Exposure of biological chromophores to ultraviolet radiation can lead to photochemical damage. However, the role of visible light, particularly in the blue region of the spectrum, has been largely ignored. To test the hypothesis that blue light is toxic to non-pigmented epithelial cells, confluent cultures of human primary retinal epithelial cells were exposed to visible light (390 -550 nm at 2.8 milliwatts/cm 2 ) for up to 6 h. A small loss of mitochondrial respiratory activity was observed at 6 h compared with dark-maintained cells, and this loss became greater with increasing time. To investigate the mechanism of cell loss, the damage to mitochondrial and nuclear genes was assessed using the quantitative PCR. Light exposure significantly damaged mitochondrial DNA at 3 h (0.7 lesion/10 kb DNA) compared with darkmaintained controls. However, by 6 h of light exposure, the number of lesions was decreased in the surviving cells, indicating DNA repair. Isolated mitochondria exposed to light generated singlet oxygen, superoxide anion, and the hydroxyl radical. Antioxidants confirmed the superoxide anion to be the primary species responsible for the mitochondrial DNA lesions. The effect of lipofuscin, a photoinducible intracellular generator of reactive oxygen intermediates, was investigated for comparison. Exposure of lipofuscin-containing cells to visible light caused an increase in both mitochondrial and nuclear DNA lesions compared with non-pigmented cells. We conclude that visible light can cause cell dysfunction through the action of reactive oxygen species on DNA and that this may contribute to cellular aging, age-related pathologies, and tumorigenesis.
The presence of the age pigment lipofuscin is associated with numerous age-related diseases. In the retina lipofuscin is located within the pigment epithelium where it is exposed to high oxygen and visible light, a prime environment for the generation of reactive oxygen species. Although we, and others, have demonstrated that retinal lipofuscin is a photoinducible generator of reactive oxygen species it is unclear how this may translate into cell damage. The position of lipofuscin within the lysosome infers that irradiated lipofuscin is liable to cause oxidative damage to either the lysosomal membrane or the lysosomal enzymes. We have found that illumination of lipofuscin with visible light is capable of extragranular lipid peroxidation, enzyme inactivation, and protein oxidation. These effects, which were pH-dependent, were significantly reduced by the addition of the antioxidants, superoxide dismutase and 1,4-diazabicyclo(2,2,2)-octane, confirming a role for both the superoxide anion and singlet oxygen. We postulate that lipofuscin may compromise retinal cell function by causing loss of lysosomal integrity and that this may be a major contributory factor to the pathology associated with retinal light damage and diseases such as age-related macular degeneration.The age pigment lipofuscin accumulates within the lysosomal system of a variety of postmitotic cells throughout life and is considered to be a biomarker of cell aging. Evidence has shown that the rate of lipofuscin accumulation corresponds to the aging rates in different species, being influenced by both metabolic activity and extent of oxidative stress (1). However, a causal role for lipofuscin in the aging process or development of age-related diseases such as neuronal ceroid lipofuscinosis, age-related macular degeneration, Ménière's disease, and cardiac hypertrophy has yet to be established (see Refs. 2 and 3). Unlike other cells in the body, in which lipofuscin occurs through the autophagic breakdown of intracellular organelles (2), the major substrate for lipofuscin in the retinal pigment epithelium (RPE) 1 of the eye is the undegradable end product resulting from the phagocytosis of photoreceptor outer segments (4, 5) that are rich in polyunsaturated fatty acids and vitamin A.Ocular lipofuscin may have a unique role to play in aging of the RPE, a tissue that is continually exposed to visible light (400 -700 nm) and high oxygen tensions (ϳ70 mm Hg). Studies have shown this type of lipofuscin to be a photoinducible generator of superoxide ions, singlet oxygen, hydrogen peroxide, and lipid peroxides (6 -9), all of which are reactive oxygen species implicated in general aging processes. These species can adversely affect cell function by damaging proteins, carbohydrates, DNA, and lipids (10). The position of lipofuscin within the lysosome infers that the first site of oxidative damage will be either the lysosomal membrane or the lysosomal enzymes. To test this hypothesis we have assessed the effect of photoactivated lipofuscin on (i) lipid peroxidat...
The purpose of this study was to determine whether an age-related increase in photoreactivity of human retinal melanosomes (MS) can cause phototoxicity to retinal pigment epithelium (RPE) cells. MS were isolated post mortem from young (20-30 years, young human melanosomes [YHMs]) and old (60-90 years, old human melanosomes [OHMs]) human eyes and from young bovine eyes (bovine melanosomes [BMs]). Confluent cultured ARPE-19 cells were fed equivalent numbers of OHMs or BMs and accumulated similar amounts of melanin as determined by electron paramagnetic resonance assay. Cells with and without MS were either maintained in the dark or exposed to blue light for up to 96 h and assessed for alterations in cell morphology, cell viability and lysosomal integrity. Incubation of cells in dark in the presence of internalized MS or irradiation of cells with blue light in the absence or presence of BMs did not significantly affect cell viability. However, exposures to blue light in the presence of OHMs resulted in abnormal cell morphology, up to approximately 75% decrease in mitochondrial activity, loss of lysosomal pH and cell death. OHMs contained significantly less melanin than YHMs, supporting the hypothesis that melanin undergoes degradation during RPE aging. Our results demonstrate that aged MS can be phototoxic to human RPE cells and support a contributing role of MS in RPE aging and in the pathogenesis of age-related macular degeneration.
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.
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