BACKGROUND: A fluorescent compound of lipofuscin, A2-E, has been shown to impair lysosomal function and to increase the intralysosomal pH of human retinal pigment epithelial (RPE) cells. This study addressed the phototoxic potential of A2-E on RPE cells. METHODS: A2-E accumulation was confirmed by fluorescence microscopy and fluorescence-activated cell sorter analysis. Acridine orange staining allowed assessment of lysosomal integrity and intralysosomal pH. Phototoxic properties of A2-E were determined by exposing A2-E-free and A2-E-fed RPE cell cultures to short-wavelength visible light and assessing cell viability and lysosomal integrity. RESULTS: Intralysosomal accumulation of A2-E was confirmed. Acridine orange staining showed that the A2-E was located in the lysosomal compartment and induced an elevation of intralysosomal pH. Exposure of A2-E fed cells to light resulted in a significant loss of cell viability by 72 h which was not observed in either RPE cells maintained in the dark or A2-E-free cultures exposed to light. Toxicity was associated with a loss of lysosomal integrity. CONCLUSION: A2-E is detrimental to RPE cell function by a variety of mechanisms including inhibition of lysosomal degrading capacity, loss of membrane integrity, and phototoxicity. Such mechanisms could contribute to retinal aging and to retinal diseases associated with excessive lipofuscin accumulation.
BACKGROUND: A fluorescent compound of lipofuscin, A2-E, has been shown to impair lysosomal function and to increase the intralysosomal pH of humanretinal pigment epithelial (RPE) cells. This study addressed the phototoxic potential of A2-E on RPE cells. METHODS: A2-E accumulation was confirmed by fluorescence microscopy and fluorescence-activated cell sorter analysis. Acridine orange staining allowed assessment of lysosomal integrity and intralysosomal pH. Phototoxic properties of A2-E were determined by exposing A2-E-free and A2-E-fed RPE cell cultures to short-wavelength visible light and assessing cell viability and lysosomal integrity. RESULTS: Intralysosomal accumulation of A2-E was confirmed. Acridine orange staining showed that the A2-E was located in the lysosomal compartment and induced an elevation of intralysosomal pH. Exposure of A2-E fed cells to light resulted in a significant loss of cell viability by 72 h which was not observed in either RPE cells maintained in the dark or A2-E-free cultures exposed to light. Toxicity was associated with a loss of lysosomal integrity. CONCLUSION: A2-E is detrimental to RPE cell function by a variety of mechanisms including inhibition of lysosomal degrading capacity, loss of membrane integrity, and phototoxicity. Such mechanisms could contribute to retinal aging and to retinal diseases associated with excessive lipofuscin accumulation.
Authors: Nicole M Haralampus-Grynaviski; Laura E Lamb; Christine M R Clancy; Christine Skumatz; Janice M Burke; Tadeusz Sarna; John D Simon Journal: Proc Natl Acad Sci U S A Date: 2003-02-28 Impact factor: 11.205