Department of Ecosystem Analysis, Institute for Environmental Research, Aachen Biology and Biotechnology (ABBt), RWTH Aachen University, Aachen, Germany.
College of Resources and Environmental Science, Chongqing University, 1 Tiansheng Road, Beibei, Chongqing, 400715, China.
Environ Sci Pollut Res Int. 2018 Feb;25(5):4012-4022. doi: 10.1007/s11356-017-8790-2. Epub 2017 Mar 16.
The binding of the p53 tumor suppression protein to DNA response elements after genotoxic stress can be quantified by cell-based reporter gene assays as a DNA damage endpoint. Currently, bioassay evaluation of environmental samples requires further knowledge on p53 induction by chemical mixtures and on cytotoxicity interference with p53 induction analysis for proper interpretation of results. We investigated the effects of genotoxic pharmaceuticals (actinomycin D, cyclophosphamide) and nitroaromatic compounds (4-nitroquinoline 1-oxide, 3-nitrobenzanthrone) on p53 induction and cell viability using a reporter gene and a colorimetric assay, respectively. Individual exposures were conducted in the absence or presence of metabolic activation system, while binary and tertiary mixtures were tested in its absence only. Cell viability reduction tended to present direct correlation with p53 induction, and induction peaks occurred mainly at chemical concentrations causing cell viability below 80%. Mixtures presented in general good agreement between predicted and measured p53 induction factors at lower concentrations, while higher chemical concentrations gave lower values than expected. Cytotoxicity evaluation supported the selection of concentration ranges for the p53 assay and the interpretation of its results. The often used 80% viability threshold as a basis to select the maximum test concentration for cell-based assays was not adequate for p53 induction assessment. Instead, concentrations causing up to 50% cell viability reduction should be evaluated in order to identify the lowest observed effect concentration and peak values following meaningful p53 induction.
在遗传毒性应激后，p53 肿瘤抑制蛋白与 DNA 反应元件的结合可以通过基于细胞的报告基因检测来定量，作为 DNA 损伤终点。目前，环境样品的生物测定评估需要进一步了解化学混合物对 p53 的诱导作用，以及细胞毒性对 p53 诱导分析的干扰，以便对结果进行正确解释。我们使用报告基因和比色法分别研究了遗传毒性药物（放线菌素 D、环磷酰胺）和硝基芳烃化合物（4-硝基喹啉 1-氧化物、3-硝基苯并蒽酮）对 p53 诱导和细胞活力的影响。单独暴露在不存在或存在代谢激活系统的情况下进行，而二元和三元混合物仅在不存在代谢激活系统的情况下进行测试。细胞活力的降低趋势与 p53 诱导呈直接相关，诱导峰值主要出现在化学浓度导致细胞活力低于 80%的情况下。在较低浓度下，混合物通常在预测和测量的 p53 诱导因子之间表现出良好的一致性，而较高的化学浓度给出的数值低于预期。细胞毒性评估支持选择 p53 测定的浓度范围，并解释其结果。通常使用 80%活力阈值作为选择基于细胞的测定最大测试浓度的基础，对于 p53 诱导评估是不够的。相反，应评估导致细胞活力降低高达 50%的浓度，以确定最低观察到的效应浓度和有意义的 p53 诱导后的峰值。
