A contributory role of oxidative stress and protection by antioxidant nutrients have been suspected in cataract formation. Ganoderic acid A (GAA), an effective lanostane triterpene, is widely reported as an antioxidant. The aim of this study is to investigate the potential effects of GAA on cataract formation. After lens epithelial cells (LECs) were exposed to UVB radiation for different periods, cell viability, apoptosis-related protein levels, malondialdehyde (MDA) and superoxide dismutase (SOD) activities were monitored. We found that cell viability, the Bcl-2/Bax ratio and SOD activity were increased, while Cleaved caspase-3 levels and MDA activity were decreased compared with those in UVB-impaired LECs after GAA treated. Furthermore, GAA activated PI3K/AKT in UVB-impaired LECs and effectively delayed the occurrence of lens opacity in vitro. In conclusion, these findings demonstrated that GAA exhibited protective functions in SRA01/04 cells and rat lenses against UVB-evoked impairment through elevating cell viability and antioxidant activity, inhibiting cell apoptosis, activating the PI3K/AKT pathway and delaying lens opacity.
Animals ; Apoptosis ; Cataract/prevention & control* ; Cell Line ; Cell Survival ; Epithelial Cells/radiation effects* ; Heptanoic Acids/pharmacology* ; Humans ; Lanosterol/pharmacology* ; Lens, Crystalline/radiation effects* ; Malondialdehyde/metabolism* ; Rats ; Superoxide Dismutase/metabolism* ; Ultraviolet Rays/adverse effects*

OBJECTIVE: To determine the apoptosis-inducing effect of ultraviolet light (UV) on human lens epithelial cell (HLEC) and to explore the involvement of changes in ALDH1 folowing UV radiation. METHODS: HLEC was exposed to the same UV light source and was subsequently divided into 6 groups according to UV radiation time of 0 (control group), 5, 10, 15, and 30 min. Apoptosis was detected by AO/EB staining. Changes of ALDH1 in HLEC were detected by immunohistochemical staining and Western blot. RESULTS: The intensity of immunohistochemical staining and the rate of positive cells decreased with increase of UV time (P<0.05). The rate of positive ALDH1 cells was negatively correlated with the rate of apoptosis (r= -0.92, P<0.05). Western blot showed the integrated absorbance values significantly decreased with the increase of UV time (P<0.05). CONCLUSION ALDH1 in HLEC decreases with an increase of UV exposure, which may be related to UV induced apoptosis of HLEC.
Aldehyde Dehydrogenase 1 ; Apoptosis ; radiation effects ; Cells, Cultured ; Epithelial Cells ; cytology ; metabolism ; radiation effects ; Humans ; Isoenzymes ; genetics ; metabolism ; Lens, Crystalline ; cytology ; Retinal Dehydrogenase ; genetics ; metabolism ; Ultraviolet Rays ; adverse effects

OBJECTIVE: To explore the apoptosis-inducing effect of ultraviolet(UV) radiation on human lens epithelial cells (HLEC), with particular focus on changes in Bcl-2 or Bax expression as possible mechanisms. METHODS: All experimental groups were exposed to the same UV light source. HLEC were divided into 6 groups according to duration of UV radiation : 0 min group (control group), 5 min group, 10 min group,15 min group, and 30 min group. Analysis on apoptosis of HLEC was performed by flow cytometry analysis (FCA, Annexin V + PI staining). Changes of Bax and Bcl-2 expression in HLEC were detected by hybridization in situ. RESULTS: Apoptosis in HLEC increased with UV exposure time. The expression level of Bax mRNA was increased with the increase of UV exposure time, whereas the expression level of Bcl-2 mRNA decreased with the increase of UV exposure time. The proportion of apoptotic cells was negatively correlated with ratio of Bcl-2/Bax (r=-0.874, P<0.05). CONCLUSION UA radiation can induce apoptosis of HLEC in vitro. Bcl-2 and Bax genes may play an important role in regulating this apoptotic process.
Apoptosis ; radiation effects ; Cells, Cultured ; Epithelial Cells ; cytology ; metabolism ; radiation effects ; Humans ; Lens, Crystalline ; cytology ; radiation effects ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Time Factors ; Ultraviolet Rays ; adverse effects ; bcl-2-Associated X Protein ; genetics ; metabolism

PURPOSE: The purpose of this study is to understand the mechanism of apoptosis occurring on a cultured human lens epithelial cell line after exposure to ultraviolet (UV) light. We intended to confirm the presence of cellular toxicity and apoptosis and to reveal the roles of p53, caspase 3 and NOXA in these processes. METHODS: Cells were irradiated with an ultraviolet lamp. Cellular toxicity was measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Hoechst staining and fluorescent anti-caspase 3 antibodies were used for apoptosis investigation. The quantities of p53, caspase 3, and NOXA were measured by Western blotting for to investigate the apoptosis pathway. RESULTS: Cellular toxicity on the human lens epithelium markedly increased with time after UV exposure. On Hoechst staining, we found that apoptosis also remarkably increased after exposure to ultraviolet light, compared with a control group. In the immunochemical study using anti-caspase 3 antibodies, active caspase 3 significantly increased after exposure to ultraviolet light. On Western blotting, p53 decreased, while caspase 3 and NOXA increased. CONCLUSIONS: Exposure of cultured human lens epithelial cell lines to ultraviolet light induces apoptosis, which promotes the expression of NOXA and caspase 3 increases without increasing p53. This may suggest that UV induced apoptosis is caused by a p53-independent pathway in human lens epithelial cells.
Apoptosis/*physiology ; Caspase 3/metabolism ; Cell Line ; Cell Survival/radiation effects ; Epithelial Cells/radiation effects ; Humans ; Lens, Crystalline/cytology/*physiology/*radiation effects ; Proto-Oncogene Proteins c-bcl-2/metabolism ; Tumor Suppressor Protein p53/metabolism ; *Ultraviolet Rays

DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase (DNA-PK), plays an important role in the nonhomologous end-joining (NHEJ) pathway of DNA double-strand breaks (DSBs) repair. To investigate the effects of DNA-PKcs down-regulation on cell growth and sensitization to low dose radiation (LDR), we transfected DNA-PKcs siRNA into human mammary epithelia cells MCF10F, then, detected the proliferation curve of the cells and the expression of protiens in DNA repair pathways. The results showed that DNA-PKcs gene silencing, induced by the transfection of DNA-PKcs siRNA could suppress significantly the cell proliferation and inhibit the expression of the DNA repair proteins, such as Ku80, ATM and P53 after 50 cGy 137Cs gamma-irradiation.The results suggested that DNA-PKcs gene silencing could increase the sensitivity of human breast epithelial cells to the LDR, which might be relative with the decrease of the proteins.
Breast ; cytology ; Cell Line ; DNA Repair ; drug effects ; radiation effects ; DNA-Activated Protein Kinase ; genetics ; Dose-Response Relationship, Radiation ; Epithelial Cells ; metabolism ; radiation effects ; Gene Silencing ; Humans ; Nuclear Proteins ; genetics ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVETo investigate the relationship among a 50-Hz MF-induced epidermal growth factor receptor (EGFR) clustering, acid sphingomyelinase (A-SMase) and ceramide (CER), and to explore the possible mechanism of receptor clustering.

METHODSHuman amnion (FL) cells were exposed to a 50-Hz sinusoidal magnetic field at 0.4 mT for 15 min with or without imipramine, a specific inhibitor of A-SMase and ceramide pretreatment. EGF treatment served as the positive control and DMSO treatment served as the solvent control. The EGFR was labeled with polyclonal anti-EGFR antibody and the clustering of EGFR was analyzed using immunofluorescence and confocal microscopy. The percentage of cells with EGFR clustering was counted and compared.

RESULTSBoth EGF treatment and 50-Hz MF exposure could induce EGFR clustering. However, the effect could be eliminated by imipramine pretreatment for 4 hours. When FL cells were incubated with ceramide following the imipramine pretreatment for 30 min, EGFR clustering induced by 50-Hz MF exposure could be recovered.

CONCLUSIONEGFR clustering induced by 50-Hz MF depends on A-SMase activity, and ceramide, as the hydrolyzate from A-SMase might participate in the process of EGFR clustering.

Amnion ; cytology ; Cell Line ; Cell Membrane ; metabolism ; radiation effects ; Ceramides ; metabolism ; Epithelial Cells ; metabolism ; radiation effects ; Humans ; Magnetic Fields ; adverse effects ; Receptor, Epidermal Growth Factor ; metabolism ; Sphingomyelin Phosphodiesterase ; metabolism ; physiology

OBJECTIVETo investigate whether the exposure to the electromagnetic noise can block reactive oxygen species (ROS) production and DNA damage of lens epithelial cells induced by 1800 MHz mobile phone radiation.

METHODSThe DCFH-DA method and comet assay were used respectively to detect the intracellular ROS and DNA damage of cultured human lens epithelial cells induced by 4 W/kg 1800 MHz mobile phone radiation or/and 2 muT electromagnetic noise for 24 h intermittently.

RESULT1800 MHz mobile phone radiation at 4 W/kg for 24 h increased intracellular ROS and DNA damage significantly (P<0.05). However, the ROS level and DNA damage of mobile phone radiation plus noise group were not significant enhanced (P>0.05) as compared to sham exposure group.

CONCLUSIONElectromagnetic noise can block intracellular ROS production and DNA damage of human lens epithelial cells induced by 1800 MHz mobile phone radiation.

Cell Phone ; Cells, Cultured ; DNA ; radiation effects ; DNA Damage ; radiation effects ; Electromagnetic Fields ; Epithelial Cells ; metabolism ; radiation effects ; Humans ; Lens, Crystalline ; cytology ; Microwaves ; adverse effects ; Radiation ; Reactive Oxygen Species ; metabolism

OBJECTIVETo investigate the effects of 50 Hz magnetic fields (MF) on DNA double-strand breaks in human lens epithelial cells (hLECs).

METHODSThe cultured human lens epithelial cells were exposed to 0.4 mT 50 Hz MF for 2 h, 6 h, 12 h, 24 h and 48 h. Cells exposed to 4-nitroquinoline-1-oxide, a DNA damage agent, at a final concentration of 0.1 micromol/L for 1 h were used as positive controls.After exposure, cells were fixed with 4 % paraformaldehyde and for H2AX (gamma H2AX) immunofluorescence measurement. gamma H2AX foci were detected at least 200 cells for each sample. Cells were classified as positive when more than three foci per cell were observed. Mean values of foci per cell and percentage of foci positive cells were adopted as indexes of DNA double-strand breaks.

RESULTThe mean value of foci per cell and the percentage of gamma H2AX foci positive cells in 50 Hz MF exposure group for 24 h were (2.93 +/-0.43) and (27.88 +/-2.59)%, respectively, which were significantly higher than those of sham-exposure group [(1.77 +/-0.37) and (19.38+/-2.70)%, P <0.05], and the mean value of foci per cell and the percentage of gamma H2AX foci positive cells in 50 Hz MF exposure group for 48 h were (3.14 +/-0.35) and (31.00 +/-3.44)%, which were significantly higher than those of sham-exposure group (P <0.01). However there was no significant difference between 50 Hz MF exposure groups for 2 h, 6 h, 12 h and sham-exposure group for above two indexes (P >0.05).

CONCLUSION0.4 mT 50 Hz MF exposure for longer duration might induce DNA double-strand breaks in human lens epithelial cells in vitro.

Cells, Cultured ; DNA ; radiation effects ; DNA Breaks, Double-Stranded ; radiation effects ; DNA Damage ; radiation effects ; DNA Repair ; radiation effects ; Electromagnetic Fields ; Epithelial Cells ; metabolism ; radiation effects ; Humans ; Lens, Crystalline ; cytology

PURPOSE: To evaluate the protective effects of epigallocatechin gallate (EGCG) against UV irradiation of cultured human lens epithelial cells. METHODS: We irradiated cultured human lens epithelial cells with a 30-second pulse from a UV lamp with an irradiance of 0.6 mW/cm2. Five minutes and 1 hour after UV irradiation, we administered 0, 5, 10, 15, 25, 50, or 100 uM EGCG. The cell number was measured with a microscopic counting chamber and cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. RESULTS: Compared to untreated cells, the total number of cultured human lens epithelial cells was markedly higher after UV irradiation. In a dose-dependent manner, viability was also higher in EGCG-treated cells. CONCLUSIONS: EGCG increased the cell count and cell viability after UV irradiation of cultured human lens epithelial cells, indicating that EGCG can protect lens epithelium against UV damage.
Antioxidants/*pharmacology ; Catechin/*analogs & derivatives/pharmacology ; Cell Count ; Cell Survival/drug effects ; Cells, Cultured ; Coloring Agents/diagnostic use ; Dose-Response Relationship, Drug ; Epithelial Cells/radiation effects ; Humans ; Lens, Crystalline/cytology/*radiation effects ; Radiation Injuries/*prevention & control ; Radiation-Protective Agents/*pharmacology ; Tetrazolium Salts/diagnostic use ; Thiazoles/diagnostic use ; *Ultraviolet Rays

OBJECTIVETo observe the effects of low power microwave radiation on lens hydration and lens epithelial cells in vitro, and detect the expression of PKC-alpha, c-fos and c-jun in lens epithelial cells.

METHODSRabbit lens were exposed to microwave radiation with frequency of 2450 MHz and power density of 0.5, 2.0 and 5.0 mW/cm(2) in vitro. The hydration of lens was measured after 8 hours. Morphological changes of lens epithelial cells were observed using a phase-contrast microscope and Hoechst 33258 staining. Expression of PKC-alpha, c-fos and c-jun were analyzed using gel electrophoresis and western blot analysis.

RESULTSAfter 2.0 and 5.0 mW/cm(2) microwave radiation, the hydration of lens was increased compared to control groups (P<0.05), the shape of lens epithelial cells showed shrinking and disorder and cells nuclei appeared chromatin condensation. There was no change of lens and lens epithelial cells after 0.5 mW/cm(2) microwave radiation. The expression of PKC-alpha was significantly increased in cell membrane, however, decreased in cell cytoplasm after 2.0 mW/cm(2) microwave radiation for 2, 4, 6 and 8 hours. There was significantly increased expression of c-fos and c-jun protein compared with control groups (P<0.05, P<0.01).

CONCLUSIONLow power microwave radiation higher than 2.0 mW/cm(2) can activate PKC-alpha by increasing its expression in cell membrane, then induce high expression of c-fos and c-jun, which may relate to cellular signaling pathway of microwave radiation injury to lens and lens epithelial cells.

Animals ; Epithelial Cells ; metabolism ; pathology ; radiation effects ; In Vitro Techniques ; Lens, Crystalline ; metabolism ; pathology ; radiation effects ; Protein Kinase C-alpha ; metabolism ; Rabbits ; Transcription Factors ; metabolism