Baseline Toxicity and Volatility Cutoff in Reporter Gene Assays Used for High-Throughput Screening

Chem Res Toxicol. 2019 Aug 19;32(8):1646-1655. doi: 10.1021/acs.chemrestox.9b00182. Epub 2019 Aug 2.

Abstract

Most studies using high-throughput in vitro cell-based bioassays tested chemicals up to a certain fixed concentration. It would be more appropriate to test up to concentrations predicted to elicit baseline toxicity because this is the minimal toxicity of every chemical. Baseline toxicity is also called narcosis and refers to nonspecific intercalation of chemicals in biological membranes, leading to loss of membrane structure and impaired functioning of membrane-related processes such as mitochondrial respiration. In cells, baseline toxicity manifests as cytotoxicity, which was quantified by a robust live-cell imaging method. Inhibitory concentrations for baseline toxicity varied by orders of magnitude between chemicals and were described by a simple quantitative structure activity relationship (QSAR) with the liposome-water partition constant as a sole descriptor. The QSAR equations were remarkably similar for eight reporter gene cell lines of different cellular origin, six of which were used in Tox21. Mass-balance models indicated constant critical membrane concentrations for all cells and all chemicals with a mean of 69 mmol·kglip-1(95% CI: 49-89), which is in the same range as for bacteria and aquatic organisms and consistent with the theory of critical membrane burden of narcosis. The challenge of developing baseline QSARs for cell lines is that many confirmed baseline toxicants are rather volatile. We deduced from cytotoxicity experiments with semi-volatile chemicals that only chemicals with medium-air partition constants >10,000 L/L can be tested in standard robotic setups without appreciable loss of effect. Chemicals just below that cutoff showed crossover effects in neighboring wells, whereas the effects of chemicals with lower medium-air partition constants were plainly lost. Applying the "volatility cut-off" to >8000 chemicals tested in Tox21 indicated that approximately 20% of Tox21 chemicals could have partially been lost during the experiments. We recommend applying the baseline QSARs together with volatility cut-offs for experimental planning of reporter gene assays, that is, to dose only chemicals with medium-air partition constants >10,000 at concentrations up to the baseline toxicity level.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biological Assay*
  • Cell Line, Tumor
  • Cell Survival / drug effects
  • Dose-Response Relationship, Drug
  • Genes, Reporter / drug effects*
  • Genes, Reporter / genetics
  • HEK293 Cells
  • Hep G2 Cells
  • High-Throughput Screening Assays*
  • Humans
  • MCF-7 Cells
  • Organic Chemicals / adverse effects*
  • Organic Chemicals / chemistry
  • Quantitative Structure-Activity Relationship

Substances

  • Organic Chemicals