水消毒副产物卤代苯醌的结构与毒性分析

Analysis on structure and toxicity of water disinfection byproducts halobenzoquinones

目的:分析卤代苯醌(HBQs)对人肝癌HepG2细胞的半数抑制浓度(IC_(50))与结构参数间的定量构效关系,探讨可能影响HBQs细胞毒性的结构参数。方法:体外培养HepG2细胞,采用不同浓度HBQs溶液作用细胞,同时设空白对照组[10%胎牛血清(FBS)+DMEM培养基]和阴性对照组[10%FBS+DMEM培养基+1.33%甲醇(MeOH)]。MTS法和中性红摄取试验(NRU)法确定HBQs的作用浓度,采用MTS法和NRU法检测各组细胞存活率并计算HBQs对人肝癌HepG2细胞的IC_(50)。采用量子化学计算模块MOPAC计算每种HBQs的结构参数。采用单因素线性回归分析方法,根据HBQs的IC_(50)和HBQs结构参数构建定量结构-毒性关系(QSTR)模型。结果:MTS法检测2,6-二氯-1,4-苯醌(2,6-DCBQ)的作用浓度为75、100、125、150、175、200、225和250μmol·L^(-1),2,6-二氯-3-甲基-1,4-苯醌(DCMBQ)的作用浓度为175、200、225、250、275、300和325μmol·L^(-1),2,3,6-三氯-1,4-苯醌(TCBQ)的作用浓度为200、225、250、275、300和325μmol·L^(-1),2,5-二溴-1,4-苯醌(2,5-DBBQ)的作用浓度为75、100、125、150、175和200μmol·L^(-1),2,6-二溴-1,4-苯醌(2,6-DBBQ)的作用浓度为200、225、250、275、300和325μmol·L^(-1);NRU法检测,2,6-DCBQ的作用浓度为25、50、75、100和125μmol·L^(-1),DCMBQ的作用浓度为125、150、175、200、225和250μmol·L^(-1),TCBQ的作用浓度为175、200、225、250、275和300μmol·L^(-1),2,5-DBBQ的作用浓度为75、100、125、150和175μmol·L^(-1),2,6-DBBQ的作用浓度为125、150、175、200、225和250μmol·L^(-1)。各组HepG2细胞存活率随HBQs浓度的升高明显降低,呈现明显剂量-反应关系。MTS法检测HBQs对HepG2细胞毒性作用为2,6-DCBQ>2,5-DBBQ>DCMBQ>TCBQ>2,6-DBBQ;NRU法检测HBQs对HepG2细胞毒性作用为2,6-DCBQ>2,5-DBBQ>DCMBQ>2,6-DBBQ>TCBQ。单因素多元线性回归分析,MTS法和NRU法检测HBQs的IC_(50)与HBQs结构参数间相关性分析比较差异均无统计学意义(P>0.05)。结论:卤代原子的取代位置、种类和数量可影响HBQs的毒性作用,且呈现卤素取代个数越多其毒性越小的趋势。

Objective:To analyze the quantitative constitutive relationship between the median-inhibitory concentration(IC_(50))of halobenzoquinones(HBQs)to the human hepatocellular carcinoma HepG2 cells and structural parameters,and to explore the structural parameters that may affect the cytotoxicity of HBQs.Methods:The HepG2 cells were cultured in vitro and cultured with different contentrations of HBQs solutions,at the same time,blank control group[10%fetal bovine serum(FBS)+DMEM culture medium]and negative control group[10%FBS+DMEM culture medium+1.33%methanol(MeOH)]were set up.The action concentrations of HBQs were comfirmed with MTS and neural red uptake(NRU)methods,respectively.The survival rates of cells in various groups were detected by MTS and NRU methods,and the IC_(50) of HBQs was calculated.The quantum chemical calculation module MOPAC was used to calculate the structural parameters of every kind of HBQs.Single-factor linear regression analysis was used to construct the quantitative structure-toxicity relationship(QSTR)models based on the IC_(50) and structural parameters of the HBQs.Results:The MTS method determination results showed that the action concentrations of 2,6-dichloro-1,4-benzoquinone(2,6-DCBQ)were 75,100,125,150,175,200,225,and 250μmol·L^(-1),the action concentrations of 2,6-dichloro-3-methyl-1,4-benzoquinone(DCMBQ)were 175,200,225,250,275,300,and 325μmol·L^(-1),the action concentrations of 2,3,6-trichlorocyclohexa-1,4-benzoquinone(TCBQ)were 200,225,250,275,300,and 325μmol·L^(-1),the action concentrations of 2,5-dibromo-1,4-benzoquinone(2,5-DBBQ)were 75,100,125,150,175,and 200μmol·L^(-1),and the action concentrations of 2,6-dibromo-1,4-benzoquinone(2,6-DBBQ)were 200,225,250,275,300,325μmol·L^(-1).The NRU method determination results showed that the action concentrations of 2,6-DCBQ were 25,50,75,100,and 125μmol·L^(-1),the action concentrations of DCMBQ were 125,150,175,200,225,and 250μmol·L^(-1),the action concentrations of TCBQ were 175,200,225,250,275,and 300μmol·L^(-1),the action concentrations of 2,5-DBBQ were 75,100,125,150,and 175μmol·L^(-1),the action concentrations of 2,6-DBBQ were 125,150,175,200,225,and 250μmol·L^(-1).The survival rates of HepG2 cells in various groups were significantly decreased with the increase of HBQs concentration,showing a dose-response relationship.The cytotoxic effects of HBQs on the HepG2 cells detected by MTS method were 2,6-DCBQ>2,5-DBBQ>DCMBQ>TCBQ>2,6-DBBQ;and the cytotoxic effects of HBQs on HepG2 cells detected by NRU method were 2,6-DCBQ>2,5-DBBQ>DCMBQ>2,6-DBBQ>TCBQ.The single-factor multiple linear regression analysis results showed that the correlation analysis between IC_(50) and structural parameters of the HBQs detected by MTS and NRU methods had no statistically significant differences(P>0.05).Conclusion:The substitution position,type and number of halogen atoms can affect the toxic effects of HBQs,and there is a trend that the more halogen substitution,the less toxic it is.