The Roles Of JNK Signaling Pathway In Dimethylarsinic Acid-Induced DNA Damage And Apoptosis In Human Embryo Lung Fibroblast Cells

Arsenic is a known carcinogeon in human. Arsenic intoxication occurs in many areas through the consumption of contaminated well water or foods containing inorganic arsenicals. On the other hand, inorganic arsenite has emerged as an outstanding chemotherapeutic agent with remarkable efficacy for certain human cancers. Thus, apparently environmental and iatrogenic exposures to arsenic will continue to be common. Dimethylarsinic acid (DMA) is the major metabolite formed after exposure to inorganic arsenic and it has been used as an herbicide. It has been thought that the methylation of inorganic arsenic is a mechanism to facilitate the detoxification and excretion of arsenic. However, more and more studies have showed that DMA can induce DNA strand damage, DNA-protein cross-links and apoptosis, which suggested that DMA has genotoxicity and carcinogenicity. Recently, more and more investigations suggest that the methylation of inorganic arsenic not only is not a universal detoxification mechanism but also plays an important role in arsenide-induced carcinogenesis. Much less information is available on the in vitro genotoxic potential or mechanisms of methylated arsenicals. The molecular mechanism for DMA-induced DNA damage is not clear. C-Jun NH₂-terminal kinase (JNK) is one of the mitogen activated kinase (MAPK) super family, which takes part in cellular proliferation, differentiation and apoptosis and plays an important role in cell damage induced by inorgan arsenic. Therefore, the study on DMA-induced cell damage and its molecular mechanisms has important significance in the molecular mechanism of arsenic-induced carcinogenesis and the prevention of arsenic poisoning.In this study, we investigated the effects of DMA on cell prolifeation, DNA damage, cell cycle, cell apoptosis and JNK signaling pathway in human embryo lung fibroblast (HELF) cells. In order to investigate the roles of JNK signaling pathway in DNA-induced cell DNA damage and apoptosis, we blocked JNK signaling patheay by useing JNK inhibitor and constructing JNK protein deficient cell strains. The results of this investigation would provide the sciencetific foundation to understand the molecular mechanism for DMA-induced cell damage, the mechanism of action for arsenic carcinogenicity and the effects of arsenic on human health.Methods1. The effects of DMA on cell proliferation in HELF cellsTo evaluate the dose-effect and time-effect relationships of cell proliferation induced by DMA in HELF cells, cell proliferation was evaluated by MTT assay and CCK-8 assay after HELF cells were treated with DMA of control, 2.5, 5.0, 10.0, or 20.0μmol/L for exposure time of 12, 24, or 48 h, respectively.2. The effects of DMA on DNA damage in HELF cellsAfter HELF cells were treated with DMA of control, 2.5, 5.0, 10.0, or 20.0μmol/L for exposure time of 12, 24, or 48 h, the total protein of HELF cells was extracted andγ-H2AX levels were detected with Western blot assay and were compared withβ-actin levels3. The effects of DMA on cell cycle in HELF cellsThe cell-cycle distribution was detected by flow cytometric analysis After HELF cells were treated with DMA of control, 2.5, 5.0, 10.0, or 20.0 (μmol/L for exposure time of 48 h or with DMA of 20.0μmol/L for exposure time of 12, 24, or 48 h.4. The Effects of DMA on HELF cell apoptosis To evaluate the dose-effect and time-effect relationships of cell apoptosis induced by DMA in HELF cells, cell apoptosis was analyzed by measuring apoptotic cells with Hoechst staining assay and the levels of cleaved caspase-3 detected by Western blot assay after HELF cells were treated with DMA of control, 2.5, 5.0, 10.0, or 20.0 umol/L for exposure time of 12, 24, or 48 h, respectively.5. The Effects of DMA on JNK phosphorylation in HELF cells.After HELF cells were treated with DMA of control, 2.5, 5.0, 10.0, or 20.0 umol/L for exposure time of 12, 24, or 48 h, the total protein of HELF cells was extracted and the levels of JNK,phospho-JNK were detected with Western blot assay.6. Construction of JNK deficient cell strainsAfter extracting total RNA from HELF cells, the target DNA fragment was amplified by reverse transcript-PCR (RT-PCR). The JNK recombinant vectors of antisense RNA were constructed. HELF cells were transfected with eukaryotic expression vectors of JNK gene antisense RNA. Transfected cells were screened by G418. The level of JNK protein in transfected cells was detected by Western blot assay to verify the JNK deficient cell (asJNK-HELF) and vector control cell (C1-HELF) .7. The effects of JNK pathway blocking on DMA-induced DNA damage and apoptosis in HELF cells.Cells were left untreated or pre-treated with 20.0μmol/L JNK inhibitor, SP600125, for 30 min before exposure to control or 20.0μmol/L DMA for 48 h. Cells were then subjected to Hoechst staining and detecting the level of cleaved caspase-3 for determination of apoptosis, detecting the level ofγ-H2AX for determination of DNA damage. We successfully constructed the JNK knockdown cell line ( asJNK-HELF ) . After asJNK-HELF and C1-HELF were treated with DMA of control or 20.0μmol/L for exposure time of 48 h, the indices of DNA damage and apoptosis were detected.Results1. The effects of DMA on cell proliferation in HELF cellsCell proliferation was significantly decreased in cells incubated with 10.0 or 20.0μmol/L DMA at 24 h. At 24 and 48 h, there were markedly decreased in cell proliferation in 5.0, 10.0, or 20.0μmol/L DMA. The results showed that DMA induced a significant inhibition of HELF cell proliferation by concentration- and time-dependent manner.2. The Effects of DMA on DNA damage in HELF cellsDMA, at concentrations of 5.0, 10.0, or 20.0μmol/L, increasedγ-H2AX levels at 12 h. After exposure to DMA for 24 h,γ-H2AX levels were elevated at concentrations of 2.5, 5.0, 10.0, or 20.0μmol/L. After 48 h, DMA, at concentrations of 2.5, 5.0, 10.0, or 20.0μmol/L, up-regulated the levels ofγ-H2AX. These data suggest that, in HELF cells, DMA caused marked DNA damage.3. The Effects of DMA on cell cycle in HELF cellsDuring the 48 h time period, DMA increased the percentage of S and G2/M phases and degraded the percentage of G0/G1 phase with increasing concentrations. The G2/M arrest was found to be time dependent after HELF cells were treated with DMA of 20.0μmol/L for exposure time of 12, 24, or 48 h. Flow cytometric analysis showed that DMA modulated cell cycle progression through inducing cells to accumulate at S and G2/M-phases with concurrent decrease of cells at G0/G1 phase.4. The Effects of DMA on cell apoptosis in HELF cellsApoptotic cells, with condensed and fragmented fluorescent nuclei, were observed in DMA-treated samples, whereas non-apoptosis cells presented round, uniformly colored nuclei. Cell apoptosis was significantly increased in cells incubated with 5.0, 10.0, or 20.0μmol/L DMA at 12h. At 24 and 48 h, there were markedly increased in cell apoptosis in 2.5, 5.0, 10.0, or 20.0μmol/L DMA. DMA, at concentrations of 10.0 or 20.0μmol/L, increased the levels of cleaved caspase-3 at 12 h. After exposure to DMA for 24 h, the cleaved caspase-3 levels were elevated at concentrations of 5.0, 10.0, or 20.0μmol/L. After 48 h, DMA, at concentrations of 2.5, 5.0, 10.0, or 20.0μmol/L, up-regulated the levels of cleaved caspase-3. These data suggested that the degree of HELF cell apoptosis was increased with the increase of concentration and time exposed to DMA.5. The Effects of DMA on JNK phosphorylation in HELF cellsPhospho-JNK levels were significantly increased with 10.0 and 20.0μmol/L DMA at 12 h. Phospho-JNK levels were significantly elevated with 5.0, 10.0, or 20.0μmol/L DMA at 24 h. After 48 h, DMA, at concentrations of 2.5, 5.0, 10.0 or 20.0μmol/L, up-regulated the levels of JNK phosphorylation. These data suggested that DMA induced JNK activation in HELF cells.6. Construction of JNK deficient cell strains(1) Construction of eukaryotic expression vectors of JNK gene antisense RNA.326bp target DNA fragments were observated after pEGFP-C1-asJNK vector were analysised by restriction endonucleases, respctively. The sequencing results showed that the target DNA sequence accorded with that of JNK gene cDNA in Genbank by 100.0%. Data indicated that the eukaryotic expression vector of JNK gene antisense RNA was successfully constructed.(2) Identification of JNK protein deficient cell strainsThe results of Western blot showed that JNK protein levels in asJNK-HELF cells was 28% of that in C1-HELF cells, or JNK protein levels in asJNK-HELF cells was decreased 72%, which indicated that the cell strains of JNK protein deficience was successfully constructed.7. Blocking of JNK pathway on DMA-induced DNA damage and cell apoptosis in HELF cells(1) Effects of JNK inhibitor on DMA-induced DNA damage and cell apoptosis in HELF cellsIn cells pre-treated with inhibitor, SP600125, 20.0μmol/L DMA significantly degraded DNA damage and cell apoptosis in HELF cells. SP600125 significantly blocked increases ofγ-H2AX level and cleaved caspase-3 level induced by 20.0μmol/L DMA compared to control. The increases in cell apoptosis induced by 20.0μmol/L DMA were significantly reduced from 70.8% to 36.3% compared to control by SP600125. These results indicated that Blocking of JNK pathway with SP600125 markedly degraded DMA-induced DNA damage and cell apoptosis in HELF cells.(2) Effects of JNK-knockdown on DMA-induced DNA damage and cell apoptosis in HELF cellsγ-H2AX level and cleaved caspase-3 level induced by 20μmol/L DMA in asJNK-HELF cells were markedly lower compared to in C1-HELF cells, respectively. The percentages of cell apoptosis induced by 20.0μmol/L DMA were 69.9% and 34.7% in C1-HELF cells and asJNK-HELF cells, respectively. These results indicated that JNK knockdown markedly reduced DMA- induced DNA damage and cell apoptosis in HELF cells.Conclusions1. DMA inhibits cell proliferation, induces DNA damage, cell cycle disorder and cell apoptosis in HELF cells.2. JNK signaling pathway plays an important role in DMA-induced DNA damage and cell apoptosis in HELF cells, blocking of JNK pathway markedly degraded DMA-induced DNA damage and cell apoptosis in HELF cells.3. The JNK protein deficient HELF cell strain was successfully constructed.