The Role Of Oxidative Stress On Adaptive Response Induced By Sodium Arsenite In Human HaCaT Keratinocytes

Objective：To explore the role of oxidative stress and Keap1/Nrf2-ARE pathway on adaptiveresponse induced by low dose sodium arsenite in immortalized humankeratinocytes(HaCaT cells).Method：(1) To evaluate the half maximal inhibitory concentration (IC50) of sodiumarsenite(NaAsO2) in HaCaT cells, the proliferation of HaCaT cells was detected by3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test after HaCaT cellswere treated with NaAsO2of0,0.1,0.15,0.6,2.5,5.0,10.0,20.0and40.0μM for24hours, respectively.Then, according to the rates of proliferation, IC50of sodiumarsenite(NaAsO2) in HaCaT cells is calculate with the GraphPad Prism6.(2) To set up pretreatment group and non-pretreated group. HaCaT cells inpretreatment group were pre-exposed to0.15μM NaAsO2for24h and then subjected to10μM of NaAsO2for0,4,8,12,24and36h.HaCaT cells in non-pretreated group wereexposured to a final concentration of10μM of NaAsO2for0,4,8,12,24and36h.(3) The adaptive response of HaCaT cells to the oxidative stress of NaAsO2wasobserved through the reactive oxygen species (ROS) level and the malondialdehyde(MDA) levels.At the end of NaAsO2exposure, HaCaT cells in pretreatment group andnon-pretreated group were harvested. The reactive oxygen species (ROS) level, themalondialdehyde (MDA) in HaCaT cells were detected respectively. DCFH-DA was used to study the content of ROS. MDA content were detected with Thibabituric Acid(TBA) test.(3) In order to understand the change of Keap1/Nrf2-ARE pathway in the adaptiveresponse induced by NaAsO2. At the end of NaAsO2exposure, HaCaT cells inpretreatment group and non-pretreated group were harvested. The cytosolic and nuclearprotein of HaCaT cells was extracted and Nrf2, Keap1, BACH1levels were determinedby Western blotting.The total protein of HaCaT cells was extracted and HO-1levelswere determined by Western blotting, then the vitality unite of superoxidedismutase(SOD) was measured with total superoxide dismutase assay kit.Results：1.The effect of sodium arsenite on cell proliferationMTT test reveal that NaAsO2can enhance proliferation of cells when the doses isbelow2.5μM. The survival rates of HaCaT cells are higher than that of the normalcontrol group(P<0.05). when the doses is above2.5μM, The survival rates of HaCaTcells are lower than that of the normal control group. The IC50of sodiumarsenite(NaAsO2) in HaCaT cells is9.077μM.2. The effect of low dose sodium arsenite on the changes of ROS levels andMDAcontent in HaCaT cellsIn order to understand the oxidative effects of arsenite, cells were incubated withDCFH-DA and the results showed increased oxidation and subsequent uorescentemission in arsenite treated cells. After exposure of sodium arsenite, the basal cellularROS level in HaCaT cells increased gradually, and reached the peak value at the8thhour in non-pretreated group, at the12th hour in pretreated group， the differencebetween the non-pretreated and pretreated group was statistically significant (P <0.05).With sodium arsenite exposure time prolonged，the intracellular ROS levels in HaCaTcells continue to decreased after the peak value time.Sodium arsenite elevated MDA contents activities in the non-pretreated group andpretreated group after0h, then the basal cellular MDA level in HaCaT cells increased gradually, and reached the peak value at the12th hour in non-pretreated group, at the24th hour in pretreated group，the difference between the non-pretreated and pretreatedgroup was statistically significant (P<0.05). The results showed that sodium arseniteinduced oxidative stress as evidenced by lipid peroxidation at high doses, andlow-dose(0.15μM) sodium arsenite pretreated can resistant to the oxidative stressinduced by subsequent sodium arsenite in high doses(10μM).3. The effect of low dose sodium arsenite on the activation of Nrf2-mediatedanti-oxidant response in HaCaT cellsArsenite activates the Nrf2-mediated anti-oxidant response. To determine whetherpretreatment with low dose arsenite and prolonged arsenite exposure activates theNrf2-mediated antioxidant response, we measured nuclear accumulation of Nrf2protein,induction of Nrf2target genes and Keap1protein, BACH1protein in thearsenite-exposed cells. INrf2(inhibitor of Nrf2) or Keap1(Kelch-like ECH-associatedprotein1), a homodimeric protein, retains Nrf2in the cytoplasm. Under normalconditions, NRF2, constitutively expressed at a low level, is primarily in the cytoplasmand mainly controlled by the Kelch-like ECH-associated protein1(KEAP1) throughubiquitination and proteasomal degradation. BACH1, the negative regulator of Nrf2anda transcriptional repressor of the heme oxygenase gene, localizes in the nucleus undernormal conditions, competes with Nrf2for binding to the ARE in the human NQO1promoter.Arsenite exposure markedly increased the protein levels of Nrf2in nuclearfractions and the protein levels of BACH1in cytosolic fractions in a time-dependentfashion, and decreased the protein levels of Keap1in cytosolic fractions. Expression ofmany Nrf2-target genes such as heme oxygenase1(HO-1) and SOD Activity weresignificantly induced. Accumulation of Nrf2in nuclear fractions and signifcantinduction of Nrf2-target genes indicate an activation of Nrf2-mediated adaptiveresponse in the arsenite-exposed cells.Exposure to inorganic arsenic resulted in Nrf2protein accumulation in the nuclearfraction of HaCaT cells in a time-dependent fashion that reached a peak at8h. To evaluate the effects of the nuclear Nrf2accumulation induced by arsenic ontranscriptional activation through the ARE, the expression of the ARE-controlled genesHO-1and SOD was assessed. Both HO-1and SOD expression were inducedconcomitantly with arsenic induction of Nrf2(Fig.5and6).Importantly,the transcriptlevels of these two genes were clearly correlated with nuclear Nrf2levels.It should benoted that although the predicted molecular mass of Nrf2is66kDa, it is shown twoimmunoreactive bands at approximately100-120kDa as in the present study. Consistentwith the Nrf2protein, expression of many INrf2(inhibitor of Nrf2) or Keap1weresignificantly induced.We tried to observe the induction of HO-1protein in HaCaT cells by arsenictreatment. To explore whether pretreatment with low dose arsenic could activate HO-1protein through Nrf2expression, we exposed HaCaT cells to the same conditions thatsodium arsenite induced Nrf2activation, and examined HO-1protein expressions byWestern blot analysis. The expressions of HO-1protein were signi cantly increasedupon arsenic exposure.The induction was time-dependent. Compared with controlgroup, the intracellular HO-1levels in HaCaT cells, exposued with10μM of sodiumarsenite for4、8、12、24and36h, were higher,the difference was statistically significant(P<0.05). With sodium arsenite exposure time prolonged，the intracellular HO-1level inHaCaT cells continue to rise, peaking at12h in pretreatment group and at24h innon-pretreated group，then decreased after the peaking hour. In pretreatment group，theHO-1level in HaCaT cells is higher than that in non-pretreated group，the differenceswere statistically significant (P <0.05).SOD activities in pretreatment group group were significantly higher than that ofnon-pretreated group at0、8、12and24h in HaCaT cells. In contrast, sodium arsenitedecreased SOD activities at10μM at4h. With sodium arsenite exposure timeprolonged，the intracellular SOD activities in HaCaT cells continue to rise, peaking at12h in pretreatment group and at8h in non-pretreated group，then decreased after thepeaking hour. In pretreatment group，the SOD activities in HaCaT cells at peak time ishigher than that in non-pretreated group，the differences were statistically significant (P <0.05). Data indicate that sodium arsenite enhanced SOD activities after pretreatmentwith low concentrations sodium arsenite, but inhibited the enzyme at highconcentrationsConclusionActivation of Keap1/Nrf2-ARE in the adaptive response process induced by lowdose of sodium arsenite decreased the oxidative stress levels induced by the followedhigher dose of sodium arsenite.