Quantitative High-Throughput Screening for Chemical Toxicity in a Population-Based In Vitro Model

University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Toxicological Sciences (Impact Factor: 3.85). 01/2012; 126(2):578-88. DOI: 10.1093/toxsci/kfs023
Source: PubMed


A shift in toxicity testing from in vivo to in vitro may efficiently prioritize compounds, reveal new mechanisms, and enable predictive modeling. Quantitative high-throughput screening (qHTS) is a major source of data for computational toxicology, and our goal in this study was to aid in the development of predictive in vitro models of chemical-induced toxicity, anchored on interindividual genetic variability. Eighty-one human lymphoblast cell lines from 27 Centre d'Etude du Polymorphisme Humain trios were exposed to 240 chemical substances (12 concentrations, 0.26nM-46.0μM) and evaluated for cytotoxicity and apoptosis. qHTS screening in the genetically defined population produced robust and reproducible results, which allowed for cross-compound, cross-assay, and cross-individual comparisons. Some compounds were cytotoxic to all cell types at similar concentrations, whereas others exhibited interindividual differences in cytotoxicity. Specifically, the qHTS in a population-based human in vitro model system has several unique aspects that are of utility for toxicity testing, chemical prioritization, and high-throughput risk assessment. First, standardized and high-quality concentration-response profiling, with reproducibility confirmed by comparison with previous experiments, enables prioritization of chemicals for variability in interindividual range in cytotoxicity. Second, genome-wide association analysis of cytotoxicity phenotypes allows exploration of the potential genetic determinants of interindividual variability in toxicity. Furthermore, highly significant associations identified through the analysis of population-level correlations between basal gene expression variability and chemical-induced toxicity suggest plausible mode of action hypotheses for follow-up analyses. We conclude that as the improved resolution of genetic profiling can now be matched with high-quality in vitro screening data, the evaluation of the toxicity pathways and the effects of genetic diversity are now feasible through the use of human lymphoblast cell lines.

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    • "The resulting data could enable an informed and focused approach to the problem of assessing hazard in risk-relevant manner in human populations that are exposed to mixtures. Furthermore, with an experimental in vitro design that represents a human population, we are able to explore not only the hazard, but also its intrinsic variability across different concentration ranges (Lock et al., 2012; O'Shea et al., 2011). Such information would be valuable to inform regulatory decisions that could more fully protect public health and sensitive subpopulations (Abdo et al., 2015). "
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    ABSTRACT: Population-based human in vitro models offer exceptional opportunities for evaluating the potential hazard and mode of action of chemicals, as well as variability in responses to toxic insults among individuals. This study was designed to test the hypothesis that comparative population genomics with efficient in vitro experimental design can be used for evaluation of the potential for hazard, mode of action, and the extent of population variability in responses to chemical mixtures. We selected 146 lymphoblast cell lines from 4 ancestrally and geographically diverse human populations based on the availability of genome sequence and basal RNA-seq data. Cells were exposed to two pesticide mixtures - an environmental surface water sample comprised primarily of organochlorine pesticides and a laboratory-prepared mixture of 36 currently used pesticides - in concentration response and evaluated for cytotoxicity. On average, the two mixtures exhibited a similar range of in vitro cytotoxicity and showed considerable inter-individual variability across screened cell lines. However, when in vitro-to-in vivo extrapolation (IVIVE) coupled with reverse dosimetry was employed to convert the in vitro cytotoxic concentrations to oral equivalent doses and compared to the upper bound of predicted human exposure, we found that a nominally more cytotoxic chlorinated pesticide mixture is expected to have greater margin of safety (more than 5 orders of magnitude) as compared to the current use pesticide mixture (less than 2 orders of magnitude) due primarily to differences in exposure predictions. Multivariate genome-wide association mapping revealed an association between the toxicity of current use pesticide mixture and a polymorphism in rs1947825 in C17orf54. We conclude that a combination of in vitro human population-based cytotoxicity screening followed by dosimetric adjustment and comparative population genomics analyses enables quantitative evaluation of human health hazard from complex environmental mixtures. Additionally, such an approach yields testable hypotheses regarding potential toxicity mechanisms.
    No preview · Article · Sep 2015 · Environment international
    • "Examples for possible applications include high-throughput screening assays or the use of complex in vitro systems as tools for testing and mechanistic toxicology. Concomitantly, there is an intensive discussion on the implementation and integration of these methods into a regulatory framework (Arnot et al. 2012; Kavlock et al. 2012; Lilienblum et al. 2008; Lock et al. 2012; SCCS et al. 2012a; Tralau et al. 2012; Wetmore et al. 2011). This document puts the respective discussion into context of current regulatory practice with the aim to Abstract The advent of new testing systems and " omics " -technologies has left regulatory toxicology facing one of the biggest challenges for decades. "
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    ABSTRACT: The advent of new testing systems and "omics"-technologies has left regulatory toxicology facing one of the biggest challenges for decades. That is the question whether and how these methods can be used for regulatory purposes. The new methods undoubtedly enable regulators to address important open questions of toxicology such as species-specific toxicity, mixture toxicity, low-dose effects, endocrine effects or nanotoxicology, while promising faster and more efficient toxicity testing with the use of less animals. Consequently, the respective assays, methods and testing strategies are subject of several research programs worldwide. On the other hand, the practical application of such tests for regulatory purposes is a matter of ongoing debate. This document summarizes key aspects of this debate in the light of the European "regulatory status quo", while elucidating new perspectives for regulatory toxicity testing.
    No preview · Article · Mar 2015 · Archives of Toxicology
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    • "Combined in vitro and computational approaches have been proposed to characterize toxicokinetic variability (Wetmore et al. 2013), but these are limited to first-order kinetics and characterization of variability in parent compound dosimetry. Other in vitro approaches to evaluating the extent of and molecular mechanisms for interindividual variability using genetically diverse cell lines have also been proposed (Lock et al. 2012; O’Shea et al. 2011). However, these and other in vitro approaches that do not capture the complexity of whole body toxicokinetics would not be successful for compounds such as TCE. "
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    ABSTRACT: Quantitative estimation of toxicokinetic variability in the human population is a persistent challenge in risk assessment of environmental chemicals. Traditionally, inter-individual differences in the population are accounted for by default assumptions or, in rare cases, are based on human toxicokinetic data. To evaluate the utility of genetically diverse mouse strains for estimating toxicokinetic population variability for risk assessment, using trichloroethylene (TCE) metabolism as a case study. We used data on oxidative and glutathione conjugation metabolism of TCE in 16 inbred and one hybrid mouse strains to calibrate and extend existing physiologically-based pharmacokinetic (PBPK) models. We added one-compartment models for glutathione metabolites and a two-compartment model for dichloroacetic acid (DCA). A Bayesian population analysis of interstrain variability was used to quantify variability in TCE metabolism. Concentration-time profiles for TCE metabolism to oxidative and glutathione conjugation metabolites varied across strains. Median predictions for the metabolic flux through oxidation was less variable (5-fold range) than that through glutathione conjugation (10-fold range). For oxidative metabolites, median predictions of trichloroacetic acid production was less variable (2-fold range) than DCA production (5-fold range), although uncertainty bounds for DCA exceeded the predicted variability. Population PBPK modeling of genetically diverse mouse strains can provide useful quantitative estimates of toxicokinetic population variability. When extrapolated to lower doses more relevant to environmental exposures, mouse population-derived variability estimates for TCE metabolism closely matched population variability estimates previously derived from human toxicokinetic studies with TCE, highlighting the utility of mouse interstrain metabolism studies for addressing toxicokinetic variability.
    Full-text · Article · Feb 2014 · Environmental Health Perspectives
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