Patton W P, Chakravarthy U, Davies R J, Archer D B
Division of Vision Sciences, Queen's University, Belfast, Northern Ireland, United Kingdom.
Invest Ophthalmol Vis Sci. 1999 Dec;40(13):3268-75.
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.
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 degrees C for either 1 or 24 hours before processing for the comet assay.
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.
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.
介导光对视网膜色素上皮（RPE）细胞损伤的分子机制尚不清楚。本研究检测了宽带UVA和UVB辐射在RPE细胞中引起的DNA损伤的定性和定量方面。
将培养的牛RPE细胞以悬浮液或贴壁单层的形式暴露于0至0.9 J/cm²的UVA剂量或0至0.09 J/cm²的UVB剂量下。通过彗星试验评估导致单链断裂的DNA损伤，在该试验中，受损的DNA从细胞核中迁移出来形成尾巴，并使用图像分析对其进行量化。进行了两项测量：尾巴DNA的平均百分比，反映细胞群体中DNA损伤的总体水平；以及橄榄尾矩，代表迁移程度，从而反映单个细胞中DNA损伤的模式。在急性实验中，细胞在紫外线照射后立即通过彗星试验进行处理。为了研究DNA修复的发生情况，RPE细胞首先暴露于UVB，然后在37℃下孵育1或24小时，再进行彗星试验处理。
暴露于UVA和UVB的细胞显示尾巴DNA的平均百分比显著高于未暴露的细胞。暴露于较大剂量UVB的细胞中橄榄尾矩更高。在暴露于UVB 24小时后评估时，该参数也显示出双峰分布，表明存在尾巴矩小和大的两个不同亚群细胞。在剂量低于0.045 J/cm²时未观察到尾巴矩非常大的细胞。
相对低剂量的UVA和UVB可诱导培养的RPE中DNA链断裂的形成。UVB照射后孵育24小时的细胞的尾矩分布与大多数暴露于低剂量的细胞中发生DNA修复以及暴露于高剂量的细胞亚群中发生凋亡一致。
