December 1999
Volume 40, Issue 13
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Retina  |   December 1999
Comet Assay of UV-Induced DNA Damage in Retinal Pigment Epithelial Cells
Author Affiliations
  • William P. Patton
    From the Division of Vision Sciences, and the
  • Usha Chakravarthy
    From the Division of Vision Sciences, and the
  • R. Jeremy H. Davies
    School of Biology and Biochemistry, Queen’s University, Belfast, Northern Ireland, United Kingdom.
  • Desmond B. Archer
    From the Division of Vision Sciences, and the
Investigative Ophthalmology & Visual Science December 1999, Vol.40, 3268-3275. doi:
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      William P. Patton, Usha Chakravarthy, R. Jeremy H. Davies, Desmond B. Archer; Comet Assay of UV-Induced DNA Damage in Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 1999;40(13):3268-3275.

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

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Abstract

purpose. The molecular mechanisms mediating photic injury to the retinal pigment epithelial (RPE) cell are not clearly understood. This study examined qualitative and quantitative aspects of DNA damage caused by broadband UVA and UVB radiation in RPE cells.

methods. Cultured bovine RPE cells were exposed to doses of between 0 and 0.9 J/cm2 UVA or 0 and 0.09 J/cm2 UVB, as either a suspension or as an attached monolayer. The damage to DNA resulting in single-strand breaks was assessed by means of the comet assay in which the damaged DNA migrates out of the nucleus forming a tail, and this was quantified using image analysis. Two measurements were taken: the mean percentage of tail DNA, which reflects the overall level of DNA damage in the group of cells, and the Olive tail moment, which represents the extent of migration and thus the pattern of DNA damage in individual cells. Cells were processed by the comet assay immediately after UV exposure in acute experiments. To study the occurrence of DNA repair, RPE cells were first exposed to UVB and then incubated at 37°C for either 1 or 24 hours before processing for the comet assay.

results. UVA- and UVB-exposed cells showed a mean percentage of tail DNA that was significantly greater than in unexposed cells. Olive tail moment was higher in cells exposed to larger doses of UVB. This parameter also showed a bimodal distribution when assessed 24 hours after exposure to UVB indicating the presence of two distinct subpopulations of cells with small and large tail moments. Cells with very large tail moments were not seen with doses below 0.045 J/cm2.

conclusions. Relatively low doses of UVA and UVB induce the formation of DNA strand breaks in cultured RPE. The tail moment profiles for cells incubated for 24 hours after UVB irradiation are consistent with the occurrence of DNA repair in most cells exposed to low doses and apoptosis in a subpopulation of the cells exposed to high doses.

The neural retina is the most complex structure in the eye, processing light signals from the environment into patterns in the visual cortex through the optic nerve. 1 The energy of light is related to the wavelength, and a discriminate point appears to lie at 510 nm. Below this point there appears to be a direct relationship between retinal damage and the wavelength of light, with lower wavelengths causing greater damage. 2 Only a small proportion of light reaching the retina is required for the visual signal, and the excess light energy is thought to be absorbed mainly by the retinal pigment epithelium (RPE). 3 The RPE plays an extremely important role in maintaining functional photoreceptors, including degradation of photoreceptor outer segments. 4 Thus, agents that damage the RPE may have adverse affects on the photoreceptors and ultimately, on vision. 5  
UV radiation, which forms part of the electromagnetic spectrum, lies between visible light and ionizing radiation and is subdivided into three main categories according to wavelength (and thus energy). 6 Exposure to UV radiation is invariably damaging to living cells, with DNA being a major target for photochemical modification. UVC (100–280 nm) and UVB (280–315 nm) photons that can be absorbed directly by DNA are most deleterious, but UVA (315–400 nm) can also damage DNA indirectly through the action of photosensitizers. 5 However, the fluence of UVA required to induce a mutagenic or cytotoxic effect can be up to 1000 times greater than that required at the maximally effective wavelength in the UVB range. 6  
Although the sun emits radiation across the UV spectrum, the UVC component and UVB wavelengths of less than 290 nm are completely absorbed by the ozone layer in the upper atmosphere. 6 7 Longer wavelength UVB radiation, together with UVA, can penetrate to the Earth’s surface and thereby interact with the eye. However, the filtering effects of the cornea and the lens of the eye markedly attenuate any UV radiation that enters the eye, so that only a very small fraction reaches the retina. Consequently, UV radiation has not been considered to be a major factor in the induction of retinal damage. 6 Depletion of the ozone layer of the stratosphere has, however, led to increased UVB irradiance at the Earth’s surface. 7 This, in combination with the increase in the average life span of people, may result in an increase in the cumulative lifetime exposure of the retina to UV radiation. Thus, a role for UV radiation in the induction of age-related diseases of the retina becomes a distinct possibility. 
The cytotoxic effects of optical radiation on various cells and tissues including retina are well recognized from many in vivo and in vitro studies. 1 8 9 10 11 These have mostly assessed the viability of cells and tissues by dye-exclusion studies in vitro or by histologic appearances in vivo. However, the molecular mechanisms mediating cytotoxic photic injury are less well understood. Notably, UV radiation can generate free radicals including oxygen-derived species, 12 which are known to cause lipid peroxidation 9 13 14 15 of cellular membranes, and it can also induce photosensitized damage in DNA leading to single-strand breaks. 12 16 17 Although lipid peroxidation has been suggested as a possible mechanism for phototoxic effects, 14 the role of UV-induced DNA damage in retinal cell injury has not been examined. 
Nuclear DNA is known to be a target for UV radiation damage in cells. 18 Most of the associated lesions can give rise to DNA strand breaks either directly or under alkaline conditions. In the present study, the comet assay was used to examine qualitative and quantitative aspects of DNA damage sustained by RPE cells when they are exposed to broadband UVB or UVA radiation. The comet assay (also termed the single-cell gel electrophoresis assay) is a rapid and sensitive method used for the detection of DNA strand breaks in individual cells. The assay can also be used to assess the efficacy of enzymatic DNA repair processes, which involve the incision, and subsequent rejoining of damaged strands. 12 It can therefore provide valuable information regarding the molecular mechanisms that counteract radiation insult. In the present study, the occurrences of DNA repair and of apoptosis have been investigated in RPE cells after UVB irradiation. In addition, the sensitivity of RPE cells to UVB radiation has been compared with that of retinal vascular endothelial (RVE) cells. 
Materials and Methods
Low-melting-point agarose, EDTA, sodium chloride, and sodium hydroxide were obtained from Sigma (Poole, UK). Normal-melting-point agarose and minimal essential medium (MEM) were obtained from Gibco (Paisley, Scotland, UK). Ethidium bromide was purchased from Bio-Rad, (Herts, UK). 
Cell Culture
Bovine RPE cells were isolated using the method of Flood et al., 19 with some adaptations. Fresh bovine eyes were obtained from an abattoir and kept on ice until dissection. Excess fatty tissue and muscle were removed, and the surface of the globe was rinsed with 70% alcohol. The eye was dissected across the posterior pole, and the vitreous body and neural retina were removed. The remaining eyecup was washed with phosphate-buffered saline (PBS), and a solution of trypsin/EDTA (0.025%) was applied to the cup for 10 minutes at 37°C. The cells were then gently scraped off the posterior layer of the eyecup into trypsin, and an equal volume of cell culture medium (MEM supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 1mM l-glutamine) was added to neutralize enzyme activity. The resultant cell suspension was transferred to a 10-ml centrifuge tube and centrifuged at 1000g for 10 minutes. The pellet was then resuspended in 3 ml fresh growth medium and cultured in 25 cm3 flasks (Falcon, UK) at 37°C in a mixture of air and 5% CO2. Cells used for all the experiments were between passages two and eight. When required, cell suspensions were prepared from the cultures propagated in flasks by washing in PBS, trypsinization, and low-speed centrifugation. Bovine RVE cells were provided by Pauline Linton, Department of Ophthalmology, Queen’s University of Belfast. 
UV Irradiation of Cells
For all experiments, excepting those in which the comet assay was performed 24 hours after exposure, the RPE cells were irradiated as freshly harvested suspensions. The cells were resuspended in PBS after counting, to attain the desired concentration (1 × 105 cells in 40 μl). UVB irradiation was performed using a broad-spectrum lamp (PL-S 9W/12; Philips, Aylesbury, UK), having peak emission at 306 nm. The UVA source was a single-tube lamp (TL-20W/08; Philips) with maximal emission at 365 nm. Incident fluence rate was measured using a radiometer (UVX; UVP, Upland, CA) with a UVX-31 sensor for UVB and a UVX-36 sensor for UVA. Irradiation was performed in an inverted Eppendorf (Fremont, CA) cap. The UV source was positioned directly above the cell suspension to avoid any absorption of UV by the plastic. Dose was monitored by measuring fluence rates for each set of experiments. The dose of UV was adjusted by varying the duration of exposure. For UVA irradiation, exposure times ranged from 0 to 30 minutes corresponding to doses between 0 and 0.9 J/cm2. For UVB irradiation, exposure times ranged from 0 to 2 minutes corresponding to doses between 0.0 and 0.09 J/cm2. In acute experiments, after exposure to UV was completed, cells were immediately processed for the comet assay. Cells used as controls were similarly trypsinized, centrifuged, and placed in the plastic container but not exposed to UV irradiation. 
In experiments measuring postirradiation effects on DNA, cell suspensions were maintained at 37°C for 1 hour after exposure before processing by the comet assay. When radiation effects were to be studied 24 hours after UV exposure, cells propagated in petri dishes were washed with PBS and irradiated as a confluent monolayer without prior trypsinization. Fresh growth medium was then added and the cells returned to the incubator for 24 hours, after which the cells were trypsinized, centrifuged, and resuspended at the correct concentration and processed by the comet assay. 
Comet Assay
This technique permits the detection of DNA damage in single cells 20 when performed at high pH (alkaline conditions) and reveals the presence of double-strand breaks, single-strand breaks, and alkali-labile sites. 21 The cells under study are sandwiched in agarose on a slide and subjected to lysis followed by electrophoresis under specified conditions. Under lytic conditions the cells lose their proteins and are believed to be incapable of repairing damaged DNA. During electrophoresis, the damaged and fragmented DNA migrates away from the nucleus and into the gel toward the anode. The amount of migrated DNA is a measure of the extent of damage. To detect DNA, the slides are stained with ethidium bromide and examined for fluorescence emission using an excitation filter of 515 to 560 nm and a barrier filter of 590 nm. Cells containing damaged DNA have the appearance of a comet with a bright head and tail. In contrast, undamaged DNA (in unaffected cells) remains tightly coiled and appears after the electrophoresis as an intact nucleus with no tail, thus allowing the cells’ differentiation from those that have incurred DNA damage. 
The technique is sensitive, simple, and inexpensive, and quantification of DNA damage is possible using computer-assisted image analysis (Komet analysis software, ver. 3.1; Kinetic Imaging, Liverpool, UK) to integrate fluorescence intensity. It is recommended by the manufacturers that 50 cells on each slide be chosen at random and analyzed using the proprietary software. The mean percentage of tail DNA, reflecting the proportion of DNA that has migrated from the head, is then calculated as an average for the 50 cells selected for measurement. In addition, a function known as the Olive tail moment is obtained for each cell analyzed. This parameter essentially represents the product of the percentage of total DNA in the tail and the distance between the centers of mass of the head and tail regions. 22 This parameter takes into account the distance that the DNA has migrated and thus provides an indication of the pattern of DNA damage in an individual cell. 
Comet Assay Method
Fully frosted slides were prewarmed to 37°C, and 100 μl (0.75%) normal-melting-point agarose was pipetted onto each one. Coverslips were applied immediately to the slides and the gels allowed to set for approximately 20 minutes. Low-melting-point 0.75% agarose (100 μl) was added to 40 μl of each cell suspension. The suspension was gently mixed to ensure even dispersion of cells and gently pipetted onto the prepared slides after removing the coverslips. The coverslips were then replaced for a few minutes while the second layer of gel containing the cells was allowed to set. The coverslips were again removed and the slides placed in freshly prepared lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, adjusted to pH 10 with NaOH and containing 1% Triton X-100) at 4°C for l hour. After this time, the slides were removed and placed into alkaline electrophoresis solution (300 mM NaOH, 1 mM EDTA) for 20 minutes to allow the DNA to unwind. Electrophoresis was performed at 20 V and 300 mA for exactly 20 minutes. The slides were removed and neutralization with 0.4 M Tris-HCl (pH 7.4) was performed for a further 20 minutes. The slides were drip dried, stained with 50 μl ethidium bromide (20 μg/ml), and viewed within 48 hours using an epifluorescence microscope (Nikon UK Ltd, Surrey, UK). 
Results
When cells processed for the comet assay were examined by fluorescence microscopy, fluorescent structures corresponding to the ethidium bromide–stained nuclear DNA of the RPE cells were revealed. In undamaged cells the DNA was tightly compressed and maintained the circular disposition of the normal nucleus (Fig. 1A ). After exposure of the cells to UV radiation, the profile of the nuclear DNA was altered with the appearance of a fluorescent streak extending from the nucleus (Fig. 1B) , comprising the comet. The background level of processing damage that is inevitable because of centrifugation, trypsinization, and pipetting of cells is reflected by minor degrees of comet formation. This was seen in unirradiated controls of suspension and monolayer experiments in which mean percentage of tail DNA was consistently in the range of 3% to 6%. Comparison of the mean percentage of tail DNA between cells irradiated either as a suspension or as a monolayer showed higher levels of DNA damage in the latter for each dose (Fig. 2)
Dose Studies
These experiments which were designed to measure DNA damage before the onset of DNA repair, were performed with RPE cells in suspension, and showed that nuclear DNA was damaged in a dose-dependent manner by UVA and UVB irradiation. As the dose of UVA radiation increased, the mean percentage of tail DNA also increased in a nonlinear fashion (Fig. 3) . The mean percentage of tail DNA was 10.67% after exposure of RPE cells for 15 minutes to the UVA source (dose of 0.45 J/cm2). Increasing exposure to 30 minutes, thus doubling the dose to 0.9 J/cm2, caused an increase to 14.96%. Exposure times in excess of 30 minutes to achieve higher levels of DNA damage were not possible, because the cells clumped together and adhered to the surface of the plastic container in which they were irradiated. Both the number of cells showing damage and the extent of damage in individual cells were greater in cells exposed to UVB than in UVA-irradiated cells. Even the lowest dose of UVB tested (0.012 J/cm2) caused the appearance of a mean of 10.73% of tail DNA. Higher doses of UVB caused marked increases in the mean percentage of tail DNA (Fig. 4A ). 
When the distribution of DNA damage in the population of RPE cells exposed to UVB was analyzed, all the cells in the control population exhibited Olive tail moments of 3 or less. A dose of 0.012 J/cm2 of UVB resulted in most of the cells having an Olive tail moment between 2 and 4. Further increments in dose up to 0.09 J/cm2 led to an increasing number of cells showing greater Olive tail moments (Fig. 4B) , seen as a shift of the bars in the chart to the right. 
Repair Studies
When RPE cells were exposed to 0.09 J/cm2 UVB and left to repair DNA damage for 1 hour at 37°C, the mean percentage of tail DNA increased significantly by a factor of 1.46 when compared with cells exposed to the same dose but lysed immediately for comet assay (n = 3, Fig. 5A ). DNA damage in RPE cells exposed to doses of UVB of 0.045 J/cm2 or less, incubated at 37°C for 24 hours before comet analysis, was not notably different from that in cells exposed to similar doses in suspension or monolayer and lysed immediately, but with the highest dose of 0.09 J/cm2 UVB, mean percentage of tail DNA was significantly increased (data not shown). The effect of repair on tail moment distribution after 0.09 J/cm2 UVB was analyzed in more detail to assess whether some of the DNA damage seen after irradiation stems from the onset of apoptotic mechanisms. Figure 5B shows the distribution of tail moment in a single population of cells when damage was assessed at 0, 1, and 24 hours after irradiation with 0.09 J/cm2 of UVB. At both 1 and 24 hours after irradiation, there was an increase in the number of cells showing larger tail moments. Within the population of cells examined at 24 hours, a bimodal distribution of DNA damage was clearly evident consistent with the presence of two distinct groups of cells. The group of cells with tail moments in excess of 12 were considered to be undergoing apoptosis, 23 whereas those with tail moments below 10 were considered to be undergoing DNA repair. 
Comparison of RPE and RVE
When the sensitivity of RPE and RVE cells to UVB when irradiated in suspension was compared by the comet assay, the former exhibited considerably more DNA damage than the latter. In these experiments (n = 3) a dose of 0.09 J/cm2 resulted in a mean of 28.14% ± 2.38% of tail DNA in RPE and 13.39% ± 1.13% in RVE cells (analysis of variance; P = 0.00001; n = 3; Fig. 6 ). 
Discussion
Most studies on the effects of near UV or visible radiation on ocular tissues have focused on functional and morphologic changes in the photoreceptors and retinal pigment epithelium in vivo. 8 24 25 26 27 Broadband blue light (400–520 nm) has been found to disrupt the blood–retinal barrier properties of the retinal pigment epithelium 28 of the rabbit eye at a dose of 18 J/cm2. The threshold dose for funduscopic 8 evidence of damage after exposure to short-wavelength UVA radiation (320 nm) has been observed to lie at 0.35 J/cm2. On histologic examination, the damaged tissue was shown to consist of cells with pyknotic photoreceptor nuclei. Other studies that examined the lethal effect of near UV radiation peaking at 365 nm showed that killing of RPE cells was dependent on the total energy dose of irradiation and the state of confluence of the culture. 26  
By means of the comet assay, the present study has elucidated some of the molecular changes subsequent to UV radiation. In the comet assay a damaged cell takes on the appearance of a comet, with head and tail regions. A variety of geometric and densitometric parameters are provided by the image analysis software, which allows an estimation of the amount of DNA in the head and tail regions and the extent of migration into the tail region. Because the tail length and density reflect the number of single-strand breaks in the DNA, the percentage of DNA in the tail provides a quantitative measure of the damaged DNA. As seen in the dose–response experiments of the present study, the extent of DNA damage increased proportionately with increasing doses of either UVA or UVB. Possibly, owing to processing artifact or the low level of DNA excision and repair that cells are constantly undergoing, unirradiated control cells also consistently exhibited a small amount of DNA in the tail that was never in excess of 6% of the total DNA in the cell. 
Because the broadband UVB source (which emits some UVA) was more effective in producing DNA damage, it was used to examine the RPE cells’ response to UV irradiation in more detail. To assess acute DNA damage induced by UVB, it is essential to perform cell lysis as soon as practically possible after exposure, thus minimizing the introduction of strand breaks arising from DNA repair by the cells enzymes. In addition to photochemically generated strand breaks, UV induces the formation of a variety of nucleobase photoproducts that are subject to excision repair. 12 During this phase of repair, the process produces new strand breaks that are detected by the comet assay leading to a concomitant increase in mean percentage of tail DNA. For this reason comet analysis was performed in dose–response experiments with freshly harvested suspensions of RPE cells that were lysed as soon as possible after irradiation. In the subsequent studies of DNA repair, these data were compared with the damage profiles observed when the suspended RPE cells were held at 37°C for 1 hour before lysis and comet assay. This treatment caused no increase in the percentage of tail DNA of unirradiated controls. 
Because the metabolic behavior of anchorage-dependent cells such as RPE may be atypical when they are maintained in suspension, the occurrence of DNA repair was also investigated under more physiologically relevant conditions by exposing cells to UVB while still attached as a monolayer, incubating them in growth medium for 24 hours, and then subjecting them to lysis and comet assay. It is noteworthy that for the same incident dose of UVB, comets from RPE cells irradiated as a monolayer displayed a considerably higher mean percentage of tail DNA than did comets from cells irradiated in suspension. This difference, which does not affect the main conclusions of this study, can be attributed at least in part to the extra time taken to process (trypsinization, incubation at 37°C, and centrifugation, which are necessary steps to prepare the cells after irradiation) monolayer irradiated cells before lysis. It may also reflect some variation in the average amount of UVB absorbed per cell during the two different irradiation protocols. 
As mentioned previously, when RPE cells, exposed to the highest dose of UVB (0.09 J/cm2), were allowed to repair DNA damage for 1 hour or 24 hours before analysis by the comet assay, it led to a significant increase in mean percentage of tail DNA when compared with those lysed immediately after irradiation. Although these measurements constitute clear evidence for the occurrence of strand incision by repair enzymes, they provide limited information concerning the fate of the cell subsequent to injury. However, assessing the Olive tail moment distribution allows greater insight in this regard. After acute injury, the Olive tail moment in individual members of a population of cells is dependent on the intensity of the injury and assumes a reasonably normal distribution. When cells are able to repair sublethal DNA damage this distribution changes so that most cells show decreasing tail moments. However, in the event that extensive DNA damage is incurred, those cells that are unable to repair damage become committed to the process of apoptosis. These cells develop very large tail moments, because DNA in cells undergoing apoptosis is cleaved into fragments with sizes in the range of 20 to 300 kbp or 50 to 200 bp. 29 In such cells, after electrophoresis, there is migration of the fragments into a distinctive comet tail with a dense region separate from the main nuclear region. Cells displaying such damage exhibit tail moments in excess of 12, whereas cells that have undergone normal DNA repair have low comet tail moments. It is considered that a bimodal distribution with both very low and very high comet tail moments within a population of cells indicates the onset of apoptosis. 23 Stimuli that result in necrosis do not cause such a bimodal distribution of tail moments. 
The present study clearly showed that exposure of RPE cells to the highest dose of 0.09 J/cm2 UVB resulted in a bimodal distribution of Olive tail moment only when the cells were allowed to repair UV-induced damage by incubation for 24 hours. This bimodal distribution was not seen when cells were processed for comet assay immediately after irradiation. As in previous studies, the cutoff point separating cells with small tail moments from those with larger tail moments was found to lie at approximately 12. 23 At the lower UVB dose of 0.045 J/cm2, comet tail moments measured 24 hours after irradiation also showed evidence of a bimodal distribution, although fewer cells exhibited large tail moments (results not shown). At the lowest dose used in this study, 0.012 J/cm2, most cells appeared to be able to repair the DNA damage, and no bimodal distribution was observed. Thus, there appears to be a level of UVB insult that the RPE can tolerate and above which the proportion of cells going into programmed cell death increases markedly. 
RPE cells showed a greater level of acute damage, represented by mean percentage of tail DNA, than RVE cells after exposure to the same dose (0.09 J/cm2). The reason for this is not clear, although it may be related to the cytoplasmic components of each cell type. The RPE are known to contain a variety of pigments, 1 including melanin, carotenes, and xanthophylls, some of which have the capacity to act as photosensitizers. 
The present study confirmed that DNA single-strand breaks occurring either directly or from alkali-labile lesions are present in the RPE DNA after exposure to UV radiation. The study also provided evidence that DNA repair processes, involving strand incision, were initiated soon after irradiation and that apoptosis was induced in a subpopulation of cells when the dose of UVB was 0.045 J/cm2 or more. Finally, the study identified higher levels of DNA damage in RPE cells compared with endothelial cells, when the same dose of UVB was used. 
 
Figure 1.
 
(A) Typical nuclear profile of undamaged RPE cells processed by comet assay. The nuclear chromatin is tightly packed into a circular shape. (B) Nuclear profile along with a tail illustrating a typical comet immediately after 0.09 J/cm2 of UVB.
Figure 1.
 
(A) Typical nuclear profile of undamaged RPE cells processed by comet assay. The nuclear chromatin is tightly packed into a circular shape. (B) Nuclear profile along with a tail illustrating a typical comet immediately after 0.09 J/cm2 of UVB.
Figure 2.
 
The relationship between mean percentage of tail DNA and dose of UVB. Cells were irradiated as a suspension or as monolayer, as indicated. For each dose of UVB, cells irradiated as a monolayer and then processed for comet analysis show a correspondingly higher percentage of tail DNA than cells irradiated in suspension. Values are ±SEM; Suspension: unexposed control 2.52% ± 0.42%; 0.012 UVB, 7.10% ± 0.89%; 0.045 UVB, 11.94% ± 1.14%; 0.09 UVB, 24.92% ± 1.62%. Monolayer: Unexposed control, 2.49% ± 0.49%; 0.012 UVB, 13.98% ± 1.53%; 0.045 UVB, 19.37% ± 1.83%; 0.09 UVB, 42.00% ± 1.72%
Figure 2.
 
The relationship between mean percentage of tail DNA and dose of UVB. Cells were irradiated as a suspension or as monolayer, as indicated. For each dose of UVB, cells irradiated as a monolayer and then processed for comet analysis show a correspondingly higher percentage of tail DNA than cells irradiated in suspension. Values are ±SEM; Suspension: unexposed control 2.52% ± 0.42%; 0.012 UVB, 7.10% ± 0.89%; 0.045 UVB, 11.94% ± 1.14%; 0.09 UVB, 24.92% ± 1.62%. Monolayer: Unexposed control, 2.49% ± 0.49%; 0.012 UVB, 13.98% ± 1.53%; 0.045 UVB, 19.37% ± 1.83%; 0.09 UVB, 42.00% ± 1.72%
Figure 3.
 
Mean percentage of tail DNA in a population of cells exposed to 0.45 and 0.9 J/cm2 of UVA (n = 2, with 100 observations). Control cells not exposed to UV radiation showed minimal levels of DNA in the tail (∼5%). Increasing doses of UVA caused larger comet tails. Values are ±SEM. Unexposed control, 3.57% ± 0.35%; 0.45 UVA, 10.67% ± 1.06%; 0.9 UVA, 14.96% ± 1.49%.
Figure 3.
 
Mean percentage of tail DNA in a population of cells exposed to 0.45 and 0.9 J/cm2 of UVA (n = 2, with 100 observations). Control cells not exposed to UV radiation showed minimal levels of DNA in the tail (∼5%). Increasing doses of UVA caused larger comet tails. Values are ±SEM. Unexposed control, 3.57% ± 0.35%; 0.45 UVA, 10.67% ± 1.06%; 0.9 UVA, 14.96% ± 1.49%.
Figure 4.
 
(A) Mean percentage of tail DNA in a population of cells exposed to a range of doses of broadband UVB (n = 3, with 150 observations). X-axis shows the dose in joules per square centimeter; y-axis represents mean percentage of DNA in the tail. Values are ±SEM. Unexposed control, 4.00% ± 0.33%; 0.012 UVB, 10.73% ± 0.88%; 0.025 UVB, 16.44% ± 1.64%; 0.045 UVB, 22.89% ± 1.87%; 0.09 UVB, 32.93% ± 2.69%. (B) Olive tail moment distribution (50 observations) in RPE cells treated with UVB as in Figure 3A . Tail moments in excess of 10 indicating moderate to severe damage were observed only in cells receiving 0.045 or 0.09 J/cm2 UVB.
Figure 4.
 
(A) Mean percentage of tail DNA in a population of cells exposed to a range of doses of broadband UVB (n = 3, with 150 observations). X-axis shows the dose in joules per square centimeter; y-axis represents mean percentage of DNA in the tail. Values are ±SEM. Unexposed control, 4.00% ± 0.33%; 0.012 UVB, 10.73% ± 0.88%; 0.025 UVB, 16.44% ± 1.64%; 0.045 UVB, 22.89% ± 1.87%; 0.09 UVB, 32.93% ± 2.69%. (B) Olive tail moment distribution (50 observations) in RPE cells treated with UVB as in Figure 3A . Tail moments in excess of 10 indicating moderate to severe damage were observed only in cells receiving 0.045 or 0.09 J/cm2 UVB.
Figure 5.
 
(A) Mean percentage of tail DNA in RPE exposed to 0.09 J/cm2 UVB radiation and processed for comet assay immediately after irradiation (t = 0) and 1 hour (t = 1) after exposure. (n = 3, with 150 observations). Tail DNA increases with increasing time after irradiation. Values are ±SEM. Unexposed control, 4.16% ± 0.34%; 1 hour control, 3.99 ± 0.45; UV acute, 27.45% ± 2.24%; 1 hour after irradiation, 40.02% ± 3.27%. (B) Distribution of Olive tail moment for the same experiment as in Figure 4A with 50 observations. A bimodal distribution of damage is seen 24 hours after exposure with peaks at tail moments of 9 and 15.
Figure 5.
 
(A) Mean percentage of tail DNA in RPE exposed to 0.09 J/cm2 UVB radiation and processed for comet assay immediately after irradiation (t = 0) and 1 hour (t = 1) after exposure. (n = 3, with 150 observations). Tail DNA increases with increasing time after irradiation. Values are ±SEM. Unexposed control, 4.16% ± 0.34%; 1 hour control, 3.99 ± 0.45; UV acute, 27.45% ± 2.24%; 1 hour after irradiation, 40.02% ± 3.27%. (B) Distribution of Olive tail moment for the same experiment as in Figure 4A with 50 observations. A bimodal distribution of damage is seen 24 hours after exposure with peaks at tail moments of 9 and 15.
Figure 6.
 
Response of RVE and RPE cells after exposure to 0.09 J/cm2 UVB radiation (n = 3, with 140 observations). RPE cells show greater mean percentage of tail DNA than the RVE cells for the same dose. Values are ±SEM. Unexposed control RVE cells, 5.24% ± 0.44%; unexposed control RPE, 4.96% ± 0.42%; exposed RVE cells, 13.39% ± 1.57%; exposed RPE, 28.14% ± 2.38%.
Figure 6.
 
Response of RVE and RPE cells after exposure to 0.09 J/cm2 UVB radiation (n = 3, with 140 observations). RPE cells show greater mean percentage of tail DNA than the RVE cells for the same dose. Values are ±SEM. Unexposed control RVE cells, 5.24% ± 0.44%; unexposed control RPE, 4.96% ± 0.42%; exposed RVE cells, 13.39% ± 1.57%; exposed RPE, 28.14% ± 2.38%.
The authors thank Pauline Linton and Gerry Mahon for their help and expertise with tissue culture; Valerie McKelvey-Martin, University of Ulster, Coleraine, for her valuable help and advice with the comet assay; the staff from the Andrology Laboratory, Royal Victoria Hospital, Belfast, the School of Biomedical Sciences, University of Ulster, Jordanstown, for assistance and for valuable advice and guidance on the use of the Comet analysis system. 
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Figure 1.
 
(A) Typical nuclear profile of undamaged RPE cells processed by comet assay. The nuclear chromatin is tightly packed into a circular shape. (B) Nuclear profile along with a tail illustrating a typical comet immediately after 0.09 J/cm2 of UVB.
Figure 1.
 
(A) Typical nuclear profile of undamaged RPE cells processed by comet assay. The nuclear chromatin is tightly packed into a circular shape. (B) Nuclear profile along with a tail illustrating a typical comet immediately after 0.09 J/cm2 of UVB.
Figure 2.
 
The relationship between mean percentage of tail DNA and dose of UVB. Cells were irradiated as a suspension or as monolayer, as indicated. For each dose of UVB, cells irradiated as a monolayer and then processed for comet analysis show a correspondingly higher percentage of tail DNA than cells irradiated in suspension. Values are ±SEM; Suspension: unexposed control 2.52% ± 0.42%; 0.012 UVB, 7.10% ± 0.89%; 0.045 UVB, 11.94% ± 1.14%; 0.09 UVB, 24.92% ± 1.62%. Monolayer: Unexposed control, 2.49% ± 0.49%; 0.012 UVB, 13.98% ± 1.53%; 0.045 UVB, 19.37% ± 1.83%; 0.09 UVB, 42.00% ± 1.72%
Figure 2.
 
The relationship between mean percentage of tail DNA and dose of UVB. Cells were irradiated as a suspension or as monolayer, as indicated. For each dose of UVB, cells irradiated as a monolayer and then processed for comet analysis show a correspondingly higher percentage of tail DNA than cells irradiated in suspension. Values are ±SEM; Suspension: unexposed control 2.52% ± 0.42%; 0.012 UVB, 7.10% ± 0.89%; 0.045 UVB, 11.94% ± 1.14%; 0.09 UVB, 24.92% ± 1.62%. Monolayer: Unexposed control, 2.49% ± 0.49%; 0.012 UVB, 13.98% ± 1.53%; 0.045 UVB, 19.37% ± 1.83%; 0.09 UVB, 42.00% ± 1.72%
Figure 3.
 
Mean percentage of tail DNA in a population of cells exposed to 0.45 and 0.9 J/cm2 of UVA (n = 2, with 100 observations). Control cells not exposed to UV radiation showed minimal levels of DNA in the tail (∼5%). Increasing doses of UVA caused larger comet tails. Values are ±SEM. Unexposed control, 3.57% ± 0.35%; 0.45 UVA, 10.67% ± 1.06%; 0.9 UVA, 14.96% ± 1.49%.
Figure 3.
 
Mean percentage of tail DNA in a population of cells exposed to 0.45 and 0.9 J/cm2 of UVA (n = 2, with 100 observations). Control cells not exposed to UV radiation showed minimal levels of DNA in the tail (∼5%). Increasing doses of UVA caused larger comet tails. Values are ±SEM. Unexposed control, 3.57% ± 0.35%; 0.45 UVA, 10.67% ± 1.06%; 0.9 UVA, 14.96% ± 1.49%.
Figure 4.
 
(A) Mean percentage of tail DNA in a population of cells exposed to a range of doses of broadband UVB (n = 3, with 150 observations). X-axis shows the dose in joules per square centimeter; y-axis represents mean percentage of DNA in the tail. Values are ±SEM. Unexposed control, 4.00% ± 0.33%; 0.012 UVB, 10.73% ± 0.88%; 0.025 UVB, 16.44% ± 1.64%; 0.045 UVB, 22.89% ± 1.87%; 0.09 UVB, 32.93% ± 2.69%. (B) Olive tail moment distribution (50 observations) in RPE cells treated with UVB as in Figure 3A . Tail moments in excess of 10 indicating moderate to severe damage were observed only in cells receiving 0.045 or 0.09 J/cm2 UVB.
Figure 4.
 
(A) Mean percentage of tail DNA in a population of cells exposed to a range of doses of broadband UVB (n = 3, with 150 observations). X-axis shows the dose in joules per square centimeter; y-axis represents mean percentage of DNA in the tail. Values are ±SEM. Unexposed control, 4.00% ± 0.33%; 0.012 UVB, 10.73% ± 0.88%; 0.025 UVB, 16.44% ± 1.64%; 0.045 UVB, 22.89% ± 1.87%; 0.09 UVB, 32.93% ± 2.69%. (B) Olive tail moment distribution (50 observations) in RPE cells treated with UVB as in Figure 3A . Tail moments in excess of 10 indicating moderate to severe damage were observed only in cells receiving 0.045 or 0.09 J/cm2 UVB.
Figure 5.
 
(A) Mean percentage of tail DNA in RPE exposed to 0.09 J/cm2 UVB radiation and processed for comet assay immediately after irradiation (t = 0) and 1 hour (t = 1) after exposure. (n = 3, with 150 observations). Tail DNA increases with increasing time after irradiation. Values are ±SEM. Unexposed control, 4.16% ± 0.34%; 1 hour control, 3.99 ± 0.45; UV acute, 27.45% ± 2.24%; 1 hour after irradiation, 40.02% ± 3.27%. (B) Distribution of Olive tail moment for the same experiment as in Figure 4A with 50 observations. A bimodal distribution of damage is seen 24 hours after exposure with peaks at tail moments of 9 and 15.
Figure 5.
 
(A) Mean percentage of tail DNA in RPE exposed to 0.09 J/cm2 UVB radiation and processed for comet assay immediately after irradiation (t = 0) and 1 hour (t = 1) after exposure. (n = 3, with 150 observations). Tail DNA increases with increasing time after irradiation. Values are ±SEM. Unexposed control, 4.16% ± 0.34%; 1 hour control, 3.99 ± 0.45; UV acute, 27.45% ± 2.24%; 1 hour after irradiation, 40.02% ± 3.27%. (B) Distribution of Olive tail moment for the same experiment as in Figure 4A with 50 observations. A bimodal distribution of damage is seen 24 hours after exposure with peaks at tail moments of 9 and 15.
Figure 6.
 
Response of RVE and RPE cells after exposure to 0.09 J/cm2 UVB radiation (n = 3, with 140 observations). RPE cells show greater mean percentage of tail DNA than the RVE cells for the same dose. Values are ±SEM. Unexposed control RVE cells, 5.24% ± 0.44%; unexposed control RPE, 4.96% ± 0.42%; exposed RVE cells, 13.39% ± 1.57%; exposed RPE, 28.14% ± 2.38%.
Figure 6.
 
Response of RVE and RPE cells after exposure to 0.09 J/cm2 UVB radiation (n = 3, with 140 observations). RPE cells show greater mean percentage of tail DNA than the RVE cells for the same dose. Values are ±SEM. Unexposed control RVE cells, 5.24% ± 0.44%; unexposed control RPE, 4.96% ± 0.42%; exposed RVE cells, 13.39% ± 1.57%; exposed RPE, 28.14% ± 2.38%.
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