| Arsenic is a documented human carcinogen with multiple targets. Epidemiological evidence shows the association between the exposure to inorganic arsenic and increases of lung, bladder and skin cancers. In addition to acting as a carcinogen, arsenite also is used to as a chemotherapeutic agent. However, the mechanisms underlying arsenite in both carcinogenesis and anticancer are still not well understand. The relationship between the reactive oxygen species (ROS) and the exposure to concentration of sodium arsenite presents positive correlation relationship. In addition to be toxic substance, ROS, which result in oxidative damages, cell degeneration and apoptosis, play a key role in activating transcription factor and changing gene expression and so on. The role of p53 is to maintain cell harmony and to provide specific and adapted responses to stresses by coordinating the expression of many effector genes. In addition to its transcriptional regulation, transcriptionally independent functions of p53 has been extensively studied. For example, p53 can directly interact with some protein participating in DNA repair.In the present study, we investigated the effect of sodium arsenite at low concentration on ROS, DNA double-strand breaks (DSBs) damages, cell apoptosis and p53 expression in human embryo lung fibroblasts cells (HELF) and human keratinocyte (HaCaT) cells; And based on the effect of methyl methanesulfonate (MMS) on DSBs damages, we investigated the effects of sodium arsenite at low concentration on DSBs damages induced by MMS; We also investigated the roles of ROS and p53 induced by sodium arsenite in preventing DNA double-strand break from MMS. The results of this investigation would provide the foundation to understand effects of sodium arsenite on human and molecule mechanisms.Methods1. The effects of sodium arsenite on ROS level in HELF cells and HaCaT cells The ROS level was examined by 2', 7'-dichlorofluorescin diacetate (DCFH-DA) assay after HELF and HaCaT cells were treated with sodium arsenite of 0, 0.1 or 0.5μM for 6 hour 2. The effect of sodium arsenite at low concentration on cell apoptosis and DSBs damages in HELF and HaCaT cellsAfter HELF and HaCaT cells were treated by 0, 0.1, 0.5μM sodium arsenite. the levels of Cleaved-caspase-3 andγ-H2AX were detected with Western blot, Cell apoptosis was detected with Hoechst33258 staining assay.3.The effects of MMS on DSBs damages in HELF and HaCaT cellsAfter HELF and HaCaT cells were treated with 0,0.1,0.2,0.4,0.6,0.8 and1.0 mM MMS,,γ-H2AX levels were detected with Western blot assay.4. The effects of sodium arsenite at low concentration on expression of p53 and the relationship between p53 expression and ROS levelAfter HELF and HaCaT cells were treated by 0, 0.1 or 0.5μM sodium arsenite or in combined with catalase (10,000U/L) for 6 h, p53 levels were detected with Western blot assay and immunofluorescence.5. The effect of sodium arsenite at low concentrartion on MMS-induced DSBs damages and level of p53HELF and HaCaT cells were treated alone with 0, 0.1 or 0.5μM of sodium arsenite for 6 h, and then treated with 0.1, 0.2, 0.4, 0.6, 0.8, 1.0mM of MMS for 6 h, the levels ofγ-H2AX and p53 were detected by Western blot assay..6. The effects of p53 inhabition on DSBs induced by MMS and sodium arsenite at low concentrationHELF and HaCaT cells were pretreated with 10μM Wortmannin and/or sodium arsenite, and then treated MMS 0.6mM. The levels ofγ-H2AX, p53 were detected by Western blot.Results1. Effects of arsenite at low concentration on ROS level in HELF cells and HaCaT cellsA 6 hour treatment of HELF and HaCaT cells with arsenite increased ROS levels, and the difference between control and treatment groups were statistically significant (p<0.05).2. Effect of arsenite at low concentration on cell apoptosis and DSBs damages in HELF and HaCaT cellsTo assess the effects of low concentration sodium arsenite on cell apoptosis and DSBs damages in HELF and HaCaT cells, Western blot and Hoechst staining were used. As shown in result, the significant difference were not found when cell apoptosis were examined by Hoechst staining assay following treatment with sodium arsenite (0, 0.1, 0.5μM), and cleaved-caspase-3 andγ-H2AX expression treated with arsenite (0, 0.1, 0.5μM) were undetected. These data suggested that treatment with sodium arsenite (0, 0.1, 0.5μM) did not induced apoptosis and DSBs damages in HELF and HaCaT cells.3. Effects of MMS on DSBs damages in HELF and HaCaT cellsTo assess the effects of different concentration of MMS on DSBs damages in HELF and HaCaT cells, cell lysates were analyzed by western immunobloting following treatment of cells with 0, 0.1,0.2, 0.4, 0.6, 0.8, 1.0mM MMS for 6 hour.. MMS induced significantlyγ-H2AX expression in does-dependent manner in HELF and HaCaT cells. These data suggested that MMS exposure result in DSBs damages.4. Effects of sodium arsenite at low concentration on p53expression and relationship between p53 and ROSTo asssee whether ROS induced by arsenite at low concentration results in the expression of p53 in HELF cells and HaCaT cells, cell lysates were analyzed by western blot after treatment with arsenite only, or in combination with Catalase (10,000U/L). The expression of p53 was inhibited by Catalase. The results indicated that the increases of p53 expression was induced by ROS after treatment with sodium arsenite.5.Effects of sodium arsenite at low concentration pre-treatment on MMS induced DSBs damages and p53 level in HELF and HaCaT cellsTo assess the effects of sodium arsenite at low concentration pre-treatment on MMS inducing DSBs damages in HELF and HaCaT cells, the levels ofγ-H2AX and p53 were detected by immunofluorescence and/or Western blot following pre-treatment of cells with 0, 0.1, 0.5μM arsenite for 6 hour, and then 0.6 mM MMS treatment for 6 hour. 0.1, 0.5μM Sodium arsenite pretreatment decreased the levels ofγ-H2AX induced by MMS. And p53 levels were markedly increased when cells were treated with 0.6 mM MMS after exposure to 0.1, 0.5μM sodium arsenite. Data suggested that DSBs damages induced by MMS were prevented by sodium arsenite at low concentration and p53 played a key role in this process.6. Effects of Wortmannin and sodium arsenite at low concentration on DSBs damages induced by MMSTo assess the relationship between p53 and DSBs damages in HELF and HaCaT cells, the levels ofγ-H2AX and p53 were detected by western blot after pretreatment with Wortmannin and/or sodium arsenite for 6h before MMS treatment for 6h. The levsl of p53 was the weakest butγ-H2AX was the greatest when treatment with pre-treatment with Wortmannin and/or sodium arsenite. These data suggested that p53 induced by sodium arsenite preventing DNA double-strand break induced by MMS.Conclusions1. Sodium arsenite at low concentration induces the increases of ROS levels, which does not causes DSBs damage and cell apoptosis in HELF and HaCaT cells. 2. ROS, which is inducd by low concentration of sodium arsenite, improves p53 express in HELF and HaCaT cells.3. Sodium arsenite at low concentration antagonizes to MMS-induced DNA double-strand break damages.4. ROS and p53 play an important role in antagonism to DNA double-strand break damage induced by MMS.
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