利用HepG2细胞毒性评估化学品急性经口毒性的实验研究

Study on using HepG2 cytotoxicity test method to evaluate acute oral toxicity of chemicals

目的评估人HepG2细胞毒性试验预测化学品急性经口毒性的可行性,为急性经口毒性试验初始剂量的确定提供依据。方法采用中性红摄取方法(neutral red uptake,NRU)检测8种化学品的HepG2细胞毒性。对HepG2细胞活性半数抑制浓度IC_(50)值与急性经口毒性动物实验LD_(50)值进行线性回归分析,并与3T3 NRU试验的RC(Registry of Cytotoxicity)回归模型进行比较。将细胞IC_(50)代入不同的回归模型推算急性毒性LD_(50)预测值,并纳入化学品毒性危害分级标准进行GHS分类比较。结果8种受试物浓度对数与HepG2细胞活性之间与存在良好的剂量—反应关系。HepG2细胞毒性IC_(50)值与急性经口毒动物实验LD_(50)值存在相关性(Pearson r=0.903,P<0.01)。通过HepG2细胞试验建立回归模型1:y(logLD_(50),mmol/kg)=0.397×(logIC_(50),mmol/L)+1.065,(R^(2)=0.815,F=26.48,P<0.01),该线性方程回归系数(斜率)的95%可信区间包含有RC模型回归系数。两种回归模型对急性经口LD_(50)的预测结果及GHS毒性分级与动物实验毒性分级的一致率分别为87.5%(7/8,模型1)和62.5%(5/8,RC模型)。提示在本实验条件下,HepG2细胞试验对化学品急性经口毒性的预测能力有所提高。结论HepG2细胞中性红摄取试验可以用于化学品急性经口毒性LD_(50)的预测。该方法为化急性毒性替代方法评价提供技术参考,有望提高对有毒化学品急性经口初始剂量和毒性分级判断的准确性。

Objective To evaluate the possibility of human HepG2 cytotoxicity assay in predicting the acute oral toxicity of chemicals,which perhaps provide the evidences of the starting dose of acute oral toxicity.Methods Eight chemicals were detected by HepG2 neutral red uptake(NRU)cytotoxicity assay.The IC_(50) of HepG2 NRU assay and LD_(50) of acute oral toxicity were analyzed by linear regression and compared with registry of cytotoxicity(RC)regression model.The two regression models were used to calculate the predictive value of LD_(50) with IC_(50) of HepG2 NRU assay.Furthermore,there was toxicity classification comparison between from predictive value of LD_(50) and from LD_(50) of acute oral toxicity.Results There was a good dose-response relation between the concentration logarithm of 8 chemicals and the activity of HepG2 cells.Pearson’s correlation analysis result showed that there was a positive correlation between IC_(50) from HepG2 NRU cytotoxicity assay and LD_(50) from acute oral toxicity(Pearson r=0.903,P<0.01).The regression model(model 1)was established by IC_(50) from cytotoxicity assay and LD_(50) from acute oral toxicity and then the simple linear regression equation:y(logLD_(50),mmol/kg)=0.397×(logIC_(50),mmol/L)+1.065(R^(2)=0.815,F=26.48,P<0.01)was obtained.The parameter(slope)of the linear equation was within 95%confidence interval of RC regression model.The predicted values of LD_(50) were calculated by two regression model equations and toxicity levels were classified.The consistent rate of classification derived from the predicted value of LD_(50) and from LD_(50) of acute oral toxicity was 87.5%(7/8,model 1)and 62.5%(5/8,RC model),respectively.Under the conditions of this cytotoxicity assay,the ability of HepG2 NRU assay to predict the acute oral toxicity of chemicals was perhaps enhanced slightly.Conclusion HepG2 NRU assay may be used to predict LD_(50)of the acute oral toxicity.This method providesthe technical support for acute toxicity assessment of chemicals and maybe improve the judgment accuracy of acute oral starting dose and toxicity classification of toxic chemicals.