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

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.

Cytotoxicity Burst? Differentiating Specific from Nonspecific Effects in Tox21 in Vitro Reporter Gene Assays

“…Lipophilic chemicals that have high sorption affinities to phospholipid membranes trigger baseline toxicity at lower dosed concentrations than hydrophilic chemicals, but the cytotoxic concentrations in the cell membranes do not differ much between different chemicals causing baseline toxicity (van Wezel and Opperhuizen 1995). We confirmed for eight reporter gene cell lines that chemicals triggered baseline toxicity when reaching a critical membrane concentration of approximately 70 mmol × L −1 lip (Escher et al 2019). We developed quantitative structure-activity relationships (QSARs) for these cell lines to predict the 10% inhibitory concentrations (IC 10 ) based on one chemical parameter, the liposome-water partition constant (K lip=w ) (Escher et al 2019).…”

“…We confirmed for eight reporter gene cell lines that chemicals triggered baseline toxicity when reaching a critical membrane concentration of approximately 70 mmol × L −1 lip (Escher et al 2019). We developed quantitative structure-activity relationships (QSARs) for these cell lines to predict the 10% inhibitory concentrations (IC 10 ) based on one chemical parameter, the liposome-water partition constant (K lip=w ) (Escher et al 2019). Known baseline toxicants (Vaes et al 1998) were used to derive the QSARs.…”

“…In our experience the typically used cell viability assays using fluorometric methods to detect metabolic activity or loss of cell membrane integrity are prone to artifacts and, as observed by Hsieh et al (2017), produce inconsistent results. We are therefore routinely screening cytotoxicity during reporter gene assays measurements by assessing confluence with a cell imager directly after dosing and prior to activity measurement of the reporter protein (Escher et al 2019). Although, for most chemicals, this imaging method provided effect concentrations similar to those of the fluorometric cell viability assay, artifacts of apparent high metabolic activity while cells have already disappeared under the microscope can be avoided.…”

New Approach Methods to Evaluate Health Risks of Air Pollutants: Critical Design Considerations for In Vitro Exposure Testing

“…For volatile compounds, both dynamics and kinetics in vitro can be confounded if not properly accounted for. For example, high-throughput testing designs often make use of multi-well plates for the testing of multiple chemicals per plate that are typically not sealed, allowing volatile chemicals to not only escape a test well (therefore reducing effective concentration) but also to contaminate nearby wells [164][165][166]. However, given appropriate handling, in vitro data can be very useful for informing in vivo predictions: Quick and Shuler (1999) demonstrated a predictive toxicokinetic model for the volatile compound naphthalene that could be constructed using in vitro data on rate constants for metabolic kinetics [167].…”

“…To date, in vitro measures of toxicokinetics for volatile compounds have not been included in chemical risk prioritizations [163,169,170] because both bioactivity and kinetic data cannot be obtained using the same methods as previously implemented for the non-volatile and semi-volatile compounds that make up the majority of high-throughput screening libraries [164,171]. The development of a generic PBTK modeling framework that can concurrently handle higher throughput data on semi-and non-volatile chemicals with lower throughput data on volatile chemicals would enable comparison of chemical risk rankings across these diverse chemistries.…”