DNA is a target of the photodynamic effects elicited in A2E-laden RPE by blue-light illumination.
OBJECTIVE
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.
Sparrow
Janet R
JR
Department of Ophthalmology, Columbia University, New York, New York 10032, USA. jrs88@columbia.edu
Zhou
Jilin
J
Cai
Bolin
B
D000080242:8-Hydroxy-2'-Deoxyguanosine; D002470:Cell Survival / Q000528:radiation effects; D002478:Cells, Cultured; D020552:Comet Assay; D004247:DNA / Q000528:radiation effects*; D004249:DNA Damage; D004260:DNA Repair / Q000187:drug effects; D043382:DNA-Formamidopyrimidine Glycosylase; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000378:metabolism; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000494:pharmacology; D029968:Escherichia coli Proteins; D006801:Humans; D007124:Immunoenzyme Techniques; D008027:Light; D009699:N-Glycosyl Hydrolases / Q000494:pharmacology; D010778:Photochemotherapy; D010857:Pigment Epithelium of Eye / Q000378:metabolism / Q000528:radiation effects*; D011726:Pyridinium Compounds / Q000528:radiation effects*; D012176:Retinoids / Q000528:radiation effects*; D014764:Viral Proteins
C112040:A2-E (N-retinylidene-N-retinylethanolamine); D029968:Escherichia coli Proteins; D011726:Pyridinium Compounds; D012176:Retinoids; D014764:Viral Proteins; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D004706:Endodeoxyribonucleases; C470698:endonuclease V, phage T4; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; D003849:Deoxyguanosine