焦炉逸散物有机提取物致HL-60细胞DNA损伤及NF-κB蛋白表达改变

DNA damage and changes in NF-κB protein expression in HL-60 cells treated with organic extracts of coke oven emissions

[目的]焦炉逸散物(COE)是主要的工业和环境污染物,其中含有大量致癌和致突变的多环芳烃(PAHs)。外源化学物经吸收后进入血液系统,有可能引起血细胞的损伤。但目前对于COE致血液系统损伤的研究尚不多见,其血液毒性机制亦尚不明了。因此,本研究拟探讨焦炉逸散物有机提取物(OE-COE)连续暴露对血液细胞的损伤效应及生物学机制。[方法]采集并提取焦炉炉顶颗粒物,制备OE-COE染毒液。取对数生长期HL-60细胞,用0、2.5、5、10、20 mg/L浓度的OE-COE连续染毒4 d,建立细胞连续暴露模型。染毒结束后,CCK-8法检测细胞存活率,流式细胞术检测细胞凋亡和坏死情况,彗星试验检测细胞DNA损伤情况,Western blot法检测细胞核转录因子κB(NF-κB)抑制蛋白(IκBα)、磷酸化IκBα、NF-κB p65、磷酸化NF-κB p65蛋白表达水平。[结果]经OE-COE 4 d连续染毒处理后,随着剂量的增加,HL-60细胞相对存活率下降(P<0.001),20 mg/L组中HL-60细胞存活率下降至38.63%。5、10、20 mg/L组HL-60细胞的相对凋亡率分别是对照组的1.22倍(P>0.05)、2.51倍(P>0.05)和13.97倍(P<0.05),细胞的相对坏死率分别是对照组的1.23倍(P>0.05)、1.16倍(P>0.05)和2.88倍(P<0.05)。10、20 mg/L组细胞尾部DNA相对百分含量分别上升至对照组的2.05倍(P<0.05)和2.40倍(P<0.05),且高于2.5、5 mg/L组(P<0.05)。5、10 mg/L组细胞的IκBα、磷酸化IκBα与NF-κB p65、磷酸化NF-κB p65蛋白相对表达水平均高于对照组(P<0.05)。[结论] OE-COE连续暴露可致HL-60细胞DNA损伤,NF-κB活化可能是OE-COE造成血液细胞DNA损伤的机制之一。

[Objective]Coke oven emissions(COE)are primary industrial and environmental pollutant,including large amounts of carcinogenic and mutagenic polycyclic aromatic hydrocarbons(PAHs).Exogenous chemicals are absorbed into hematological system first,and may cause blood cell damage.However,few studies have reported the damage to hematological system induced by COE and related mechanism.This study is designed to investigate the damage and biological mechanism of hematological cells exposed to organic extracts of coke oven emissions(OE-COE).[Methods]COE were collected at the top area of a coke oven and extracted to prepare OECOE.HL-60 cells were collected in logarithmic growth phase and cultured with OE-COE for 4 days at concentrations of 0,2.5,5,10,and 20 mg/L to establish continuous exposure models.After exposure,cell survival rate was detected by CCK-8,cell apoptosis and necrosis by flow cytometry,DNA damage by comet assay,and the expression levels of inhibitor of nuclear factor-kappa B(NF-κB)(IκBα),phosphorylation IκBα,NF-κB p65,and phosphorylation NF-κB p65 by Western blot.[Results]After 4 days of continuous exposure,with the dosage increase of OE-COE,the relative survival rate of HL-60 cells was decreased significantly(P<0.001),especially the relative survival rate of the 20 mg/L group which was decreased to 38.63%.The relative apoptosis rates of HL-60 cells in the 5,10,and 20 mg/L groups were 1.22(P>0.05),2.51(P>0.05),and 13.97(P<0.05)times higher than that of the control group;the relative necrosis rates of HL-60 cells in the 5,10,and 20 mg/L groups were 1.23(P>0.05),1.16(P>0.05),and 2.88(P<0.05)times higher than that of the control group.The relative percentages of tail DNA of HL-60 cells in the 10 mg/L and 20 mg/L groups were 2.05(P<0.05)and 2.40(P<0.05)times higher than that of the control group,and were also higher than those of the 2.5 and 5 mg/L groups(P<0.05).The relative expression levels of IκBα,phosphorylation IκBα,NF-κB p65,and phosphorylation NF-κB p65 proteins in the 5 and 10 mg/L groups were higher than those of control group(P<0.05).[Conclusion]OE-COE continuous exposure could induce DNA damage in HL-60 cells,and the activation of NF-κB might be one of the mechanisms of DNA damage after OE-COE exposure.