July 2019
Volume 60, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2019
Phototoxicity of rhodopsin photobleaching products in ARPE-19 cells
Author Affiliations & Notes
  • Tadeusz J Sarna
    Biophysics, Jagiellonian University, Krakow, Poland
  • Magdalena M Olchawa
    Biophysics, Jagiellonian University, Krakow, Poland
  • Olga Krzysztynska-Kuleta
    Biophysics, Jagiellonian University, Krakow, Poland
    Malopolska Centre for Biotechnology, Jagiellonian University, Krakow, Poland
  • Barbara Czuba-Pelech
    Biophysics, Jagiellonian University, Krakow, Poland
  • Footnotes
    Commercial Relationships   Tadeusz Sarna, None; Magdalena Olchawa, None; Olga Krzysztynska-Kuleta, None; Barbara Czuba-Pelech, None
  • Footnotes
    Support  MAESTRO4 2013/08/A/NZ1/00194 (TS) and SYMFONIA 2 2013/08/W/NZ3/00700 (TS) from the Poland National Science Centre
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 1673. doi:
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      Tadeusz J Sarna, Magdalena M Olchawa, Olga Krzysztynska-Kuleta, Barbara Czuba-Pelech; Phototoxicity of rhodopsin photobleaching products in ARPE-19 cells. Invest. Ophthalmol. Vis. Sci. 2019;60(9):1673.

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

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Abstract

Purpose : A growing body of experimental evidence suggests that rhodopsin photobleaching products, such as all-trans-retinal (ATR), may be involved in light-induced damage to the retina. Here we asked if ATR, formed in photoreceptor outer segments (POS), after in vitro exposure to strong visible light, may contribute to dysfunction of cultured retinal pigment epithelium (RPE) cells.

Methods : ARPE-19 cells pre-loaded with rhodopsin-rich POS isolated from bovine retinas were irradiated with green light (520-580 nm, 7 mW/cm2) to photobleach rhodopsin, and subsequently with blue light (425 nm, 10 mW/cm2) to excite ATR, for selected time intervals. Survival of cells was determined by MTT assay and propidium iodide staining. Changes in mitochondrial membrane potential (ΔΨm) were assessed by JC-1 staining. Cells and model systems, subjected to photic stress mediated by ATR, were also analyzed for the presence of protein hydroperoxides using the fluorogenic coumarin boronic acid (CBA) indicator. The effect of photic stress on specific and non-specific phagocytic activity of the cells was measured by flow cytometry using bovine POS labelled with fluoresceine-5-isothiocyanate and fluorescent polystyrene beads, respectively.

Results : Irradiation or ARPE-19 cells containing phagocytized rhodopsin-rich POS with green light and subsequently with blue light induced dose dependent loss in cell viability accompanied by a reduction in the cell mitochondrial membrane potential. Sub-lethal doses of photic stress, mediated by rhodopsin-rich POS, significantly inhibited the cell specific and non-specific phagocytic activity. In both cases, the inhibition of phagocytosis was transient and largely recoverable by 24 hours. Photic stress mediated by rhodopsin-rich POS induced peroxidation of cellular proteins and bovine serum albumin (BSA) in model systems.

Conclusions : Our data support the hypothesis that products of rhodopsin photobleaching formed in POS, exhibit substantial photoreactivity and can contribute to oxidative stress conditions in the photoreceptor/RPE complex and deterioration of key biological functions of the RPE.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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