不同预处理消毒饮用水中非挥发性有机提取物对HepG2细胞的损伤作用

DNA Damage Caused by Non-volatile Organic Compounds Extracted from Surface Water Treated with Different Disinfection Methods

[目的]研究汉江水源水以及3种不同预处理方法(氯气、二氧化氯和臭氧)消毒水中的非挥发性有机提取物(non-volatility organic compounds,NOCs)对肝癌细胞(HepG2细胞)的损伤作用。[方法]设氯气(Cl2+Cl2)、二氧化氯(ClO2+Cl2)、臭氧(O3+Cl2)和水源水(raw water)4个处理组以及一个阴性对照组(1‰DMSO)和一个阳性对照组(Bap)。各实验组分别以0.2、1、5、25、125mL/mL培养基的浓度对体外HepG2细胞进行染毒。以单细胞凝胶电泳(single cell gel electrophoresis,SCGE)、细胞松弛素B阻断核质分裂微核法(cytokinesis-block micronucleus)和MTT试验分别检测其对细胞DNA断裂损伤、染色体损伤和细胞存活率的影响。[结果]①彗星试验显示,3种不同预处理方法消毒水中NOCs在5、25、125mL/mL培养基均可明显导致DNA单、双链断裂,与阴性对照组相比差异有统计学意义(P<0.05)。相同浓度各处理组间的差异也具有统计学意义(P<0.05);碱性彗星试验显示致DNA损伤最明显的是二氧化氯组;中性彗星试验显示致DNA损伤最明显的为氯气组。②微核试验显示,3种不同预处理方法消毒水和水源水NOCs在5、25、125mL/mL培养基均可致细胞微核的形成,与阴性对照组相比差异有统计学意义(P<0.05);与同浓度的臭氧组相比,氯气组在1mL/mL培养基的细胞微核率升高(P<0.05),二氧化氯组在1mL/mL和5mL/mL培养基的细胞微核率均明显升高(P<0.05);水源水组在1mL/mL和25mL/mL培养基时的细胞微核率显著低于同浓度的二氧化氯、氯气和臭氧组(P<0.05),在125mL/mL培养基则显著高于二氧化氯、氯气和臭氧组(P<0.05)。③与对照组相比,3种不同预处理方法消毒水和水源水NOCs对体外HepG2细胞存活率有随浓度的增加而降低的趋势,在25、125mL/mL培养基差异均有统计学意义(P<0.05);④相关性研究表明,碱性OTM和中性OTM与微核率均呈正相关(r=0.697,P=0.001;r=0.575,P=0.012);细胞生存率与碱性OTM和微核率均呈负相关(r=-0.763,P=0.000;r=-0.635,P=0.005)。[结论]3种不同预处理方法消毒水中NOCs均可以导致体外HepG2细胞DNA单链和双链断裂。氯气预处理消毒水中的NOCs主要导致DNA双链的断裂,二氧化氯预处理消毒水中的NOCs主要导致DNA单链断裂。

[Objective] To evaluate the in vitro toxicity of non-volatility organic compounds（NOCs）extracted from Hanjiang water disinfected by different sequential treatments.[Methods] Hanjiang water was disinfected using ozone,chlorine dioxide or chlorine as the primary disinfectant followed by chlorine as the secondary disinfectant. HepG2 cells were exposed to NOCs extracts of these different treated water samples corresponding to concentrations of 0.2,1.0,5.0,25.0 and 125.0 mL/mL medium. DNA single-strand breaks,chromosome damage and cell survival rates induced by NOCs were investigated via single cell gel electrophoresis（SCGE）,cytokinesis-blocked micronucleus（CBMN）test and MTT test respectively.[Results] ① Significant and dose-dependent increase of DNA damage induced by NOCs extracted from before and after chlorination,chlorine dioxide and ozone disinfected drinking water were found both in alkaline and neutral comet assay from the doses of 5.0 to 125.0 mL/mL medium （P 0.05）,especially at high dosages（25.0 and 125.0 mL/mL medium）（P 0.01）. NOCs extracted from water sample of chlorine dioxide group caused most serious DNA damage in alkaline comet assay and that of ozone group caused the lowest damage. Extraction from chlorination group caused DNA damage most serious in neutral comet assay. ② NOCs extracted from chlorination,chlorine dioxide and ozone disinfected drinking water caused significant and dose-dependent increase of MN frequencies from the doses of 5.0 to 125.0 mL/mL medium（P 0.05）in HepG2 cells; ③ NOCs extracted from chlorination,chlorine dioxide and ozone disinfected drinking water caused a significant and dose-dependent decrease of cell viability from the doses of 25.0 to 125.0 mL/mL medium（P 0.05）in HepG2 cells; ④ There were better correlations among cell survival rates,DNA damage（both alkaline and neutral Comet assay）and chromosome damage,either positive or negative.[Conclusion] According to Comet assay and Micronucleus assay,chlorination,chlorine dioxide and ozone could cause DNA breaks,both single-and double-strand,but chlorination caused DNA double-strand breaks most seriously and chlorine dioxide caused DNA single-strand breaks most seriously.