In vitro phototoxicity of rhodopsin photobleaching products in the retinal pigment epithelium (RPE)

Abstract

Although the primary biological function of retinal photoreceptors is to absorb light and provide visual information, extensive exposure to intense light could increase the risk of phototoxic reactions mediated by products of rhodopsin bleaching that might accumulate in photoreceptor outer segments (POS). The phototoxicity of POS, isolated from bovine retinas, was examined in cultured retinal pigment epithelium cells (ARPE-19) containing phagocytised POS and in selected model systems by determining POS ability to photogenerate singlet oxygen, and photoinduce oxidation of cholesterol and serum albumin. Bleaching of rhodopsin-rich POS with green light resulted in the formation of retinoid products exhibiting distinct absorption spectra in the near-UV. Irradiation of POS-fed ARPE-19 cells with blue light reduced their survival in a dose-dependent manner with the effect being stronger for cells containing prebleached POS. The specific and non-specific phagocytic activity of ARPE-19 cells was inhibited by sub-lethal photic stress mediated by phagocytised POS. The oxidising ability of POS photobleaching products was demonstrated both in a model system consisting of serum albumin and in ARPE-19 cells. Distinct photooxidation of proteins, mediated by POS, was observed using coumarin boronic acid as a sensitive probe of protein hydroperoxides. Irradiation of POS with blue light also induced oxidation of liposomal cholesterol as determined by HPLC-EC(Hg). Time-resolved singlet oxygen phosphorescence demonstrated the efficiency of retinoids, extracted from POS by chloroform-methanol treatment, to photogenerate singlet oxygen. The results indicate that photic stress mediated by POS photobleaching products could inhibit phagocytic efficiency of RPE cells and, ultimately, compromise their important biological functions.

Keywords:

• Bleached rhodopsin
• phagocytosis
• photic stress
• photoreceptor outer segments
• retinoids

Acknowledgments

The authors thank Institute of Applied Radiation Chemistry, Lodz University of Technology for providing us with the CBA probe.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

Faculty of Biochemistry, Biophysics and Biotechnology of Jagiellonian University is a partner of the Leading National Research Center (KNOW) supported by the Ministry of Science and Higher Education. This work was supported by research grant SYMFONIA 1 2013/08/W/NZ3/00700 from the Poland National Science Center. The Faculty of Biochemistry, Biophysics, and Biotechnology of the Jagiellonian University is a beneficiary of structural funds from Error! Hyperlink reference not valid. Grant POIG.02.01.00-12-064/08 – “Molecular biotechnology for health”.