| PrefaceArsenic is a kind of human carcinogen, arsenicosis can be induced by ingesting excess amount of arsenic via industrial, environmental pathways. The chronic endemic arsenicosis attack millions of Chinese in Inner Mongolia, Shan Xi province et al. Skin is one identified target tissue for arsenic toxicity. Arsenic is associated with hyperkeratosis, abnormal pigmention and skin cancer. In addition to its skin symptoms, arsenic exposure has been suggested to play roles in multi-organ damages and tumors.Although various damages are induced by arsenic, the mechanism of arsenicosis remains unclear. It has been confirmed by epidemiological data that arsenic exposure can induce oxidative stress in human cells, cause the breaks of DNA chain or the production of adducts and also affect the antioxidative defense system in the cells by reactive oxygen species (ROS).In recent years, arsenic has been used to treat leukaemia and the effects on cell cycles and apoptosis in malignant cells are under investigations in recent years. While few studies are on normal cells.In the present study, the human keratinocyte cell line HaCaT were treated with sodium arsenite ( NaAsO2) , the effects of arsenic on cytotoxicity, oxidative damage, antioxidative system, cell cycle and apoptosis in HaCaT cell line were demonstrated. It provide the basic data to diagnosis, prevention and treatment of arsenicosis.Methods1. Cell cultureHaCaT cell line was cultured in complete medium in a fully humidified atmosphere with 5% C02 at 37T!. Cells were trypsined and passaged when grown in logarithmic course.2. Cell treatmentsWhen the cells were in logarithmic phase, they were trypsined and seeded in 96 well plates with the density of 1 x lOVml, or 5 X lOVml in 6 well plates and 100ml culture flasks with the density of 1 lOVml. When grown in 80% confluence , the cells were treated with various concentrations of NaAsO2.3. Morphologic observationWhen formed a confluent layer, the target cells were resuspended and plated in the 6 well plates with the density of 5 x 10 /ml. The cells were grown to 80% confluence and then exposed to lml of 0, 5, 10, 20 and 50jxmol/L NaAsO2for 24h. Cell cultures were viewed and photographed with the phase-contrast microscope.4. Detection of viability of cellsCells were cultured in 96 well plates with the density of 1 x 105/ml for 24 hours, incubated with 0,2. 5,5,10 and 20jxmol/L sodium arsenite for another 24 hours before Alamarblue was added to the medium. After 4 hours, the OD of the medium was detected at 540nm and 620nm. The reduction rate of Alamarblue was calculated.5. Evaluation of DNA damageComet assay was conducted to evaluate the DNA damage. Briefly, sandwich layer was made before slides were immersed in lysis solution for 1. 5h, DNA was unfolded and electrophoresis was conducted for 20 min at 25 V and 300 mA. Subsequently, slides were washed in neutralization buffer. Ethidium bromide was added to each slide, DNA migration was measured with a scaled ocular as the total image length (including head and tail length). For the evaluation of DNA migration, 100 cells were scored for each group.6. Detection of ROS content in HaCaTDCFH-DA staining was performed and Flowcytometry ( FCM) and invert fluorescence microscope were used for ROS detection.7. Detection of antioxidative enzymes and GSHBradford assay was used to detect the protein content in cells. Nitrite method was used to detect the SOD activity, the activity of CAT was detected by ultraviolet direct velocity assay and fluorescence method was performed to determine the GSH content in cells.8. Detection of mRNA expression of CATRT-PCR was used to detect the mRNA expression. Total RNAs were extracted, ultraviolet spectrophotometer was used to detect the OD of the RNA sample. RNAs were reversed transcripted to cDNA followed by PCR amplification. After electrophoresis and scan of the products, IDV were calculated by the imaging analysis system.9. Detection of protein expression of CAT and ERK1/7Western blot was conducted to detect the protein expression. Cells were harvested by use of a rubber policeman and lysed in RIPA buffer for 15 min on ice. After centrifugation, supernatants were collected, and the protein content was measured. The protein were subjected to SDS-PAGE, transferred to PVDF membranes, and incubated with first and second antibodies. Signals were detected by use of an ECL kit. After exposal, the IDVs were calculated by the imaging analysis system.10. Assay of cell cyclePI was added after the treatment. FCM was used to detect the DNA content, 10 thousand cells were collected with the excition wavelength 488 nm and the emission wavelength 600 nm.11. Assay of apoptosisCells were resuspended in Annexin V-FITC and PI reaction mixture. FCM was used to collect 10 thousand cells with the excition wavelength 488 nm and the emission wavelength 525 nm.12. Statistical analysisSPSS11. 0 software was used to analyze the data. Values of mean andstandard deviation from the repeat of independent experiments were calculated. ANOVA was used to test whether the treated cultures were significant from the controls. P value of <0.05 was considered significant in all cases.Results1. Morphologic changes in HaCaTWhen cells were treated with 10|xmol/L sodium arsenite, a small part of cells became rounded and the shapes were irregular. While in the cells treated with 20jxmol/L sodium arsenite, the number of detached cells increased, cells were smaller and rounder, the toxic effects were more serious while treated with 50pjnol/L sodium arsenite, few attached cells could be seen.2. Effect of NaAsO2 on the viability of HaCaTWhen cells were exposed to =$5fimol/L NaAsO2 for 24 hours, the reduction rates of Alamarblue were increased ( P < 0. 05 ). While in 20|xmol/L groups, the reduction rates of Alamarblue decreased significantly ( P <0. 05). When pre-treated with 5mmol/L of NAC for 24 hours, the reduction rate in the cells increased ( P < 0. 05 ), while pre-treated with both 2. 5 mmol/L and 5mmol/L of BSO for 24 hours, the reduction rate was decreased markedly ( P < 0.05).3. DNA damage in HaCaT induced by NaAsO2When treated with ^2. 5[xmol/L of NaAsO2, The comet tails increased in a dose-dependent fashion.4. ROS content in HaCaTThe ROS content increased significantly ( P <0. 05) with the dose-dependent fashion in all treated groups.5. Effect of NaAs02on antioxidative systemSOD activity was not influenced by arsenite. The activity and expression of CAT decreased markedly when the arsenite were ^10 jxmol/L. GSH contents increased in all cells treated with various concentrations of arsenite. GSSG contents increased when the concentrations of arsenite were >2.5 |xmol/L.6. Effect of NaAsO2 on cell cycle and apoptosis in HaCaT... |
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