxicant on rat retinal neurosensory (R28) cells and human microvascular endothelial cells (HMVECs).

Methods: : R28 cells were cultured in Modified Eagle Medium with 10% fetal bovine serum. HMVECs were cultured in Medium 131 with growth supplement. Cells were treated for 24 hours with 1000, 400, 200 and 100µM of B(e)P. Cell viability was measured by trypan blue exclusion assay. Caspase-3/7, -8, -9 and -12 activities were measured by Fluorochrome assays. DNA laddering was evaluated by gel electrophoresis.

Results: : The mean cell viability of R28 cells after exposure to B(e)P 1000, 400, 200 and 100 µM was 39.93% ± 1.90 (P<0.001), 69.17% ± 2.75 (P<0.01), 86.37% ± 2.63 (P 0.05), respectively, compared to 1000 µM DMSO-equivalent control (98.13% ± 1.02). There was a 2-fold increase in caspase-3/7 and -8 activities at 200 µM B(e)P compared with the DMSO-equivalent control. However, this increase was not seen at 1000 and 400 µM B(e)P concentrations. There was no increase in caspase-9 and -12 activities at any B(e)P concentrations. DNA laddering revealed bands at 200 bp intervals at the 200 µM B(e)P concentration but not at higher concentrations.The mean cell viability of HMVEC after exposure to B(e)P 1000, 400, 200 and 100µM was 33.53% ± 1.8 (P<0.001), 45.23% ± 0.83 (P<0.001), 65.93% ± 2.87 (P 0.05) compared to 1000 µM DMSO-equivalent controls (84.1% ± 2.87). No increase of caspase-3/7 activity and DNA laddering was found at any of the B(e)P concentrations studied.

Conclusions: : Cigarette smoke is a strong environmental factor associated with AMD. B(e)P is a major polycyclic aromatic hydrocarbon (PAH) of cigarette smoke. We show B(e)P causes caspase dependent apoptosis in R28 cells at lower concentrations (200µM) and necrosis at higher concentrations (1000, 400µM). B(e)P causes caspase independent necrosis in HMVEC at all B(e)P concentrations. These findings may be important molecular mechanisms in the onset and progression of AMD.

Keywords: apoptosis/cell death • retina • inner retina dysfunction: biochemistry and cell biology 
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