Effects of Chemicals in Reporter Gene Bioassays with Different Metabolic Activities Compared to Baseline Toxicity

High-throughput cell-based bioassays are used for chemical screening and risk assessment. Chemical transformation processes caused by abiotic degradation or metabolization can reduce the chemical concentration or, in some cases, lead to the formation of more toxic transformation products. Unaccounted loss processes may falsify the bioassay results. Capturing the formation and effects of transformation products is important for relating the in vitro effects to in vivo. Reporter gene cell lines are believed to have low metabolic activity, but inducibility of cytochrome P450 (CYP) enzymes has been reported. Baseline toxicity is the minimal toxicity a chemical can have and is caused by the incorporation of the chemical into cell membranes. In the present study, we improved an existing baseline toxicity model based on a newly defined critical membrane burden derived from freely dissolved effect concentrations, which are directly related to the membrane concentration. Experimental effect concentrations of 94 chemicals in three bioassays (AREc32, ARE-bla and GR-bla) were compared with baseline toxicity by calculating the toxic ratio (TR). CYP activities of all cell lines were determined by using fluorescence-based assays. Only ARE-bla showed a low basal CYP activity and inducibility and AREc32 showed a low inducibility. Overall cytotoxicity was similar in all three assays despite the different metabolic activities indicating that chemical metabolism is not relevant for the cytotoxicity of the tested chemicals in these assays. Up to 28 chemicals showed specific cytotoxicity with TR > 10 in the bioassays, but baseline toxicity could explain the effects of the majority of the remaining chemicals. Seven chemicals showed TR in vitro bioassay. The new baseline model can be used not only to identify specific cytotoxicity mechanisms but also to identify potential problems in the experimental performance or evaluation of the bioassay and thus improve the quality of the bioassay data.基于细胞的高通量生物检测（high-throughput cell-based bioassay）常用于化学品筛选与风险评估。由非生物降解（abiotic degradation）或代谢作用引发的化学转化过程，会降低化学品的浓度，在部分情况下还会生成毒性更强的转化产物（transformation product）。未被纳入考量的损耗过程可能会导致生物检测结果失真。捕捉转化产物的生成与效应，对于关联体外（in vitro）与体内（in vivo）实验的效应具有重要意义。报告基因细胞系（reporter gene cell line）通常被认为代谢活性较低，但已有研究报道了细胞色素P450（cytochrome P450, CYP）酶的诱导性。基线毒性（baseline toxicity）是化学品所能具有的最低毒性，其产生源于化学品嵌入细胞膜的过程。本研究基于一项新定义的临界膜负荷（critical membrane burden）改进了现有基线毒性模型，该临界负荷由与膜浓度直接相关的游离态效应浓度（freely dissolved effect concentration）推导而来。本研究通过计算毒性比（toxic ratio, TR），将94种化学品在三种生物检测法（AREc32、ARE-bla与GR-bla）中的实验效应浓度与基线毒性进行了对比。所有细胞系的CYP酶活性均通过基于荧光的检测法（fluorescence-based assay）完成测定。仅ARE-bla表现出较低的基础CYP酶活性与诱导性，而AREc32仅表现出较低的诱导性。尽管三种检测法的代谢活性存在差异，但整体细胞毒性水平较为相似，这表明在本研究的检测体系中，化学品代谢与受试化学品的细胞毒性并无关联。在这些生物检测中，多达28种化学品表现出TR>10的特异性细胞毒性，但基线毒性可解释绝大多数剩余化学品所产生的效应。另有7种化学品在体外生物检测中呈现出TR值。这款新型基线毒性模型不仅可用于识别特异性细胞毒性机制，还可用于排查生物检测实验操作或结果评估中存在的潜在问题，进而提升生物检测数据的质量。