May 2003
Volume 44, Issue 5
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Retinal Cell Biology  |   May 2003
DNA Is a Target of the Photodynamic Effects Elicited in A2E-Laden RPE by Blue-Light Illumination
Author Affiliations
  • Janet R. Sparrow
    From the Department of Ophthalmology, Columbia University, New York, New York.
  • Jilin Zhou
    From the Department of Ophthalmology, Columbia University, New York, New York.
  • Bolin Cai
    From the Department of Ophthalmology, Columbia University, New York, New York.
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 2245-2251. doi:10.1167/iovs.02-0746
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      Janet R. Sparrow, Jilin Zhou, Bolin Cai; DNA Is a Target of the Photodynamic Effects Elicited in A2E-Laden RPE by Blue-Light Illumination. Invest. Ophthalmol. Vis. Sci. 2003;44(5):2245-2251. doi: 10.1167/iovs.02-0746.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

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.

Aging is thought to involve progressive damage to cellular macromolecules. 1 2 3 Although proteins, lipids, and DNA are all vulnerable, DNA damage is considered to be fundamental in the age-related decline in cellular function. 1 4 5 Although cells have mechanisms for repairing these lesions, repair is often incomplete, and the persistence of lesions can gradually lead to transcription decay and may eventually elicit cellular responses such as apoptosis. A variety of reactive species can mediate DNA damage, with oxidative reactions being considered a significant cause of age-related damage. 2 6 The range of lesions that can be induced include damage to the deoxyribose-phosphate backbone, specific chemical modifications of purine and pyrimidine bases, and DNA–protein cross-links. 7 8 A common oxidative DNA lesion is 8-oxo-deoxyguanosine (8-oxo-dG), a modification that forms by the insertion of oxygen at the C-8 position of guanine. 7 9  
For tissues that retain the capacity to proliferate, apoptosis provides a mechanism for eliminating a dysfunctional cell, 1 10 but for nonreplicating cells such as the RPE, cell death has dire implications. Indeed, in atrophic age-related macular degeneration (AMD), it is the loss of RPE cells that leads to photoreceptor cell degeneration and impairment of vision. One factor that may jeopardize the health of aging RPE cells is the lifelong accumulation of lipofuscin fluorophores. 11 12 In studies aimed at examining a causal link between lipofuscin accumulation and RPE cell death, we and others have shown that a hydrophobic constituent of RPE lipofuscin, the pyridinium bisretinoid A2E, confers a susceptibility to blue light-mediated cell death. 13 14 15 The propensity of blue-light–illuminated A2E-laden RPE to undergo apoptosis in these culture models 15 is concordant with epidemiologic studies suggesting a relationship between blue-light–exposure and atrophic AMD. 16 17 18 Moreover, accentuated deposition of A2E is now known to occur as a byproduct of reduced function of ABCR, 19 20 the transporter in photoreceptor outer segments responsible for autosomal recessive Stargardt disease, 21 some forms of recessive retinitis pigmentosa and cone–rod dystrophy 22 23 24 and perhaps atrophic AMD, in a subset of patients. 25 26 27 28 29  
It has been shown that lipofuscin isolated from RPE can serve as photoinducible generator of singlet oxygen, superoxide anion, and H2O2. 30 31 32 So, too, we have demonstrated that blue light illumination of A2E leads to the generation of singlet oxygen. 33 In turn, these singlet oxygens become inserted into carbon-carbon double bonds of the retinoid-side arms of A2E to generate highly reactive epoxides. 33 34 Because an enhancer (D2O) and quenchers (histidine, DABCO, azide) of singlet oxygen can influence the incidence of cell death after blue light illumination, 34 it is likely that these photochemical events are involved in the mechanism by which blue-light–irradiated A2E-laden RPE cells are injured. 
Which subcellular structures are the targets of photodamage can depend on the location of the photosensitizer at the time of illumination but can include the nucleus, mitochondria, lysosomes, and cellular membranes. 35 36 37 To begin to determine the intracellular sites at which RPE cells are damaged by the interaction of A2E and blue light, we have studied DNA damage in individual A2E-laden RPE cells after 430 nm illumination. 
Methods
A2E Accumulation in Culture
Human adult RPE cells (ARPE-19; American Type Culture Collection, Manassas, VA) which do not contain endogenous A2E 38 were grown as previously described, 13 38 and nonconfluent cultures were allowed to amass synthesized A2E 39 (20 μM; 24 hours). The intracellular localization of A2E in this culture model was described earlier. 13 38  
Illumination
Culture medium was replaced by phosphate-buffered saline with calcium, magnesium, and glucose (PBS-CMG) and cells (0.72 cm2 area) were exposed to blue light (430 ± 30 nm) delivered from a tungsten halogen source for the indicated periods. The power (26 mW) was calibrated with a power meter (Scientech, Boulder, CO). 
Single-Cell Gel Electrophoresis Assay
DNA damage was detected by single-cell gel electrophoresis (CometAssay; Trevigen, Gaithersburg, MD). 40 Fifteen minutes after exposure to blue light (0.19 mW/mm2; 5 minutes) the cells were detached by minimal trypsinization and after being added to low-melting-point agarose (1 × 105 cells/mL; 1/10 dilution; 37°C), 75-μL aliquots of the mixture were pipetted onto slides and allowed to gel in the dark. The slides were subsequently incubated in lysis solution at 4°C for 90 minutes followed by washing and placement in an alkali buffer for 45 minutes at room temperature in the dark. The slides were then transferred to a horizontal chamber for electrophoresis (1 V/cm; 30 minutes) in alkali solution (0.3 M NaOH, 1 mM EDTA, pH >13) on ice. After immersion in ethanol for 5 minutes, the slides were thoroughly air dried, and replicate preparations were stained with green fluorescent dye (SYBR Green; Applied Biosystems, Foster City, CA). 
To test for the presence of base-specific changes, the DNA glycolases formamidopyrimidine DNA N-glycosylase (Fpg), Escherichia coli endonuclease III (endo III), and T4-endonuclease V (T4-endo V; Trevigen) were used. 41 In each case, enzyme was applied to the agarose-embedded nuclei after cell lysis, incubation was performed in a humidified chamber at 37°C for 1 hour, and electrophoresis was performed. 
DNA repair after exposure to blue light (5–60 minutes) was evaluated by incubating the cells for periods of different duration (30 minutes and 1, 3, 6, and 10 hours) at 37°C to allow for removal of the damage. Repair capacity, defined as relative decrease (%) in comet tail moment, was calculated as [(tail moment immediately after illumination – tail moment after repair)/(tail moment immediately after illumination − tail moment of untreated cells)] × 100. Repair during a 20-minute period of illumination and for the 30 minutes after illumination incubation were also evaluated after preincubation (30 minutes) in aphidicolin (20 μM; Sigma Chemical Co., St. Louis, MO) followed by its continuous presence in the medium. 42 43 44 Aphidicolin interferes with the repair process by binding to and inhibiting DNA polymerases. Untreated cells, A2E-containing cells not exposed to blue light, and A2E-deficient cells exposed to blue light were included as the controls in these assays. 
Single-Cell Gel Assay Analysis
Slides were coded and examined blindly by epifluorescence microscopy. Comets were photographed with 460 to 500 nm excitation, 510 to 560 nm emission, and a ×10 objective. Images were captured by charge-coupled device (CCD) camera and 50 randomly selected fluorescent comets were analyzed per slide using software for analysis of the fluorescent comets (Euclid Analysis, St. Louis, MO). Comet tail moment was determined as the length of the comet’s tail multiplied by the percentage of total DNA in the tail, the latter being resolved from pixel intensities. Means were compared using the nonparametric Kruskal-Wallis test followed by the Dunn multiple comparison test (Instat; GraphPad Software, San Diego, CA). Normalized data were analyzed by ANOVA and Student-Newman-Keuls multiple comparison test. The level of significance was 0.05. 
Detection of 8-Oxo-dG
Oxidative modification of the nucleoside deoxyguanosine to generate 8-oxo-dG was detected by immunostaining with monoclonal antibody to 8-oxo-dG (Trevigen). Accordingly, after fixation in 70% ethanol (−20°C, 10 minutes) and denaturing of DNA (4 N HCl, 7 minutes), the cells were incubated in blocking serum (10% fetal bovine serum in 10 mM Tris-HCl [pH 7.5]; 1 hour, 37°C) followed by anti-8-oxo-dG diluted 1:300 in blocking serum (4°C, overnight). Visualization was by the biotin-avidin immunoperoxidase method (ABC, Vector Laboratories, Burlingame, CA) to yield a red reaction product (NovaRed; Vector Laboratories). Oxidative DNA damage was also assayed using a fluorescence assay (OxyDNA; Biotrin International, Dublin, Ireland) that is based on the detection of 8-oxo-dG by specific binding of FITC-conjugated avidin. 45 Briefly, blue-light–irradiated A2E-laden ARPE-19 cells were fixed in 4% paraformaldehyde on ice for 15 minutes. After dehydration (70% and 95% methanol, −20°C, 3 minutes), permeabilization (99% methanol, −20°C, 30 minute), and rehydration, the cells were incubated with FITC-conjugated avidin (1:12.5 dilution) for 1 hour at 37°C. After washing, the slides were mounted and examined by fluorescence microscopy. Methylene-blue–treated (20 mM, incubation for 30 minutes) RPE cells exposed to white light (30 W lamp; 30 minutes) served as the positive control. Photoactivated methylene blue is a known generator of singlet oxygen. 46  
Detection of Nonviable Cells
Cell viability was quantified after labeling nuclei of nonviable cells with the membrane impermeant dye (Dead Red; Molecular Probes, Eugene, OR) and the nuclei of all cells with 4′,6′-diamino-2-phenylindole (DAPI), as previously described. 34 Counting was performed from digital images, and nonviable cells were expressed as a proportion of the total number of cells in an illuminated field. 
Results
When analyzed by alkaline comet assay, A2E-containing RPE exposed to blue light were associated with the formation of distinct comet tails indicative of DNA damage (Fig. 1) . With a 20 minute exposure, 95% to 100% of the nuclei exhibited comets (100 nuclei/experiment, five experiments). Conversely, in preparations from control cells (untreated cells, cells exposed to blue light only or A2E-laden cells not exposed to blue light) nuclei appeared as condensed spherical bodies resembling normal nuclei. Under these conditions, the occasional nucleus exhibiting a discrete corona, that likely represented the nonspecific effects of trypsinization and centrifugation during the processing of cells for the comet assay. Tail moment, a parameter which increases in proportion to the number of DNA strand breaks, 44 47 48 was dramatically elevated in A2E-containing cells which had been exposed to blue light (Fig. 2) . With blue-light–irradiated cells, the magnitude of the increase in tail moment was found to be dependent on the time of exposure (Fig. 2A) . Conversely, measurements of tail moment obtained from nonilluminated A2E-laden cells, and cells exposed only to light, were not different from untreated control cells (Fig 2B) . We previously showed that the death of blue-light–illuminated A2E-containing RPE occurs through oxygen-associated mechanisms. 34 Moreover, we have demonstrated that on irradiation with blue light, A2E undergoes singlet-oxygen mediated photooxidation resulting in the formation of highly reactive epoxides. 33 Thus, in the current work we examined the ability of sodium azide, an efficient quencher of singlet oxygen, to prevent DNA damage in our model. Illumination of A2E-laden RPE in the presence of 1, 5, and 10 mM azide, resulted in a concentration-dependent decrease in mean comet tail moment with reductions of 20% (1 mM), 37% (5 mM), and 58% (10 mM) being observed compared with illumination in its absence (Fig. 3)
To begin to characterize the types of DNA damage induced in blue-light–irradiated A2E-laden RPE, the alkaline assay was also used in association with the DNA repair enzymes Fpg, endo III, and T4-endo V. Addition of these enzymes converts modified bases into strand breaks that can be detected by quantitative analysis of the comet, an increase in comet tail moment reflecting the presence of the enzyme-sensitive lesion. 41 44 49 Accordingly, when 430-nm–illuminated A2E-laden RPE were assayed in the presence of Fpg, a bacterial enzyme that recognizes modified purine bases (adenine, guanine) including 8-oxo-dG and formamidopyrimidines (ring-opened purines), 50 tail moment was 1.7- to 2 times greater than in the absence of the glycosylase (Fig. 4) . Tail moment in the control (A2E+Fpg) treated with Fpg was also increased in comparison to controls assayed in the absence of Fpg, a finding that indicates the presence of preexisting base changes. 51 The use of endo III to probe for oxidized pyrimidine (thymine and cytosine) bases in the context of A2E and blue light, generated a similar magnitude of increase in tail moment compared with the results in the absence of the enzyme (Fig. 4) . However, treatment of the agarose-embedded nuclei with T4-endo V, an enzyme that recognizes pyrimidine dimers, did not result in a further increase in tail moment over that observed in A2E-laden, blue light exposed cells (Fig. 4) . The latter finding is perhaps not surprising, because pyrimidine dimers are characteristic of direct UV excitation of DNA. 52 53  
The formation of 8-oxo-dG by oxidative modification of the native nucleoside deoxyguanosine, is a well known DNA base modification, and one detected by Fpg. 50 Because antibodies raised against protein-conjugated 8-oxodG have been shown to bind with high specificity, 54 we used a monoclonal antibody to 8-oxo-dG in an immunoperoxidase assay to confirm the presence of this oxidized base. Labeling of blue-light–exposed, A2E-laden RPE with anti-8-oxo-dG was confined to nuclei of the cells, whereas no nuclear staining was observable in cells treated only with A2E or only with blue light (Fig. 5) . The presence of oxidatively altered guanine was further confirmed in an epifluorescence assay based on the specific binding of FITC-conjugated avidin to 8-oxo-dG in individual cells. The sensitivity of avidin binding to 8-oxo-dG is estimated to be approximately 104 molecules of 8-oxo-dG in 106 cells or 1 base product per 100 cells. 45 With this assay, the nuclei of A2E-laden RPE cells exposed to blue light exhibited FITC fluorescence indicative of binding of FITC-avidin to the 8-oxo-dG moiety (Fig. 5) . Similar nuclear staining was observed in methylene blue–incubated cells that served as a positive control. Conversely, with blue light illumination in the absence of A2E, nuclei exhibited background fluorescence only. 
To study the capacity of the cells to repair the DNA damage we observed, we also challenged the cells with A2E and blue light (20 minutes) and then observed for repair of the damage during a period of incubation. 40 48 The time course of repair established for damage elicited by a 20-minute exposure to blue light, revealed two components (Fig. 6A) . An early rapid-repair phase involved a reduction in mean comet tail moment of approximately 50% within the first hour after damage. During a second, slower stage, the processing of DNA lesions continued but was not complete even after 10 hours. To evaluate repair capacity as a function of the extent of damage, A2E-laden RPE were exposed to 5, 10, or 20 minutes of illumination and tail moment measures obtained immediately after illumination (t = 0), were compared with tail moment after a 30 minute incubation (t = 3 hour; Fig. 6B ). Repair capacity after a 5-minute illumination reached 58% and then declined to 54% with a 10-minute exposure, and to 48% with a 20-minute exposure, a finding that indicate that the cells’ ability to repair was slightly reduced as the severity of damage increased. When the polymerization step of repair was inhibited by adding the DNA synthesis inhibitor aphidicolin, 42 43 during the period of illumination and for 30 minutes afterward, tail moment was also enhanced by 37% (56.3% ± 1.5%; without aphidicolin: 35.53% ± 1.4%; P < 0.001; three experiments). 
In some experiments in which DNA damage was quantified after various intervals of illumination, cell viability was also evaluated in companion cultures. In this case, paired cultures of A2E-laden RPE were blue light illuminated, with one set of cultures being submitted to alkaline comet assay, whereas cell viability was determined in the fellow culture after an 8-hour incubation. Plotting initial mean tail moment against the percentage of nonviable cells (Fig. 6C) demonstrated that mean tail moments that were greater than 40 were associated with the death of a subpopulation of the cells. 
Discussion
Opinions vary as to whether endogenous sources of reactive oxygen species, generated as a byproduct of mitochondrial respiration, represent a significant threat to genomic stability. 5 55 However, there is considerable evidence that exogenous elements such as UV light, ionizing radiation, and chemical agents, factors that impact tissues to differing degrees, may place considerable burden on genome maintenance. A cell such as the RPE may be susceptible to an additional unique source of damage in that it both accumulates photoreactive chromophores with age and, unlike most other cells in the body, is exposed to visible light. In keeping with this notion, we have shown that illumination of A2E-containing RPE with a 430-nm light, a wavelength that corresponds to the peak absorbance and excitation of A2E, leads to DNA injury, the extent of which is proportional to duration of exposure. The reduction in damage afforded by the singlet oxygen quencher sodium azide, revealed that at least some of the damage involves oxidative mechanisms, whereas the detection of Fpg- and endo III–sensitive DNA lesions indicated the presence of oxidized pyrimidine and purine bases. In addition, labeling with specific antibody and binding of fluorescein-labeled avidin indicated that guanine bases were oxidatively modified to generate 8-oxo-dG and perhaps other structurally related lesions. The cells’ ability to repair the DNA damage declined as the severity was increased, whereas kinetic studies revealed an initial period of rapid repair followed by a course of more slowly progressing recovery. 
The subcellular location of a photosensitizer is important in determining the types of cellular damage incurred. 35 In our tissue culture model, A2E accumulates intracellularly in lysosomes, a behavior that replicates the lysosomal compartmentalization of A2E in vivo. 38 Although A2E is not localized to the nucleus, we observed that under blue light illumination, A2E triggered DNA damage. Similar observations have been made for other photosensitizers that accumulate outside the nucleus. 36 Singlet oxygen is potentially an important cytotoxic product emerging from the photosensitization of A2E, 33 34 but its short lifetime in cells allows it to diffuse a distance of only 20 nm. 35 In terms of ravaging agents, an alternative possibility is that photoproducts induced by blue light illumination 33 34 diffuse through the nuclear membrane and damage DNA. This is a scenario that is thought to be the case for other photosensitizers 56 and one we are currently investigating. 
Although we have clearly demonstrated the presence of base oxidation, we cannot rule out the possibility that direct strand breaks were also induced. The repair of oxidized base lesions by base excision repair is a much slower process (half-time of approximately 3 hours) than strand-break ligation (half-time of approximately 30 minutes). 57 Thus, the initial period of rapid repair that we have identified (Fig. 6) may represent repair of strand breaks. Similarly, the partial inhibition of repair induced by the DNA polymerase inhibitor during the 30-minute period after illumination may represent interference with the rejoining of direct strand breaks. Although the presence of comets immediately after illumination may also indicate that strand breaks were induced as primary lesions, strand breaks measured by the comet assay immediately after illumination could just as well represent nicks arising between the incision and rejoining stages of excision repair processes 58 initiated during illumination. In addition, we cannot exclude the possibility that oxidative base modifications other than 8-oxo-dG occurred. In this context, it is well known that some base modifications, such as those occurring at the N7 position of guanine, are so destabilizing that ring-opened species are created and/or glycosidic bonds are cleaved to create abasic (apurinic, apyrimidinic) sites. 59 60 61 Both of these secondary transformations are converted to single-strand breaks under the alkali conditions of the comet assay. 62 63  
The extent to which photochemical damage from a given amount of light can affect cell viability, depends on the ability of cellular repair mechanisms to compensate. To counteract DNA damage, cells have refined DNA repair mechanisms. Base excision repair is the main excision repair pathway that removes 8-oxo-dG by a process that, in most of these lesions, is restricted to the damaged base. 64 In the initial step of the process, a DNA glycosylase recognizes and removes the damaged base, leaving an abasic site (AP site). The AP site is subsequently recognized and cleaved by AP endonuclease, thus creating a DNA strand break 5′ to the baseless sugar. After a DNA polymerase catalyzes the removal of the 5′-sugar phosphate moiety and incorporates a new nucleotide, the break is sealed by DNA ligase. The DNA glycosylase responsible for excising 8-oxo-dG from human DNA is OGG1, a protein that is the functional counterpart of Fpg. 64  
It appears that under some circumstances, the rate of damage exceeds the repair capacity. For instance, it has been shown that with chronic inflammation and malnutrition the rate of damage can exceed repair efforts. 65 66 67 Not only do cell types differ in the efficiency with which they correct DNA lesions, 41 individuals can also exhibit differences in the activity of repair enzymes acting on oxidized bases. 68 In the case of hOGG1, genetic polymorphisms and alternative splicing leads to the presence of multiple isoforms of the enzyme in human cells, and these isoforms differ in enzyme activity. 69 It is suggested that variable activity such as this, especially when placed on a background of environmental factors, may lead to interindividual differences in the capacity to repair DNA damage. It is also of interest that several studies have reported an age-associated accumulation of DNA damage that has escaped repair, 70 a finding that may reflect a decline in repair activity with age. 64 71 What is more, the levels of oxidative DNA lesions, in particular 8-oxo-dG, were found to be higher in nonreplicating cells than in slowly dividing ones, 72 possibly because DNA-damaged cells within the latter population can be combated by negative selection. 73  
We have studied DNA damage after relatively short periods of illumination. However, if A2E contributes to RPE atrophy in AMD or Stargardt disease, cellular damage may occur gradually over periods of years. A slow accretion in DNA damage could lead to a reduction in transcriptional activity and/or to the induction of new transcripts, such as those associated with cellular stress. 74 Whether gradual changes such as these could contribute to an RPE phenotype that is susceptible to atrophy remains to be determined. 
 
Figure 1.
 
Induction of DNA damage in blue-light–illuminated, A2E-laden RPE. Cells were processed by alkaline single-cell gel electrophoresis assay. A2E-laden cells exposed to blue light (A2E+blue light) exhibit comet tails indicative of damaged DNA. Comets were not formed from control cells (A2E only, blue light only, untreated cells).
Figure 1.
 
Induction of DNA damage in blue-light–illuminated, A2E-laden RPE. Cells were processed by alkaline single-cell gel electrophoresis assay. A2E-laden cells exposed to blue light (A2E+blue light) exhibit comet tails indicative of damaged DNA. Comets were not formed from control cells (A2E only, blue light only, untreated cells).
Figure 2.
 
Quantitation of DNA damage after blue light illumination (430 nm) of A2E-laden RPE. DNA damage was detected by alkaline comet assay and quantified as tail moment: the product of tail length and percentage of DNA in the tail. (A) Mean tail moment measured 15 minutes after illumination and plotted as a function of duration of illumination. Data are mean ± SEM of results in 4 to 11 independent experiments, 50 nuclei per experiment. One error bar is smaller than the corresponding symbol. (B) Tail moment is indicative of DNA damage in A2E-laden RPE exposed to blue light for 20 minutes (A2E BL) *P < 0.01 when compared to untreated (control) cells, A2E-loaded cells not exposed to blue light (A2E), or blue-light–exposed cells without A2E loading (BL). Data are mean ± SEM of results in three experiments, 50 nuclei per experiment.
Figure 2.
 
Quantitation of DNA damage after blue light illumination (430 nm) of A2E-laden RPE. DNA damage was detected by alkaline comet assay and quantified as tail moment: the product of tail length and percentage of DNA in the tail. (A) Mean tail moment measured 15 minutes after illumination and plotted as a function of duration of illumination. Data are mean ± SEM of results in 4 to 11 independent experiments, 50 nuclei per experiment. One error bar is smaller than the corresponding symbol. (B) Tail moment is indicative of DNA damage in A2E-laden RPE exposed to blue light for 20 minutes (A2E BL) *P < 0.01 when compared to untreated (control) cells, A2E-loaded cells not exposed to blue light (A2E), or blue-light–exposed cells without A2E loading (BL). Data are mean ± SEM of results in three experiments, 50 nuclei per experiment.
Figure 3.
 
Quenching of singlet oxygen suppresses blue-light–induced DNA damage in A2E-laden RPE. A2E-laden RPE were exposed to 430-nm light in the absence or presence of varying concentrations of sodium azide. DNA damage was detected by alkaline comet assay and quantified as tail moment. *P < 0.05; **P < 0.01, compared with A2E/blue light in absence of azide. Data are mean ± SEM of results in three experiments, 50 nuclei per experiment. Some error bars are below the resolution of the axis.
Figure 3.
 
Quenching of singlet oxygen suppresses blue-light–induced DNA damage in A2E-laden RPE. A2E-laden RPE were exposed to 430-nm light in the absence or presence of varying concentrations of sodium azide. DNA damage was detected by alkaline comet assay and quantified as tail moment. *P < 0.05; **P < 0.01, compared with A2E/blue light in absence of azide. Data are mean ± SEM of results in three experiments, 50 nuclei per experiment. Some error bars are below the resolution of the axis.
Figure 4.
 
Detection of base-specific DNA damage by enzyme-induced conversion to DNA breaks. A2E-laden RPE cells were exposed to a 430-nm light (A2E BL) and nuclei in agarose were treated with Fpg, which recognizes oxidized guanines; EIII, which is specific for oxidized pyrimidines; and T4-endo V (T4E), which recognizes pyrimidine dimers. Data were normalized as magnitude of increase over the untreated control, and, for each group, independent results obtained from two experiments are presented.
Figure 4.
 
Detection of base-specific DNA damage by enzyme-induced conversion to DNA breaks. A2E-laden RPE cells were exposed to a 430-nm light (A2E BL) and nuclei in agarose were treated with Fpg, which recognizes oxidized guanines; EIII, which is specific for oxidized pyrimidines; and T4-endo V (T4E), which recognizes pyrimidine dimers. Data were normalized as magnitude of increase over the untreated control, and, for each group, independent results obtained from two experiments are presented.
Figure 5.
 
Detection of 8-oxo-dG base modification. (A–C) Immunocytochemical labeling with antibody to 8-oxo-dG. A2E-laden RPE exposed to blue light (A) exhibited nuclear staining indicative of the presence of 8-oxo-dG. (B) Staining not present in nonilluminated cells (A2E). (C) Phase-contrast view of field shown in (B). (D–G) Epifluorescence visualization of binding of FITC-avidin to 8-oxo-dG. The nuclei of A2E–laden RPE cells exposed to blue light (D) exhibited FITC fluorescence indicative of binding of FITC-avidin to the 8-oxo-dG moiety. A2E autofluorescence was extranuclear and partially bleached by the illumination. With blue light exposure in the absence of A2E (E), nuclei are unstained. Nuclei were also unlabeled in A2E-laden cells not exposed to blue light (F); A2E autofluorescence was visible outside nuclei. Methylene blue-incubated cells exposed to visible light served as the positive control (G). Scale bar, 10 μm.
Figure 5.
 
Detection of 8-oxo-dG base modification. (A–C) Immunocytochemical labeling with antibody to 8-oxo-dG. A2E-laden RPE exposed to blue light (A) exhibited nuclear staining indicative of the presence of 8-oxo-dG. (B) Staining not present in nonilluminated cells (A2E). (C) Phase-contrast view of field shown in (B). (D–G) Epifluorescence visualization of binding of FITC-avidin to 8-oxo-dG. The nuclei of A2E–laden RPE cells exposed to blue light (D) exhibited FITC fluorescence indicative of binding of FITC-avidin to the 8-oxo-dG moiety. A2E autofluorescence was extranuclear and partially bleached by the illumination. With blue light exposure in the absence of A2E (E), nuclei are unstained. Nuclei were also unlabeled in A2E-laden cells not exposed to blue light (F); A2E autofluorescence was visible outside nuclei. Methylene blue-incubated cells exposed to visible light served as the positive control (G). Scale bar, 10 μm.
Figure 6.
 
Repair and lethality of DNA damage mediated by blue light illumination of A2E-laden RPE. Damaged DNA was quantified as tail moment. (A) Time course of DNA repair in blue-light–illuminated (20 minutes) A2E-laden RPE. Postillumination incubation intervals of 15 minutes to 10 hours allowed for repair. Dotted line: mean tail moment in the control. Data are mean ± SEM of results in three to four experiments. (B) Repair capacity during a 30-minute interval after irradiation for 5, 10 and 20 minutes was calculated. Data are the mean ± SEM of results in three experiments. The means are not significantly different. (C) Correlation between extent of DNA damage immediately after blue light illumination of A2E-laden RPE and the incidence of nonviable cells 8 hours later. Paired cultures of A2E-laden RPE were blue light illuminated, one culture being used to measure DNA damage by tail moment, whereas cell viability in the companion cultures was determined after an 8-hour period of incubation. Values are the mean ± SEM of results in three experiments; 50 nuclei per experiment. Some error bars are smaller than the corresponding symbols.
Figure 6.
 
Repair and lethality of DNA damage mediated by blue light illumination of A2E-laden RPE. Damaged DNA was quantified as tail moment. (A) Time course of DNA repair in blue-light–illuminated (20 minutes) A2E-laden RPE. Postillumination incubation intervals of 15 minutes to 10 hours allowed for repair. Dotted line: mean tail moment in the control. Data are mean ± SEM of results in three to four experiments. (B) Repair capacity during a 30-minute interval after irradiation for 5, 10 and 20 minutes was calculated. Data are the mean ± SEM of results in three experiments. The means are not significantly different. (C) Correlation between extent of DNA damage immediately after blue light illumination of A2E-laden RPE and the incidence of nonviable cells 8 hours later. Paired cultures of A2E-laden RPE were blue light illuminated, one culture being used to measure DNA damage by tail moment, whereas cell viability in the companion cultures was determined after an 8-hour period of incubation. Values are the mean ± SEM of results in three experiments; 50 nuclei per experiment. Some error bars are smaller than the corresponding symbols.
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Figure 1.
 
Induction of DNA damage in blue-light–illuminated, A2E-laden RPE. Cells were processed by alkaline single-cell gel electrophoresis assay. A2E-laden cells exposed to blue light (A2E+blue light) exhibit comet tails indicative of damaged DNA. Comets were not formed from control cells (A2E only, blue light only, untreated cells).
Figure 1.
 
Induction of DNA damage in blue-light–illuminated, A2E-laden RPE. Cells were processed by alkaline single-cell gel electrophoresis assay. A2E-laden cells exposed to blue light (A2E+blue light) exhibit comet tails indicative of damaged DNA. Comets were not formed from control cells (A2E only, blue light only, untreated cells).
Figure 2.
 
Quantitation of DNA damage after blue light illumination (430 nm) of A2E-laden RPE. DNA damage was detected by alkaline comet assay and quantified as tail moment: the product of tail length and percentage of DNA in the tail. (A) Mean tail moment measured 15 minutes after illumination and plotted as a function of duration of illumination. Data are mean ± SEM of results in 4 to 11 independent experiments, 50 nuclei per experiment. One error bar is smaller than the corresponding symbol. (B) Tail moment is indicative of DNA damage in A2E-laden RPE exposed to blue light for 20 minutes (A2E BL) *P < 0.01 when compared to untreated (control) cells, A2E-loaded cells not exposed to blue light (A2E), or blue-light–exposed cells without A2E loading (BL). Data are mean ± SEM of results in three experiments, 50 nuclei per experiment.
Figure 2.
 
Quantitation of DNA damage after blue light illumination (430 nm) of A2E-laden RPE. DNA damage was detected by alkaline comet assay and quantified as tail moment: the product of tail length and percentage of DNA in the tail. (A) Mean tail moment measured 15 minutes after illumination and plotted as a function of duration of illumination. Data are mean ± SEM of results in 4 to 11 independent experiments, 50 nuclei per experiment. One error bar is smaller than the corresponding symbol. (B) Tail moment is indicative of DNA damage in A2E-laden RPE exposed to blue light for 20 minutes (A2E BL) *P < 0.01 when compared to untreated (control) cells, A2E-loaded cells not exposed to blue light (A2E), or blue-light–exposed cells without A2E loading (BL). Data are mean ± SEM of results in three experiments, 50 nuclei per experiment.
Figure 3.
 
Quenching of singlet oxygen suppresses blue-light–induced DNA damage in A2E-laden RPE. A2E-laden RPE were exposed to 430-nm light in the absence or presence of varying concentrations of sodium azide. DNA damage was detected by alkaline comet assay and quantified as tail moment. *P < 0.05; **P < 0.01, compared with A2E/blue light in absence of azide. Data are mean ± SEM of results in three experiments, 50 nuclei per experiment. Some error bars are below the resolution of the axis.
Figure 3.
 
Quenching of singlet oxygen suppresses blue-light–induced DNA damage in A2E-laden RPE. A2E-laden RPE were exposed to 430-nm light in the absence or presence of varying concentrations of sodium azide. DNA damage was detected by alkaline comet assay and quantified as tail moment. *P < 0.05; **P < 0.01, compared with A2E/blue light in absence of azide. Data are mean ± SEM of results in three experiments, 50 nuclei per experiment. Some error bars are below the resolution of the axis.
Figure 4.
 
Detection of base-specific DNA damage by enzyme-induced conversion to DNA breaks. A2E-laden RPE cells were exposed to a 430-nm light (A2E BL) and nuclei in agarose were treated with Fpg, which recognizes oxidized guanines; EIII, which is specific for oxidized pyrimidines; and T4-endo V (T4E), which recognizes pyrimidine dimers. Data were normalized as magnitude of increase over the untreated control, and, for each group, independent results obtained from two experiments are presented.
Figure 4.
 
Detection of base-specific DNA damage by enzyme-induced conversion to DNA breaks. A2E-laden RPE cells were exposed to a 430-nm light (A2E BL) and nuclei in agarose were treated with Fpg, which recognizes oxidized guanines; EIII, which is specific for oxidized pyrimidines; and T4-endo V (T4E), which recognizes pyrimidine dimers. Data were normalized as magnitude of increase over the untreated control, and, for each group, independent results obtained from two experiments are presented.
Figure 5.
 
Detection of 8-oxo-dG base modification. (A–C) Immunocytochemical labeling with antibody to 8-oxo-dG. A2E-laden RPE exposed to blue light (A) exhibited nuclear staining indicative of the presence of 8-oxo-dG. (B) Staining not present in nonilluminated cells (A2E). (C) Phase-contrast view of field shown in (B). (D–G) Epifluorescence visualization of binding of FITC-avidin to 8-oxo-dG. The nuclei of A2E–laden RPE cells exposed to blue light (D) exhibited FITC fluorescence indicative of binding of FITC-avidin to the 8-oxo-dG moiety. A2E autofluorescence was extranuclear and partially bleached by the illumination. With blue light exposure in the absence of A2E (E), nuclei are unstained. Nuclei were also unlabeled in A2E-laden cells not exposed to blue light (F); A2E autofluorescence was visible outside nuclei. Methylene blue-incubated cells exposed to visible light served as the positive control (G). Scale bar, 10 μm.
Figure 5.
 
Detection of 8-oxo-dG base modification. (A–C) Immunocytochemical labeling with antibody to 8-oxo-dG. A2E-laden RPE exposed to blue light (A) exhibited nuclear staining indicative of the presence of 8-oxo-dG. (B) Staining not present in nonilluminated cells (A2E). (C) Phase-contrast view of field shown in (B). (D–G) Epifluorescence visualization of binding of FITC-avidin to 8-oxo-dG. The nuclei of A2E–laden RPE cells exposed to blue light (D) exhibited FITC fluorescence indicative of binding of FITC-avidin to the 8-oxo-dG moiety. A2E autofluorescence was extranuclear and partially bleached by the illumination. With blue light exposure in the absence of A2E (E), nuclei are unstained. Nuclei were also unlabeled in A2E-laden cells not exposed to blue light (F); A2E autofluorescence was visible outside nuclei. Methylene blue-incubated cells exposed to visible light served as the positive control (G). Scale bar, 10 μm.
Figure 6.
 
Repair and lethality of DNA damage mediated by blue light illumination of A2E-laden RPE. Damaged DNA was quantified as tail moment. (A) Time course of DNA repair in blue-light–illuminated (20 minutes) A2E-laden RPE. Postillumination incubation intervals of 15 minutes to 10 hours allowed for repair. Dotted line: mean tail moment in the control. Data are mean ± SEM of results in three to four experiments. (B) Repair capacity during a 30-minute interval after irradiation for 5, 10 and 20 minutes was calculated. Data are the mean ± SEM of results in three experiments. The means are not significantly different. (C) Correlation between extent of DNA damage immediately after blue light illumination of A2E-laden RPE and the incidence of nonviable cells 8 hours later. Paired cultures of A2E-laden RPE were blue light illuminated, one culture being used to measure DNA damage by tail moment, whereas cell viability in the companion cultures was determined after an 8-hour period of incubation. Values are the mean ± SEM of results in three experiments; 50 nuclei per experiment. Some error bars are smaller than the corresponding symbols.
Figure 6.
 
Repair and lethality of DNA damage mediated by blue light illumination of A2E-laden RPE. Damaged DNA was quantified as tail moment. (A) Time course of DNA repair in blue-light–illuminated (20 minutes) A2E-laden RPE. Postillumination incubation intervals of 15 minutes to 10 hours allowed for repair. Dotted line: mean tail moment in the control. Data are mean ± SEM of results in three to four experiments. (B) Repair capacity during a 30-minute interval after irradiation for 5, 10 and 20 minutes was calculated. Data are the mean ± SEM of results in three experiments. The means are not significantly different. (C) Correlation between extent of DNA damage immediately after blue light illumination of A2E-laden RPE and the incidence of nonviable cells 8 hours later. Paired cultures of A2E-laden RPE were blue light illuminated, one culture being used to measure DNA damage by tail moment, whereas cell viability in the companion cultures was determined after an 8-hour period of incubation. Values are the mean ± SEM of results in three experiments; 50 nuclei per experiment. Some error bars are smaller than the corresponding symbols.
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