Article
In vitro assays for evaluating the ultraviolet B-induced damage in cultured human retinal pigment epithelial cells.
School of Optometry, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1.
Journal of Photochemistry and Photobiology B Biology (impact factor:
2.81).
08/2007;
88(1):21-8.
DOI:10.1016/j.jphotobiol.2007.04.012
pp.21-8
Source: PubMed
-
Citations (0)
- Cited In (3)
-
Article: Protective effects of (-)-epigallocatechin gallate on UVA-induced damage in ARPE19 cells.
[show abstract] [hide abstract]
ABSTRACT: Oxidative injury to the retinal pigment epithelium (RPE) has been proposed to play a contributing role in age-related macular degeneration (AMD). Exposure to solar ultraviolet (UV) radiation is believed to cause the production of reactive oxygen species (ROS), which may cause oxidative damage to RPE cells. Studies have shown that (-)-epigallocatechin gallate (EGCG), an abundant and active component in green tea, can protect several cell types from oxidative stress. It may be useful in the prevention of early AMD. To determine whether EGCG protects RPE cells from UVA-induced damage, we used a cell viability assay to determine the viability of UVA-treated cells. Intracellular H(2)O(2) levels were measured by flow cytometry. Western blotting was used to detect UVA-induced signaling pathways. The results indicated that EGCG inhibits UVA-induced RPE cell death. In addition, intracellular H(2)O(2) generation in RPE cells irradiated by UVA was inhibited by EGCG in a concentration-dependent manner. EGCG also inhibited UVA-induced extracullar signal-regulated kinase (ERK) and c-jun-NH2 terminal kinase (JNK) activation in RPE cells while a higher concentration of EGCG had an inhibitory effect on UVA-induced p38 activation. Finally, we investigated cyclooxygenase-2 (COX-2) expression in RPE cells exposed to UVA radiation, and EGCG was found to also have inhibited UVA-induced COX-2 expression. Taken together, our results demonstrate that EGCG inhibits UVA-induced H(2)O(2) production, mitogen-activating protein kinase activation, and expression of COX-2. Moreover, it enhances RPE cell survival after UVA exposure. This suggests EGCG is effective in preventing UVA-induced damage in RPE cells and may be suitable for further developments as a chemoprotective factor for the primary prevention of early AMD.Molecular vision 02/2008; 14:2528-34. · 2.20 Impact Factor -
Article: ATM localization and gene expression in the adult mouse eye.
[show abstract] [hide abstract]
ABSTRACT: High levels of metabolism and oxygen consumption in most adult murine ocular compartments, combined with exposure to light and ultraviolet (UV) radiation, are major sources of oxidative stress, causing DNA damage in ocular cells. Of all mammalian body cells, photoreceptor cells consume the largest amount of oxygen and generate the highest levels of oxidative damage. The accumulation of such damage throughout life is a major factor of aging tissues. Several multiprotein complexes have recently been identified as the major sensors and mediators involved in the maintenance of DNA integrity. The activity of these complexes initially seemed to be restricted to dividing cells, given their ultimate role in major cell cycle checkpoints. However, it was later established that they are also active in post-mitotic cells. Recent findings demonstrate that the DNA damage response (DDR) is essential for the development, maintenance, and normal functioning of the adult central nervous system. One major molecular factor in the DDR is the protein, ataxia telangiectasia mutated (ATM). It is required for the rapid induction of cellular responses to DNA double-strand breaks. These cytotoxic DNA lesions may be caused by oxidative damage. To understand how ATM prevents oxidative stress and participates in the maintenance of genomic integrity and cell viability of the adult retina, we determined the ATM expression patterns and studied its localization in the adult mouse eye. Atm gene expression was analyzed by RT-PCR experiments and its localization by in situ hybridization on adult mouse ocular and cerebellar tissue sections. ATM protein expression was determined by western blot analysis of proteins homogenates extracted from several mouse tissues and its localization by immunohistochemistry experiments performed on adult mouse ocular and cerebellar tissue sections. In addition, subcellular localization was realized by confocal microscopy imaging of ocular tissue sections, with a special focus on retinal cells. Using RT-PCR, we detected a band of the expected size, with its sequence matching the amplified Atm cDNA sequence. Atm mRNA was detected in most cell bodies of the adult mouse eye by in situ hybridization of ocular tissue sections with specific digoxigenin-labeled PCR-amplified cDNA probes. Western blotting with different specific antibodies revealed bands corresponding to the expected sizes of ATM and its active forms (ATMp). These bands were not observed in the analysis of protein homogenates from Atm-deficient mouse tissues. ATM immunoreactivity was detected in the nucleus of all adult mice retinal cells and in most non-neuronal ocular cell types. The active phosphorylated form of ATM was also present in the retina as well as in non-neuronal cells of the adult mouse eye. However, its subcellular localization differed as a function of the cell type examined. A major finding of this study was that ATMp immunostaining in photoreceptor cells was exclusively in the cytoplasm, whereas ATM immunostaining was only in the nucleus of these cells. Furthermore, the specific and distinct ATM and ATMp immunolabeling patterns in photoreceptor cells were identical to those observed in the adult mouse cerebellar granule cells. We report the expression profile of Atm gene and protein in the adult mouse eye. In particular, we observed a difference between the localization patterns of the active and inactive forms of ATM in photoreceptor cells. These localization patterns suggest that ATM and its phosphorylated activated form may be involved in both the protection of cells from oxidative damage and the maintenance of ocular cell structure and function. The protection mechanisms mediated by the two forms of ATM appear to be particularly important in maintaining photoreceptor integrity.Molecular vision 02/2009; 15:393-416. · 2.20 Impact Factor -
Article: In vitro ultraviolet-induced damage in human corneal, lens, and retinal pigment epithelial cells.
[show abstract] [hide abstract]
ABSTRACT: The purpose was to develop suitable in vitro methods to detect ocular epithelial cell damage when exposed to UV radiation, in an effort to evaluate UV-absorbing ophthalmic biomaterials. Human corneal epithelial cells (HCEC), lens epithelial cells (HLEC), and retinal pigment epithelial cells (ARPE-19) were cultured and Ultraviolet A/Ultraviolet B (UVA/UVB) blocking filters and UVB-only blocking filters were placed between the cells and a UV light source. Cells were irradiated with UV radiations at various energy levels with and without filter protections. Cell viability after exposure was determined using the metabolic dye alamarBlue and by evaluating for changes in the nuclei, mitochondria, membrane permeability, and cell membranes of the cells using the fluorescent dyes Hoechst 33342, rhodamine 123, calcein AM, ethidium homodimer-1, and annexin V. High-resolution images of the cells were taken with a Zeiss 510 confocal laser scanning microscope. The alamarBlue assay results of UV-exposed cells without filters showed energy level-dependent decreases in cellular viability. However, UV treated cells with 400 nm LP filter protection showed the equivalent viability to untreated control cells at all energy levels. Also, UV irradiated cells with 320 nm LP filter showed lower cell viability than the unexposed control cells, yet higher viability than UV-exposed cells without filters in an energy level-dependent manner. The confocal microscopy results also showed that UV radiation can cause significant dose-dependent degradations of nuclei and mitochondria in ocular cells. The annexin V staining also showed an increased number of apoptotic cells after UV irradiation. The findings suggest that UV-induced HCEC, HLEC, and ARPE-19 cell damage can be evaluated by bioassays that measure changes in the cell nuclei, mitochondria, cell membranes, and cell metabolism, and these assay methods provide a valuable in vitro model for evaluating the effectiveness of UV-absorbing ophthalmic biomaterials, including contact lenses and intraocular lenses.Molecular vision 01/2011; 17:237-46. · 2.20 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed.
The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual
current impact factor.
Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence
agreement may be applicable.
Keywords
broadband UV-B-induced damage
broadband UVB radiations
cell viability
cellular viability
Confocal analysis concentrated
confocal microscopy
control cell group
control cells
cultured human retinal pigment epithelial cells
entire culture period
fluorescent microspheres
human retinal pigment epithelial cell line
morphological change
phagocytotic activity
Phagocytotic activity assay data
probe cellular viability
significant viability change
Treated cells
UV-B-exposed cells
UVB-exposed cells
