Cell-based high-content screening (HCS) assays have become an increasingly attractive alternative to traditional in vitro and in vivo testing in pharmaceutical drug development and toxicological ...safety assessment. The time- and cost-effectiveness of HCS assays, combined with the organotypic nature of human induced pluripotent stem cell (iPSC)-derived cells, open new opportunities to employ physiologically relevant in vitro model systems to improve screening for potential chemical hazards. In this study, we used two human iPSC types, cardiomyocytes and hepatocytes, to test various high-content and molecular assay combinations for their applicability in a multiparametric screening format. Effects on cardiomyocyte beat frequency were characterized by calcium flux measurements for up to 90 min. Subsequent correlation with intracellular cAMP levels was used to determine if the effects on cardiac physiology were G-protein-coupled receptor dependent. In addition, we utilized high-content cell imaging to simultaneously determine cell viability, mitochondrial integrity, and reactive oxygen species (ROS) formation in both cell types. Kinetic analysis indicated that ROS formation is best detectable 30 min following initial treatment, whereas cytotoxic effects were most stable after 24 h. For hepatocytes, high-content imaging was also used to evaluate cytotoxicity and cytoskeletal integrity, as well as mitochondrial integrity and the potential for lipid accumulation. Lipid accumulation, a marker for hepatic steatosis, was most reliably detected 48 h following treatment with test compounds. Overall, our results demonstrate how a compendium of assays can be utilized for quantitative screening of chemical effects in iPSC cardiomyocytes and hepatocytes and enable rapid and cost-efficient multidimensional biological profiling of toxicity.
Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes show promise for screening during early drug development. Here, we tested a hypothesis that in vitro assessment of multiple ...cardiomyocyte physiological parameters enables predictive and mechanistically-interpretable evaluation of cardiotoxicity in a high-throughput format. Human iPSC-derived cardiomyocytes were exposed for 30min or 24h to 131 drugs, positive (107) and negative (24) for in vivo cardiotoxicity, in up to 6 concentrations (3nM to 30uM) in 384-well plates. Fast kinetic imaging was used to monitor changes in cardiomyocyte function using intracellular Ca2+ flux readouts synchronous with beating, and cell viability. A number of physiological parameters of cardiomyocyte beating, such as beat rate, peak shape (amplitude, width, raise, decay, etc.) and regularity were collected using automated data analysis. Concentration–response profiles were evaluated using logistic modeling to derive a benchmark concentration (BMC) point-of-departure value, based on one standard deviation departure from the estimated baseline in vehicle (0.3% dimethyl sulfoxide)-treated cells. BMC values were used for cardiotoxicity classification and ranking of compounds. Beat rate and several peak shape parameters were found to be good predictors, while cell viability had poor classification accuracy. In addition, we applied the Toxicological Prioritization Index (ToxPi) approach to integrate and display data across many collected parameters, to derive “cardiosafety” ranking of tested compounds. Multi-parameter screening of beating profiles allows for cardiotoxicity risk assessment and identification of specific patterns defining mechanism-specific effects. These data and analysis methods may be used widely for compound screening and early safety evaluation in drug development.
•Induced pluripotent stem cell-derived cardiomyocytes are promising in vitro models.•We tested if evaluation of cardiotoxicity is possible in a high-throughput format.•The assay shows benefits of automated data integration across multiple parameters.•Quantitative assessment of concentration–response is possible using iPSCs.•Multi-parametric screening allows for cardiotoxicity risk assessment.
Abstract
Methods to assess environmental exposure to hazardous chemicals have primarily focused on quantification of individual chemicals, although chemicals often occur in mixtures, presenting ...challenges to the traditional risk characterization framework. Sampling sites in a defined geographic region provide an opportunity to characterize chemical contaminants, with spatial interpolation as a tool to provide estimates for non-sampled sites. At the same time, the use of in vitro bioactivity measurements has been shown to be informative for rapid risk-based decisions. In this study, we measured in vitro bioactivity in 39 surface soil samples collected immediately after flooding associated with Hurricane Harvey in Texas in a residential area known to be inundated with polycyclic aromatic hydrocarbon (PAH) contaminants. Bioactivity data were from a number of functional and toxicity assays in 5 human cell types, such as induced pluripotent stem cell-derived hepatocytes, cardiomyocytes, neurons, and endothelial cells, as well as human umbilical vein endothelial cells. Data on concentrations of PAH in these samples were also available and the combination of data sources offered a unique opportunity to assess the joint spatial variation of PAH components and bioactivity. We found significant evidence of spatial correlation of a subset of PAH contaminants and of cell-based phenotypes. In addition, we show that the cell-based bioactivity data can be used to predict environmental concentrations for several PAH contaminants, as well as overall PAH summaries and cancer risk. This study’s impact lies in its demonstration that cell-based profiling can be used for rapid hazard screening of environmental samples by anchoring the bioassays to concentrations of PAH. This work sets the stage for identification of the areas of concern and direct quantitative risk characterization based on bioactivity data, thereby providing an important supplement to traditional individual chemical analyses by shedding light on constituents that may be missed from targeted chemical monitoring.
Background: The National Academies' Standing Committee on Use of Emerging Science for Environmental Health Decisions held a meeting (21–22 September 2009 in Washington, DC) titled "Computational ...Toxicology: From Data to Analyses to Applications." This commentary reflects on the presentations and roundtable discussions from the meeting that were designed to review the state of the art in the field and the practical applications of the new science and to provide focus to the field. Objectives: The meeting considered two topics: the emerging data streams amenable to computational modeling and data mining, and the emerging data analysis and modeling tools. Discussion: Computational toxicology is a subdiscipline of toxicology that aims to use the mathematical, statistical, modeling, and computer science tools to better understand the mechanisms through which a given chemical induces harm and, ultimately, to be able to predict adverse effects of the toxicants on human health and/or the environment. The participants stressed the importance of computational toxicology to the future of environmental health sciences and regulatory decisions in public health; however, many challenges remain to be addressed before the findings from high-throughput screens and in silico models may be considered sufficiently robust and informative. Conclusions: Many scientists, regulators, and the general public believe that new and better ways to assess human toxicity are now needed, and technological breakthroughs are empowering the field of toxicity assessment. Even though the application of computational toxicology to environmental health decisions requires additional efforts, the merger of the power of computers with biological information is poised to deliver new tools and knowledge.
The potential for cardiotoxicity is carefully evaluated for pharmaceuticals, as it is a major safety liability. However, environmental chemicals are seldom tested for their cardiotoxic potential. ...Moreover, there is a large variability in both baseline and drug-induced cardiovascular risk in humans, but data are lacking on the degree to which susceptibility to chemically-induced cardiotoxicity may also vary. Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes have become an important in vitro model for drug screening. Thus, we hypothesized that a population-based model of iPSC-derived cardiomyocytes from a diverse set of individuals can be used to assess potential hazard and inter-individual variability in chemical effects on these cells. We conducted concentration-response screening of 134 chemicals (pharmaceuticals, industrial and environmental chemicals and food constituents) in iPSC-derived cardiomyocytes from 43 individuals, comprising both sexes and diverse ancestry. We measured kinetic calcium flux and conducted high-content imaging following chemical exposure, and utilized a panel of functional and cytotoxicity parameters in concentration-response for each chemical and donor. We show reproducible inter-individual variability in both baseline and chemical-induced effects on iPSC-derived cardiomyocytes. Further, chemical-specific variability in potency and degree of population variability were quantified. This study shows the feasibility of using an organotypic population-based human in vitro model to quantitatively assess chemicals for which little cardiotoxicity information is available. Ultimately, these results advance in vitro toxicity testing methodologies by providing an innovative tool for population-based cardiotoxicity screening, contributing to the paradigm shift from traditional animal models of toxicity to in vitro toxicity testing methods.
•Cardiotoxicity information is lacking for most environmental chemicals.•Cardiovascular disease risks vary greatly across the population.•We demonstrated that hiPSC-derived cardiomyocytes can address these gaps.•Potency and population variability in cardiotoxicity varied across chemicals.•This population-based model can substantially improve chemical safety evaluations.
Development of predictive in vitro assays for early toxicity evaluation is extremely important for improving the drug development process and reducing drug attrition rates during clinical ...development. High-content imaging-based in vitro toxicity assays are emerging as efficient tools for safety and efficacy testing to improve drug development efficiency. In this report we have used an induced pluripotent stem cell (iPSC)-derived hepatocyte cell model having a primary tissue-like phenotype, unlimited availability, and the potential to compare cells from different individuals. We examined a number of assays and phenotypic markers and developed automated screening methods for assessing multiparameter readouts of general and mechanism-specific hepatotoxicity. Endpoints assessed were cell viability, nuclear shape, average and integrated cell area, mitochondrial membrane potential, phospholipid accumulation, cytoskeleton integrity, and apoptosis. We assayed compounds with known mechanisms of toxicity and also evaluated a diverse hepatotoxicity library of 240 compounds. We conclude that high-content automated screening assays using iPSC-derived hepatocytes are feasible, provide information about mechanisms of toxicity, and can facilitate the safety assessment of drugs and chemicals.
•Trichloroethylene is mutagenic and carcinogenic in humans and animals.•Trichloroethylene undergoes both cytochrome P450- and glutathione-dependent metabolism.•Glutathione-derived metabolites of ...trichloroethylene are mutagenic.•Species-, sex-, and tissue-dependent differences exist in trichloroethylene metabolism.
Metabolism is critical for the mutagenicity, carcinogenicity, and other adverse health effects of trichloroethylene (TCE). Despite the relatively small size and simple chemical structure of TCE, its metabolism is quite complex, yielding multiple intermediates and end-products. Experimental animal and human data indicate that TCE metabolism occurs through two major pathways: cytochrome P450 (CYP)-dependent oxidation and glutathione (GSH) conjugation catalyzed by GSH S-transferases (GSTs). Herein we review recent data characterizing TCE processing and flux through these pathways. We describe the catalytic enzymes, their regulation and tissue localization, as well as the evidence for transport and inter-organ processing of metabolites. We address the chemical reactivity of TCE metabolites, highlighting data on mutagenicity of these end-products. Identification in urine of key metabolites, particularly trichloroacetate (TCA), dichloroacetate (DCA), trichloroethanol and its glucuronide (TCOH and TCOG), and N-acetyl-S-(1,2-dichlorovinyl)-l-cysteine (NAcDCVC), in exposed humans and other species (mostly rats and mice) demonstrates function of the two metabolic pathways in vivo. The CYP pathway primarily yields chemically stable end-products. However, the GST pathway conjugate S-(1,2-dichlorovinyl)glutathione (DCVG) is further processed to multiple highly reactive species that are known to be mutagenic, especially in kidney where in situ metabolism occurs. TCE metabolism is highly variable across sexes, species, tissues and individuals. Genetic polymorphisms in several of the key enzymes metabolizing TCE and its intermediates contribute to variability in metabolic profiles and rates. In all, the evidence characterizing the complex metabolism of TCE can inform predictions of adverse responses including mutagenesis, carcinogenesis, and acute and chronic organ-specific toxicity.
Angiogenesis is a complex process that is required for development and tissue regeneration and it may be affected by many pathological conditions. Chemicals and drugs can impact formation and ...maintenance of the vascular networks; these effects may be both desirable (e.g., anti-cancer drugs) or unwanted (e.g., side effects of drugs). A number of in vivo and in vitro models exist for studies of angiogenesis and endothelial cell function, including organ-on-a-chip microphysiological systems. An arrayed organ-on-a-chip platform on a 96-well plate footprint that incorporates perfused microvessels, with and without tumors, was recently developed and it was shown that survival of the surrounding tissue was dependent on delivery of nutrients through the vessels. Here we describe a technology transfer of this complex microphysiological model between laboratories and demonstrate that reproducibility and robustness of these tissue chip-enabled experiments depend primarily on the source of the endothelial cells. The model was highly reproducible between laboratories and was used to demonstrate the advantages of the perfusable vascular networks for drug safety evaluation. As a proof-of-concept, we tested Fluorouracil (1-1,000 μM), Vincristine (1-1,000 nM), and Sorafenib (0.1–100 μM), in the perfusable and non-perfusable micro-organs, and in a colon cancer-containing micro-tumor model. Tissue chip experiments were compared to the traditional monolayer cultures of endothelial or tumor cells. These studies showed that human in vitro vascularized micro-organ and micro-tumor models are reproducible organ-on-a-chip platforms for studies of anticancer drugs. The data from the 3D models confirmed advantages of the physiological environment as compared to 2D cell cultures. We demonstrated how these models can be translated into practice by verifying that the endothelial cell source and passage are critical elements for establishing a perfusable model.
Quantitative structure–activity relationship (QSAR) modeling and toxicogenomics are typically used independently as predictive tools in toxicology. In this study, we evaluated the power of several ...statistical models for predicting drug hepatotoxicity in rats using different descriptors of drug molecules, namely, their chemical descriptors and toxicogenomics profiles. The records were taken from the Toxicogenomics Project rat liver microarray database containing information on 127 drugs (http://toxico.nibio.go.jp/datalist.html). The model end point was hepatotoxicity in the rat following 28 days of continuous exposure, established by liver histopathology and serum chemistry. First, we developed multiple conventional QSAR classification models using a comprehensive set of chemical descriptors and several classification methods (k nearest neighbor, support vector machines, random forests, and distance weighted discrimination). With chemical descriptors alone, external predictivity (correct classification rate, CCR) from 5-fold external cross-validation was 61%. Next, the same classification methods were employed to build models using only toxicogenomics data (24 h after a single exposure) treated as biological descriptors. The optimized models used only 85 selected toxicogenomics descriptors and had CCR as high as 76%. Finally, hybrid models combining both chemical descriptors and transcripts were developed; their CCRs were between 68 and 77%. Although the accuracy of hybrid models did not exceed that of the models based on toxicogenomics data alone, the use of both chemical and biological descriptors enriched the interpretation of the models. In addition to finding 85 transcripts that were predictive and highly relevant to the mechanisms of drug-induced liver injury, chemical structural alerts for hepatotoxicity were identified. These results suggest that concurrent exploration of the chemical features and acute treatment-induced changes in transcript levels will both enrich the mechanistic understanding of subchronic liver injury and afford models capable of accurate prediction of hepatotoxicity from chemical structure and short-term assay results.
A human microfluidic four-cell liver acinus microphysiology system (LAMPS), was evaluated for reproducibility and robustness as a model for drug pharmacokinetics and toxicology. The model was ...constructed using primary human hepatocytes or human induced pluripotent stem cell (iPSC)-derived hepatocytes and 3 human cell lines for the endothelial, Kupffer and stellate cells. The model was tested in two laboratories and demonstrated to be reproducible in terms of basal function of hepatocytes, Terfenadine metabolism, and effects of Tolcapone (88 μM), Troglitazone (150 μM), and caffeine (600 μM) over 9 days in culture. Additional experiments compared basal outputs of albumin, urea, lactate dehydrogenase (LDH) and tumor necrosis factor (TNF)α, as well as drug metabolism and toxicity in the LAMPS model, and in 2D cultures seeded with either primary hepatocytes or iPSC-hepatocytes. Further experiments to study the effects of Terfenadine (10 μM), Tolcapone (88 μM), Trovafloxacin (150 μM with or without 1 μg/mL lipopolysaccharide), Troglitazone (28 μM), Rosiglitazone (0.8 μM), Pioglitazone (3 μM), and caffeine (600 μM) were carried out over 10 days. We found that both primary human hepatocytes and iPSC-derived hepatocytes in 3D culture maintained excellent basal liver function and Terfenadine metabolism over 10 days compared the same cells in 2D cultures. In 2D, non-overlay monolayer cultures, both cell types lost hepatocyte phenotypes after 48 h. With respect to drug effects, both cell types demonstrated comparable and more human-relevant effects in LAMPS, as compared to 2D cultures. Overall, these studies show that LAMPS is a robust and reproducible in vitro liver model, comparable in performance when seeded with either primary human hepatocytes or iPSC-derived hepatocytes, and more physiologically and clinically relevant than 2D monolayer cultures.
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