Purpose: When the pyridinium bisretinoid A2E, an age-related fluorophore in the retinal pigment epithelium (RPE), is irradiated with blue light, photochemical events are initiated that can ultimately provoke cell death. This study was designed to determine whether DNA is a target of the cellular damage.
Methods: ARPE-19 cells accumulated A2E before exposure to blue light. DNA damage was assayed in individual cells by alkaline gel electrophoresis (comet assay), with and without the addition of the repair enzymes formamidopyrimidine N-glycosylase (Fpg), endonuclease III (endo III) and T4-endonuclease V (T4-endo V) to characterize DNA lesions. Damage was quantified as comet tail moment. The base lesion 8-oxo-deoxyguanosine (8-oxo-dG) was detected by immunoperoxidase and histochemical methods. The singlet oxygen quencher, sodium azide, was tested for its ability to reduce DNA damage, and cell viability was quantified.
Results: DNA damage was induced in A2E-containing RPE exposed to 430-nm illumination. The extent of damage, measured as tail moment, was proportional to exposure duration and was reduced by preincubation with sodium azide. The detection of FPG- and endo III-sensitive DNA lesions revealed the presence of oxidized purine and pyrimidine bases, whereas labeling with specific antibody and binding of fluorescein-labeled avidin indicated that guanine bases were oxidatively modified to 8-oxo-dG. The ability of the cells to repair the DNA damage declined as the severity was increased, and kinetic studies disclosed rapid and slow stages of repair.
Conclusions: DNA is one of the cellular constitutents that can be damaged by the interaction of A2E and blue light. At least some of the DNA lesions are oxidative base modifications.