Scheers E M, Ekwall B, Dierickx P J
Institute of Public Health-Louis Pasteur, Laboratory Biochemical Toxicology, J. Wytsmanstraat 14, 1050, Brussels, Belgium.
Toxicol In Vitro. 2001 Apr;15(2):153-61. doi: 10.1016/s0887-2333(00)00062-x.
Within the framework of the EDIT (Evaluation guided Development of In vitro Toxicity and toxicokinetic tests) programme, the long-term cytotoxicity of 27 chemicals was investigated on Hep G2 cells. The first step in the experiments was to determine the PI50(24h) of the chemicals. This is the concentration of compound needed to reduce the total protein content by 50% after 24 h of treatment. In the long-term experiments the chemicals were tested in six different concentrations, using the PI50(24h) as maximum concentration. The cells were treated twice a week with the same concentration of test compound and were trypsinised and counted once a week (dynamic culture). The number of cells was compared to the number of cells of the control. Three major long-term cytotoxicity patterns could be distinguished. After 6 weeks, the EC50(6w)s were determined. This is the concentration of compound needed to reduce the number of cells by 50% after 6 weeks of treatment. These values were compared with the PI50(24h). A good correlation was found for the 27 chemicals (r(2)=0.860). After 6 weeks, the concentration of test compound needed to reduce the total cell protein content by 50% after 24 h after 6 weeks of pretreatment of the cells with a particular concentration of test compound was measured: the PI50(24h-6w). For the majority of compounds there is no difference between the PI50(24h) and the PI50(24h-6w). For ethanol, arsenic (III) oxide, verapamil hydrochloride and orphenadrine, the PI50(24h-6w) increased in comparison to the PI50(24h). For some compounds a doseresponse was observed, indicating that the cells have become more resistant or more sensitive. Linear regression analysis revealed a good correlation (r(2)=0.709) between the EC50(6w) and the human acute toxicity. All these data indicate that a good alternative test may be found for predicting the long-term human toxicity.
在EDIT（体外毒性和毒代动力学试验评估指导下的开发）项目框架内，研究了27种化学物质对Hep G2细胞的长期细胞毒性。实验的第一步是确定这些化学物质的PI50(24h)。这是在处理24小时后使总蛋白含量降低50%所需的化合物浓度。在长期实验中，以PI50(24h)作为最大浓度，对化学物质进行六种不同浓度的测试。细胞每周用相同浓度的测试化合物处理两次，每周进行一次胰蛋白酶消化和计数（动态培养）。将细胞数量与对照组细胞数量进行比较。可以区分出三种主要的长期细胞毒性模式。6周后，测定EC50(6w)。这是在处理6周后使细胞数量减少50%所需的化合物浓度。将这些值与PI50(24h)进行比较。发现27种化学物质具有良好的相关性（r(2)=0.860）。6周后，测量在用特定浓度的测试化合物对细胞进行6周预处理后，24小时内使总细胞蛋白含量降低50%所需的测试化合物浓度：PI50(24h - 6w)。对于大多数化合物，PI50(24h)和PI50(24h - 6w)之间没有差异。对于乙醇、三氧化二砷、盐酸维拉帕米和邻苯海明，PI50(24h - 6w)相对于PI50(24h)有所增加。对于一些化合物观察到剂量反应，表明细胞变得更具抗性或更敏感。线性回归分析显示EC50(6w)与人类急性毒性之间具有良好的相关性（r(2)=0.709）。所有这些数据表明，可能会找到一种很好的替代试验来预测长期人类毒性。
