The Effect Of JNK Signaling Pathway In Arsenite-induced DNA Strand Breaks In Human Embryo Lung Fibroblast Cells

Arsenic is a documented human carcinogen with multiple targets. Epidemiological evidence shows association between inorganic arsenic in drinking water and increased risk of lung,bladder and skin cancers.The genotoxicity of arsenic,which includes the modification of DNA,DNA break damages and DNA repair prevention,may be the most important mechanism of arsenic carcinogenesis.Many studies suggested that DNA break damage which includes single strand breaks(SSBs)and double strand breaks(DSBs)is essential in tumorigenesis.The paper investigates whether arsenic induces DNA break damage via JNK signal pathway. There are many studies about the carcinogenesis of arsenic in enviroment.But most of them only focused on what degree of DNA break damage induced by arsenic rather than how it occurs.Since scanty reports on the role of signal pathway during DNA break damage.We attempt to make out whether the signal pathway was invovled in DNA break damage. Because arsenic could induce the prevention of DNA repair process which related with the signal transduction,we presume that the DNA break damage may related with the signal transduction too.On the other hand,the final phase of apoptosis is DNA breakdown,and the chemical agents also can induce the DNA breakage directly.So we must consider the apoptosis when studying DNA break damage.In the present study,we focus our attention at early time when significant apoptosis did not occur.The form of arsenic in the environment is mainly inorganic and 3 valence.We use the sodium arenite(NaAsO2)to investigate the effects of JNK signaling pathway in arsenite-induced DNA strand breaks in human embryo lung fibroblast(HELF)cells.The paper attempted to reveal how the higher doses aresnite influence the single and double DNA break damage of human embryo lung fibroblast when JNK pathway was inhibited or not.We also obseaved the proliferation,apoptosis and JNK activation of HELF cells on arsenite exposure.Our data provided clues to further understand the cytotoxicity and carcinogenesis of arsenic. Methods:2.1 Cell proliferation assayCell proliferation was evaluated by WST-8 hydrolysis using Cell Counting Kit-8.After cells were treated,10μL of WST-8 was added to each well and the incubation continued for an additional 1-4 hr. Following this,plates were read on a spectrometer.The relative cell proliferation ratio results were plotted using non-treated controls to determine the 100%activity level.2.2 Cell apoptosis assayApoptosis was routinely measured by Hoechst staining assay. Treated cells were washed in PBS and then fixed for 10 min at room temperature.Following fixation,cells were rinsed 3 times in PBS and then incubated with the 33258 Hoechst staining for 5 rain at room temperature to visualize nuclear morphology using a fluorescent microscopy.2.3 Western blot assayAll cells prepared for immunoblotting without serum shock.Cell lysates were prepared by using the lysis buffer.The lysate proteins subjected to 10%SDS-PAGE and transferred to a nitrocellulose membrane.After blocking with non-fat dry milk,the membrane was incubated with antibodies specific for polyclonal rabbit antibodies.To confirm even loading,membranes were stripped and probed withβ-Actin antibody.2.4 Standard alkaline comet assayThe HELF cells were typysied and combined with low temperature melting agarose and spread on a slide glass already covered the solidificated normal temperature melting agarose.After agarose solidification,the slides were immersed at 4℃for 2 h in freshly lysing solution.After lysis,the slides were placed in fresh electrophoretic buffer for DNA unwinding and then electrophoresed.These measures were performed at 4℃under dim light.After electrophoresis,the slides were washed with neutralizing buffer and the cells were stained with SYBR Greenâ… at the recommended dilution.Cells were visualized and captured on a fluorescent microscopy.The oliver tail moment(OTM)value were calculated by the freeware Casp2.1.2.3.Results3.1 Effects of arsenite exposure on the proliferation of HELF cells Arsenic cytotoxicity was studied by proliferation and apoptosis for HELF cells exposed to various doses of NaAsO₂ for 12h and 24 h.Cell proliferation was markedly decreased except in cells incubated with 5μmol/L NaAsO₂ in 24 h but only the 40μmol/L NaAsO₂ can induces significant effect in 12h.3.2 Effects of arsenite exposure on the apoptosis of HELF cells There is no relevant apoptosis effect on exposure of arsenite for 12h in HELF cells.Cell apoptosis was significantly increased(P＜0.05) except 5,10μmol/L NaAsO₂ treated groups in 24h.3.3 Effects of arsenite exposure on the SSBs of HELF cellsUsing standard alkaline comet assay we measured DNA break damage,of which mainly the SSBs,induced by arsenite.Although the data demonstrate that 24 h NaAsO₂ treatment with indicated doses can induce up-regulation of OTM value,only at the 20,40μmol/L the significance can be found(P＜0.05,＜0.01 repectively).Neither of any groups in 12 h treatment of arsenite has significant effect on DNA break damage in HELF cells.Hydrogen dioxide(H₂O₂)5mmol/L treated cells for 30 min was used as positive control.3.4 Effects of arsenite exposure on the DSBs of HELF cellsThe effects of different concentrations of arsenite onγ-H2AX expression were examined by immunobloting assay following treatment of cells with 0,5,10,20,40μmol/L NaAsO₂ for 6,12,24h respectively. The results revealed that except the 6 h treatment all other groups can exhibit theγ-H2AX formation which represents DSBs.In contrast to comet assay result,theγ-H2AX expression has drastically increased in 12h group.3.5 Effects of arsenite exposure on the JNK phosphorylation of HELF cells Our previous studies have shown that arsenite induce JNK activation in a time and dose dependent manner.Here we examined the phosphorylation of JNK after various concentrations of NaAsO₂ treatment for 12h and 24h.The data showed that JNK pathway was specially activated at the 12 h.3.6 Effect of JNK inhibition on the arsenite-induced apoptosis in HELF cellsCells incubated with 0,40,40μmol/L NaAsO₂ for 12 and 24h.One of the 40μmol/L NaAsO₂ was pre-treated with the JNK inhibitor SP600125 20μmol/L for 30min,which named inhibitor group,another group which named arsenite group.The results were obtained among control,arsenite and inhibitor groups after different treatment terms.The JNK inhibitor SP600125 greatly reduced the apoptosis compared arsenite group and inhibitor group at 24h(P＜0.01).However,there is no significant effect among all three groups at 12h(P＞0.05).The data showed that JNK inhibition can influence the apoptosis effect of arsenite.3.7 Effect of JNK inhibition on the DNA break damage induced by arsenite in HELF cells3.7.1 Effect of JNK inhibition on the SSBs induced by arsenite in HELF cellsCells were treated as in 3.6.We found that the OTM value greatly attenuated at the 40μmol/L NaAsO₂ for 24 h treatment accompanied with arsenite group(P＜0.05).Interestingly,we found that the OTM value was slightly increased compared to control after JNK inhibition at 12h(P＞0.05),which appeared to contradict to apoptosis rate reduce at that time.3.7.2 Effect of JNK inhibition on the DSBs induced by arsenite in HELF cellsCells were treated as in 3.6.The SP600125 down-regulated the DSBs at 12h,24h indicated byγ-H2AX expression.The JNK inhibitor SP600125 greatly reduced theγ-H2AX compared arsenite group and inhibitor group at 12h(P＜0.05)and 24h(P＜0.05).Conclusion:Our data show that,at 12h,JNK pathway was activated by NaAsO₂ in HELF cells.NaAsO₂ induced the DSBs through JNK pathway in HELF cells which occurred at 12h when apoptosis was not significant.Further treatment with NaAsO₂ can cause cell apoptosis.Our results provide the new clues to further understand the carcinogenesis of arsenic.