Article

Compound Cytotoxicity Profiling Using Quantitative High-Throughput Screening

NIH Chemical Genomics Center, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892-3370, USA.
Environmental Health Perspectives (Impact Factor: 7.03). 04/2008; 116(3):284-91. DOI: 10.1289/ehp.10727
Source: PubMed

ABSTRACT The propensity of compounds to produce adverse health effects in humans is generally evaluated using animal-based test methods. Such methods can be relatively expensive, low-throughput, and associated with pain suffered by the treated animals. In addition, differences in species biology may confound extrapolation to human health effects.
The National Toxicology Program and the National Institutes of Health Chemical Genomics Center are collaborating to identify a battery of cell-based screens to prioritize compounds for further toxicologic evaluation.
A collection of 1,408 compounds previously tested in one or more traditional toxicologic assays were profiled for cytotoxicity using quantitative high-throughput screening (qHTS) in 13 human and rodent cell types derived from six common targets of xenobiotic toxicity (liver, blood, kidney, nerve, lung, skin). Selected cytotoxicants were further tested to define response kinetics.
qHTS of these compounds produced robust and reproducible results, which allowed cross-compound, cross-cell type, and cross-species comparisons. Some compounds were cytotoxic to all cell types at similar concentrations, whereas others exhibited species- or cell type-specific cytotoxicity. Closely related cell types and analogous cell types in human and rodent frequently showed different patterns of cytotoxicity. Some compounds inducing similar levels of cytotoxicity showed distinct time dependence in kinetic studies, consistent with known mechanisms of toxicity.
The generation of high-quality cytotoxicity data on this large library of known compounds using qHTS demonstrates the potential of this methodology to profile a much broader array of assays and compounds, which, in aggregate, may be valuable for prioritizing compounds for further toxicologic evaluation, identifying compounds with particular mechanisms of action, and potentially predicting in vivo biological response.

0 Bookmarks
 · 
132 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Large-scale pharmacogenomic high-throughput screening (HTS) studies hold great potential for generating robust genomic predictors of drug response. Two recent large-scale HTS studies have reported results of such screens, revealing several known and novel drug sensitivities and biomarkers. Subsequent evaluation, however, found only moderate interlaboratory concordance in the drug response phenotypes, possibly due to differences in the experimental protocols used in the two studies. This highlights the need for community-wide implementation of standardized assays for measuring drug response phenotypes so that the full potential of HTS is realized. We suggest that the path forward is to establish best practices and standardization of the critical steps in these assays through a collective effort to ensure that the data produced from large-scale screens would not only be of high intrastudy consistency, so that they could be replicated and compared successfully across multiple laboratories. Cancer Res; 74(15); 1-8. ©2014 AACR.
    Cancer Research 07/2014; 74(15). DOI:10.1158/0008-5472.CAN-14-0725 · 9.28 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Increasing use of plant feed ingredients may introduce contaminants not previously associated with farming of salmonids, such as pesticides and PAHs from environmental sources or from thermal processing of oil seeds. To screen for interaction effects of contaminants newly introduced in salmon feeds, Atlantic salmon primary hepatocytes were used. The xCELLigence cytotoxicity system was used to select optimal dosages of the PAHs benzo(a)pyrene and phenanthrene, the pesticides chlorpyrifos and endosulfan, and combinations of these. NMR and MS metabolic profiling and microarray transcriptomic profiling was used to identify novel biomarkers. Lipidomic and transcriptomic profiling suggested perturbation of lipid metabolism, as well as endocrine disruption. The pesticides gave the strongest responses, despite having less effect on cell viability than the PAHs. Only weak molecular responses were detected in PAH-exposed hepatocytes. Chlorpyrifos suppressed the synthesis of unsaturated fatty acids. Endosulfan affected steroid hormone synthesis, while benzo(a)pyrene disturbed vitamin D3 metabolism. The primary mixture effect was additive, although at high concentrations the pesticides acted in a synergistic fashion to decrease cell viability and down-regulate CYP3A and FABP4 transcription. This work highlights the usefulness of 'omics techniques and multivariate data analysis to investigate interactions within mixtures of contaminants with different modes of action.
    Toxicology Letters 09/2014; 229. DOI:10.1016/j.fct.2014.08.008 · 3.36 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The molecular mechanisms whereby small molecules that contaminate our environment cause physiological effects are largely unknown, in terms of both targets and mechanisms. The essential human enzyme porphobilinogen synthase (HsPBGS, a.k.a. 5-aminolevulinate dehydratase, ALAD) functions in heme biosynthesis. HsPBGS catalytic activity is regulated allosterically via an equilibrium of inactive hexamers and active octamers, and we have shown that certain drugs and drug-like small molecules can inhibit HsPBGS in vitro by stabilizing the hexamer. Here we address whether components of the National Toxicology Program library of environmental contaminants can stabilize the HsPBGS hexamer and inhibit activity in vitro. Native polyacrylamide gel electrophoresis was used to screen the library (1,408 compounds) for components that alter the oligomeric distribution of HsPBGS. Freshly purchased samples of 37 preliminary hits were used to confirm the electrophoretic results and to determine the dose-dependence of the perturbation of oligomeric distribution. Seventeen compounds were identified which alter the oligomeric distribution toward the hexamer and also inhibit HsPBGS catalytic activity, including the most potent HsPBGS inhibitor yet characterized (Mutagen X, IC50 = 1.4μM). PBGS dysfunction is associated with the inborn error of metabolism know as ALAD porphyria and with lead poisoning. The identified hexamer-stabilizing inhibitors could potentiate these diseases. Allosteric regulation of activity via an equilibrium of alternate oligomers has been proposed for many proteins. Based on the precedent set herein, perturbation of these oligomeric equilibria by small molecules (such as environmental contaminants) can be considered as a mechanism of toxicity.
    Current Chemical Biology 10/2013; 7(2). DOI:10.2174/2212796811307020011

Full-text (3 Sources)

Download
73 Downloads
Available from
Jun 1, 2014