Evaluating scientific confidence in the concordance of in vitro and in vivo protective points of departure.

To fulfil the promise of reducing reliance on mammalian in vivo laboratory animal studies, new approach methods (NAMs) need to provide a confident basis for regulatory decision-making. However, previous attempts to develop in vitro NAMs-based points of departure (PODs) have yielded mixed results, with PODs from U.S. EPA's ToxCast, for instance, appearing more conservative (protective) but poorly correlated with traditional in vivo studies. Here, we aimed to address this discordance by reducing the heterogeneity of in vivo PODs, accounting for species differences, and enhancing the biological relevance of in vitro PODs. However, we only found improved in vitro-to-in vivo concordance when combining the use of Bayesian model averaging-based benchmark dose modeling for in vivo PODs, allometric scaling for interspecies adjustments, and human-relevant in vitro assays with multiple induced pluripotent stem cell-derived models. Moreover, the available sample size was only 15 chemicals, and the resulting level of concordance was only fair, with correlation coefficients <0.5 and prediction intervals spanning several orders of magnitude. Overall, while this study suggests several ways to enhance concordance and thereby increase scientific confidence in vitro NAMs-based PODs, it also highlights challenges in their predictive accuracy and precision for use in regulatory decision making.

Risk-based prioritization of PFAS using phenotypic and transcriptomic data from human induced pluripotent stem cell-derived hepatocytes and cardiomyocytes.

Per- and polyfluoroalkyl substances (PFAS) are chemicals with important applications; they are persistent in the environment and may pose human health hazards. Regulatory agencies are considering restrictions and bans of PFAS; however, little data exists for informed decisions. Several prioritization strategies were proposed for evaluation of potential hazards of PFAS. Structure-based grouping could expedite the selection of PFAS for testing; still, the hypothesis that structure-effect relationships exist for PFAS requires confirmation. We tested 26 structurally diverse PFAS from 8 groups using human-induced pluripotent stem cell-derived hepatocytes and cardiomyocytes, and tested concentration-response effects on cell function and gene expression. Few phenotypic effects were observed in hepatocytes, but negative chronotropy was observed for 8 of the 26 PFAS. Substance- and cell type-dependent transcriptomic changes were more prominent but lacked substantial group-specific effects. In hepatocytes, we found up-regulation of stress-related and extracellular matrix organization pathways, and down-regulation of fat metabolism. In cardiomyocytes, contractility-related pathways were most affected. We derived phenotypic and transcriptomic points of departure and compared them to predicted PFAS exposures. The conservative estimates for bioactivity and exposure were used to derive bioactivity-to-exposure ratio (BER) for each PFAS, most (23 of 26) PFAS had BER > 1. Overall, these data suggests that structure-based grouping of PFAS may not be sufficient to predict their biological effects. Testing of individual PFAS may be needed for scientific-based decision-making. Our proposed strategy of using two human cell types and considering phenotypic and transcriptomic effects, combined with dose-response analysis and calculation of BER, may be used for PFAS prioritization.

Per- and polyfluoroalkyl substances (PFAS) are man-made chemicals used in many products. However, most of these substances have not been tested for safety, and concerns exist that they may be harmful to human health and/or the environment. This study aimed to use human cell-based models to investigate if some of the PFAS may exhibit hazardous properties and if similarities among substances are observed. Few effects were observed in liver cells, but a decrease in beating frequency was observed in heart cells for some PFAS. Gene expression changes were substance- and cell type-dependent. We did not find convincing structure-based similarities among PFAS; this suggests that testing of individual PFAS may be necessary in the future to inform health decisions. Overall, this study showed that a test strategy of using two human cell types, from liver and heart, may inform PFAS prioritization without a need for testing in animals.

Arrestin domain-containing protein 1-mediated microvesicles (ARMMs) protect against cadmium-induced neurotoxicity.

Exposure to environmental heavy metals such as cadmium (Cd) is often linked to neurotoxicity but the underlying mechanisms remain poorly understood. Here we show that Arrestin domain-containing protein 1 (ARRDC1)-mediated microvesicles (ARMMs)--an important class of extracellular vesicles (EVs) whose biogenesis occurs at the plasma membrane--protect against Cd-induced neurotoxicity. Cd increased the production of EVs, including ARMMs, in a human neural progenitor cell line, ReNcell CX (ReN) cells. ReN cells that lack ARMMs production as a result of CRISPR-mediated ARRDC1 knockout were more susceptible to Cd toxicity as evidenced by increased LDH production as well as elevated level of oxidative stress markers. Importantly, adding ARMMs back to the ARRDC1-knockout ReN cells significantly reduced Cd-induced toxicity. Consistent with this finding, proteomics data showed that anti-oxidative stress proteins are enriched in ARMMs secreted from ReN cells. Together our study reveals a novel protective role of ARMMs in Cd neurotoxicity and suggests that ARMMs may be used therapeutically to reduce neurotoxicity caused by exposure to Cd and potentially other metal toxicants.

Role of Heme Oxygenase-1 in Dual Stress Response of Herbicide and Micronutrient Fe in Arabidopsis thaliana.

Increasingly intensive agricultural practices are leading not only to herbicide contamination but also to nutritional stress on nontarget plants. This study evaluated the role of heme oxygenase-1 (HO-1) in the dual stress response of herbicide dichlorprop and micronutrient Fe in Arabidopsis thaliana. Our results revealed that co-treatment with 20 µM zinc protoporphyrin (a specific inhibitor of HO-1) reduced the activity of HO-1 by 21.6%, Fe2+ content by 19.8%, and MDA content by 20.0%, reducing abnormal iron aggregation and oxidative stress in response to the herbicide compared to treatment with (R)-dichloroprop alone, which has herbicidal activity. Thus, free Fe2+ released from HO-1 mediated dichlorprop-induced oxidative stress in the Fenton reaction and affected aberrant Fe aggregation, which also had an enantioselective effect. This study contributes to an in-depth understanding of the toxicity mechanism of herbicides under nutrient stresses, thus providing new strategies to control the environmental risks of herbicides.

A Population-Based Human In Vitro Approach to Quantify Inter-Individual Variability in Responses to Chemical Mixtures.

Human cell-based population-wide in vitro models have been proposed as a strategy to derive chemical-specific estimates of inter-individual variability; however, the utility of this approach has not yet been tested for cumulative exposures in mixtures. This study aimed to test defined mixtures and their individual components and determine whether adverse effects of the mixtures were likely to be more variable in a population than those of the individual chemicals. The in vitro model comprised 146 human lymphoblastoid cell lines from four diverse subpopulations of European and African descent. Cells were exposed, in concentration−response, to 42 chemicals from diverse classes of environmental pollutants; in addition, eight defined mixtures were prepared from these chemicals using several exposure- or hazard-based scenarios. Points of departure for cytotoxicity were derived using Bayesian concentration−response modeling and population variability was quantified in the form of a toxicodynamic variability factor (TDVF). We found that 28 chemicals and all mixtures exhibited concentration−response cytotoxicity, enabling calculation of the TDVF. The median TDVF across test substances, for both individual chemicals or defined mixtures, ranged from a default assumption (101/2) of toxicodynamic variability in human population to >10. The data also provide a proof of principle for single-variant genome-wide association mapping for toxicity of the chemicals and mixtures, although replication would be necessary due to statistical power limitations with the current sample size. This study demonstrates the feasibility of using a set of human lymphoblastoid cell lines as an in vitro model to quantify the extent of inter-individual variability in hazardous properties of both individual chemicals and mixtures. The data show that population variability of the mixtures is unlikely to exceed that of the most variable component, and that similarity in genome-wide associations among components may be used to accrue additional evidence for grouping of constituents in a mixture for cumulative assessments.

Enantioselective Response of Wheat Seedlings to Imazethapyr: From the Perspective of Fe and the Secondary Metabolite DIMBOA.

The responses of trace elements and secondary metabolites to stress can reflect plant adaptation to the environment. If and how the imperative trace element Fe and the defensive secondary metabolite 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazine-3(4H)-one (DIMBOA) mediate the toxicity of chiral herbicides to nontarget plants remains inconclusive. We found that the herbicidal-active imazethapyr enantiomer [(R)-IM] stimulated heme oxygenase-1 activity, triggered the release of the catalytic product Fe2+, increased reactive oxygen species production, decreased the DIMBOA content, and increased the DIMBOA-Fe content. XAFS analyses and in vitro Fenton assays demonstrated that DIMBOA could relieve phytotoxicity by chelating excessive Fe3+ to restore Fe homeostasis. The free radical scavenging ability of the chelate of DIMBOA and Fe was also involved. This work refines the dual role of DIMBOA and Fe in mediating the enantioselective phytotoxicity of chiral herbicides, which provides a new direction for improving the herbicide resistance of crops.

Chemical and biological assessments of environmental mixtures: A review of current trends, advances, and future perspectives.

Considering the chemical complexity and toxicity data gaps of environmental mixtures, most studies evaluate the chemical risk individually. However, humans are usually exposed to a cocktail of chemicals in real life. Mixture health assessment remains to be a research area having significant knowledge gaps. Characterization of chemical composition and bioactivity/toxicity are the two critical aspects of mixture health assessments. This review seeks to introduce the recent progress and tools for the chemical and biological characterization of environmental mixtures. The state-of-the-art techniques include the sampling, extraction, rapid detection methods, and the in vitro, in vivo, and in silico approaches to generate the toxicity data of an environmental mixture. Application of these novel methods, or new approach methodologies (NAMs), has increased the throughput of generating chemical and toxicity data for mixtures and thus refined the mixture health assessment. Combined with computational methods, the chemical and biological information would shed light on identifying the bioactive/toxic components in an environmental mixture.

Molecular mechanisms of environmental toxin cadmium at the feto-maternal interface investigated using an organ-on-chip (FMi-OOC) model.

Human labor is associated with feto-maternal-derived signals that coordinate to initiate delivery. Exposure to environmental chemicals can prematurely trigger labor-initiating signals at the feto-maternal interface (FMi: decidua, amniochorion), leading to spontaneous preterm birth (PTB). Testing the association between environmental chemical exposure and PTB is difficult due to many limitations in vivo or in vitro. Physiological organ-on-chips (OOCs) are potential alternatives for studying mechanisms leading to PTB. The presented study tested the effect of maternal exposure to cadmium (Cd), an environmental toxin, using the FMi-OOC that incorporates maternal decidua cells and three different fetal cells (chorion, amnion mesenchymal, and amnion epithelial cells). Cd transport through the FMi and its impact on cell cycle, cell death, and inflammation were analyzed. Cd treatment resulted in significant cell death and a pro-inflammatory environment in the maternal decidua, but had minimal effect on the fetal chorion cells, and no effect in the fetal amnion cells compared to controls. The maternal response, but lack of fetal response, indicates that Cd-mediated adverse effects originate from maternal pathophysiology rather than fetal-derived triggers of preterm labor. This study demonstrates that the FMi-OOC can indeed predict the response of FMi upon exposure to chemicals, opening the possibility for using OOC models for environmental toxin screens.

Potential Human Health Hazard of Post-Hurricane Harvey Sediments in Galveston Bay and Houston Ship Channel: A Case Study of Using In Vitro Bioactivity Data to Inform Risk Management Decisions.

Natural and anthropogenic disasters may be associated with redistribution of chemical contaminants in the environment; however, current methods for assessing hazards and risks of complex mixtures are not suitable for disaster response. This study investigated the suitability of in vitro toxicity testing methods as a rapid means of identifying areas of potential human health concern. We used sediment samples (n = 46) from Galveston Bay and the Houston Ship Channel (GB/HSC) areas after hurricane Harvey, a disaster event that led to broad redistribution of chemically-contaminated sediments, including deposition of the sediment on shore due to flooding. Samples were extracted with cyclohexane and dimethyl sulfoxide and screened in a compendium of human primary or induced pluripotent stem cell (iPSC)-derived cell lines from different tissues (hepatocytes, neuronal, cardiomyocytes, and endothelial) to test for concentration-dependent effects on various functional and cytotoxicity phenotypes (n = 34). Bioactivity data were used to map areas of potential concern and the results compared to the data on concentrations of polycyclic aromatic hydrocarbons (PAHs) in the same samples. We found that setting remediation goals based on reducing bioactivity is protective of both "known" risks associated with PAHs and "unknown" risks associated with bioactivity, but the converse was not true for remediation based on PAH risks alone. Overall, we found that in vitro bioactivity can be used as a comprehensive indicator of potential hazards and is an example of a new approach method (NAM) to inform risk management decisions on site cleanup.

New Target in an Old Enemy: Herbicide (R)-Dichlorprop Induces Ferroptosis-like Death in Plants.

Iron is an essential microelement in plants that is involved in several growth processes. The use of herbicides may cause the abnormal aggregation of iron in leaves, but the regulatory mechanisms underlying this phenomenon remain unclear. Here, we show that chiral herbicide (R)-dichlorprop ((R)-DCPP) triggers ferroptosis-like death in Arabidopsis thaliana. (R)-DCPP led to reactive oxygen species (ROS) accumulation and iron aggregation, and these processes were iron dependent. Under (R)-DCPP treatment, ROS, lipid hydrogen peroxides, and malondialdehyde were significantly accumulated. In addition, (R)-DCPP induced the depletion of glutathione, ascorbic acid, and glutathione peroxidase as well as the accumulation of toxic lipid peroxides. Thus, oxidation imbalance led to cell death, and this mode of action could be inhibited by the ferroptosis inhibitor ferrostatin-1 or ciclopirox olamine. NADPH oxidases were found to be involved in herbicide-induced ROS accumulation, and lipoxygenase and NADPH cytochrome P450 oxidase were shown to positively regulate (R)-DCPP-induced lipid peroxidation. Overall, these results indicate that the iron- and ROS-dependent signaling cascades were involved in the (R)-DCPP-induced phytotoxicity pathway, which disrupted the structure of plant cell membranes and triggered ferroptosis. Generally, this study provides new insight into the mechanisms of pesticide phytotoxicity and suggests new therapeutic directions to protect nontarget plants.

Quantitative In Vitro-to-In Vivo Extrapolation for Mixtures: A Case Study of Superfund Priority List Pesticides.

In vitro cell-based toxicity testing methods generate large amounts of data informative for risk-based evaluations. To allow extrapolation of the quantitative outputs from cell-based tests to the equivalent exposure levels in humans, reverse toxicokinetic modeling is used to conduct in vitro-to-in vivo extrapolation (IVIVE) from in vitro effective concentrations to in vivo oral dose equivalents. IVIVE modeling approaches for individual chemicals are well-established; however, the potential implications of chemical-to-chemical interactions in mixture settings on IVIVE remain largely unexplored. We hypothesized that chemical coexposures could modulate both protein binding efficiency and hepatocyte clearance of the chemicals in a mixture, which would in turn affect the quantitative IVIVE toxicokinetic parameters. To test this hypothesis, we used 20 pesticides from the Agency for Toxic Substances and Disease Registry Substance Priority List, both individually and as equimolar mixtures, and investigated the concentration-dependent effects of chemical interactions on in vitro toxicokinetic parameters. Plasma protein binding efficiency was determined by using ultracentrifugation, and hepatocyte clearance was estimated in suspensions of cryopreserved primary human hepatocytes. We found that for single chemicals, the protein binding efficiencies were similar at different test concentrations. In a mixture, however, both protein binding efficiency and hepatocyte clearance were affected. When IVIVE was conducted using mixture-derived toxicokinetic data, more conservative estimates of activity-to-exposure ratios were produced as compared with using data from single chemical experiments. Because humans are exposed to mixtures of chemicals, this study is significant as it demonstrates the importance of incorporating mixture-derived parameters into IVIVE for in vitro bioactivity data in order to accurately prioritize risks and facilitate science-based decision-making.

Relationships between constituents of energy drinks and beating parameters in human induced pluripotent stem cell (iPSC)-Derived cardiomyocytes.

Consumption of energy drinks has been associated with adverse cardiovascular effects; however, little is known about the ingredients that may contribute to these effects. We therefore characterized the chemical profiles and in vitro effects of energy drinks and their ingredients on human induced pluripotent stem cell (iPSC)-derived cardiomyocytes, and identified the putative active ingredients using a multivariate prediction model. Energy drinks from 17 widely-available over-the-counter brands were evaluated in this study. The concentrations of six common ingredients (caffeine, taurine, riboflavin, pantothenic acid, adenine, and L-methionine) were quantified by coupling liquid chromatography with a triple quadrupole mass spectrometer for the acquisition of LC-MS/MS spectra. In addition, untargeted analyses for each beverage were performed with a platform combining LC, ion mobility spectrometry and mass spectrometry (LC-IMS-MS) measurements. Approximately 300 features were observed across samples in the untargeted studies, and of these ~100 were identified. In vitro effects of energy drinks and some of their ingredients were then tested in iPSC-derived cardiomyocytes. Data on the beat rate (positive and negative chronotropy), ion channel function (QT prolongation), and cytotoxicity were collected in a dilution series. We found that some of the energy drinks elicited adverse effects on the cardiomyocytes with the most common being an increase in the beat rate, while QT prolongation was also observed at the lowest concentrations. Finally, concentration addition modeling using quantitative data from the 6 common ingredients and multivariate prediction modeling was used to determine potential ingredients responsible for the adverse effects on the cardiomyocytes. These analyses suggested theophylline, adenine, and azelate as possibly contributing to the in vitro effects of energy drinks on QT prolongation in cardiomyocytes.

Risk Characterization and Probabilistic Concentration-Response Modeling of Complex Environmental Mixtures Using New Approach Methodologies (NAMs) Data from Organotypic in Vitro Human Stem Cell Assays.

BACKGROUND: Risk assessment of chemical mixtures or complex substances remains a major methodological challenge due to lack of available hazard or exposure data. Therefore, risk assessors usually infer hazard or risk from data on the subset of constituents with available toxicity values. OBJECTIVES: We evaluated the validity of the widely used traditional mixtures risk assessment paradigms, Independent Action (IA) and Concentration Addition (CA), with new approach methodologies (NAMs) data from human cell-based in vitro assays. METHODS: A diverse set of 42 chemicals was tested both individually and as mixtures for functional and cytotoxic effects in vitro. A panel of induced pluripotent stem cell (iPSCs)-derived models (hepatocytes, cardiomyocytes, endothelial, and neurons) and one primary cell type (HUVEC) were used. Bayesian concentration-response modeling of individual chemicals or their mixtures was performed for a total of 47 phenotypes to derive point-of-departure (POD) values. Probabilistic IA or CA was conducted to estimate the mixture effects based on the bioactivity profiles from the individual chemicals and compared with mixture bioactivity. RESULTS: All mixtures showed significant bioactivity, even though some were constructed using individual chemical concentrations considered "low" or "safe." Even though CA is much more accurate as a predictor of mixture effects in comparison with IA, with CA-based POD typically within an order of magnitude of the actual mixture, in some cases, the bioactivity of the mixtures appeared to be much greater than that of their components under either additivity assumption. DISCUSSION: These results suggest that CA is a preferred first approximation for predicting mixture toxicity when data for all constituents are available. However, because the accuracy of additivity assumptions varies greatly across phenotypes, we posit that mixtures and complex substances need to be directly tested for their hazard potential. NAMs provide a practical solution that rapidly yields highly informative data for mixtures risk assessment. https://doi.org/10.1289/EHP7600.

Enantioselective effects of imazethapyr on the secondary metabolites and nutritional value of wheat seedlings.

The secondary metabolism of plants is key for mediating responses to environmental stress, but few studies have examined how the relationship between secondary metabolism and the stress response of plants is affected by exposure to chiral herbicides. Here, we studied the enantioselective disturbance of the chiral herbicide imazethapyr (IM) on the secondary metabolism and nutrient levels of wheat seedlings. The bioactive enantiomer R-IM significantly increased the contents of major secondary metabolites, including phenolic acids, flavonoids, and carotenoids but greatly inhibited the production of benzoxazine. The antioxidant system also responded strongly to R-IM; specifically, the activities of SOD, CAT, and GPX enzymes were all significantly induced, and the GSH content initially increased but then decreased. Furthermore, the nutrient levels of wheat seedlings were also affected; dietary fiber content decreased, while the contents of the microelements Fe, Mn, and Zn increased. In sum, this study provides new insight into the phytotoxic effects of IM and raises new questions on the role of secondary metabolites and nutrients in mediating enantioselective effects.

Testing the efficacy of broad-acting sorbents for environmental mixtures using isothermal analysis, mammalian cells, and H. vulgaris.

The hazards associated with frequent exposure to polycyclic aromatic hydrocarbons (PAHs), pesticides, Aroclors, plasticizers, and mycotoxins are well established. Adsorption strategies have been proposed for the remediation of soil and water, although few have focused on the mitigation of mixtures. This study tested a hypothesis that broad-acting sorbents can be developed for diverse chemical mixtures. Adsorption of common and hazardous chemicals was characterized using isothermal analysis from Langmuir and Freundlich equations. The most effective sorbents included medical-grade activated carbon (AC), parent montmorillonite clay, acid-processed montmorillonite (APM), and nutrient-amended montmorillonite clays. Next, we tested the ability of broad-acting sorbents to prevent cytotoxicity of class-specific mixtures using 3 mammalian in vitro models (HLF, ESD3, and 3T3 cell lines) and the hydra assay. AC showed the highest efficacy for mitigating pesticides, plasticizers, PAHs, and mycotoxins. Clays, such as APM, were effective against pesticides, Aroclors, and mycotoxins, while amended clays were most effective against plasticizers. Finally, a sorbent mixture was shown to be broadly active. These results are supported by the high correlation coefficients for the Langmuir model with high capacity, affinity, and free energy, as well as the significant protection of cells and hydra (p < 0.05). The protection percentages in 3T3 cells and hydra showed the highest correlation as suggested by both Pearson and Spearman with r = 0.84 and rho = 0.73, respectively (p < 0.0001). Collectively, these studies showed that broad-acting sorbents may be effective in preventing toxic effects of chemical mixtures and provided information on the most effective sorbents based on adsorption isotherms, and in vitro and aquatic organism test methods.

Risk Characterization of Environmental Samples Using In Vitro Bioactivity and Polycyclic Aromatic Hydrocarbon Concentrations Data.

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.

Human in vitro vascularized micro-organ and micro-tumor models are reproducible organ-on-a-chip platforms for studies of anticancer drugs.

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.

Rapid hazard characterization of environmental chemicals using a compendium of human cell lines from different organs

The lack of adequate toxicity data for the vast majority of chemicals in the environment has spurred the development of new approach methodologies (NAMs). This study aimed to develop a practical high-throughput in vitro model for rapidly evaluating potential hazards of chemicals using a small number of human cells. Forty-two compounds were tested using human induced pluripotent stem cell (iPSC)-derived cells (hepatocytes, neurons, cardiomyocytes and endothelial cells), and a primary endothelial cell line. Both functional and cytotoxicity endpoints were evaluated using high-content imaging. Concentration-response was used to derive points-of-departure (POD). PODs were integrated with ToxPi and used as surrogate NAM-based PODs for risk characterization. We found chemical class-specific similarity among the chemicals tested; metal salts exhibited the highest overall bioactivity. We also observed cell type-specific patterns among classes of chemicals, indicating the ability of the proposed in vitro model to recognize effects on different cell types. Compared to available NAM datasets, such as ToxCast/Tox21 and chemical structure-based descriptors, we found that the data from the five-cell-type model was as good or even better in assigning compounds to chemical classes. Additionally, the PODs from this model performed well as a conservative surrogate for regulatory in vivo PODs and were less likely to underestimate in vivo potency and potential risk compared to other NAM-based PODs. In summary, we demonstrate the potential of this in vitro screening model to inform rapid risk-based decision-making through ranking, clustering, and assessment of both hazard and risks of diverse environmental chemicals.

Disturbance of chiral ionic liquids to phototaxis of Chlamydomonas reinhardtii: regular analysis and mechanism attempt.

Given the recent extensive synthesis and application of ionic liquids (ILs), finding a sensitive and visual indicator to provide a fast-initial risk assessment of IL use has become a pressing issue. In this study, we verified that the phototaxis of Chlamydomonas reinhardtii is a valid indicator of the environmental risk associated with chiral ILs L-(+)- and D-(-)-1-butyl-3-methylimidazolium lactate (BMIM L). Briefly, C. reinhardtii was exposed to a 4000-lx side light source for varying lengths of time. Following the allotted exposure time, the algae aggregation was photographed, and then quantitative analysis was conducted using Image-J software to obtain the corresponding relationship between IL stimulation and C. reinhardtii phototaxis. The gray areas from each treatment were measured and the percentage was calculated. After 16 h of side lighting, for control, the percentage of gray areas was - 22%, while for L-(+)- and D-(-)- BMIM L were 17% and 33%, respectively. Then, after 8 h of darkness, where D-(-)-BMIM L and the control showed the positive phototaxis, but the L-(+)-BMIM L-treated group showed virtually no change. This phenomenon is consistent with excessive production of reactive oxygen species (ROS). Moreover, atomic force microscope (AFM) results indicated distinct aggregation between D-(-)- and L-(+)-BMIM L, which caused changes in cell permeability that induced a change in ROS transfer. Furthermore, relationship between phototaxis and changes in cell ultrastructure and photosynthetic efficiency was also investigated. This work demonstrates the potential of phototaxis to serve as a sensitive, convenient, and cost effective qualitative assessment of ILs' toxic impact, with the understanding that quantitative evaluation requires further improvement.

Tissue-Engineered Bone Tumor as a Reproducible Human in Vitro Model for Studies of Anticancer Drugs.

Studies of anticancer therapies in traditional cell culture models can demonstrate efficacy of direct-acting compounds but lack the 3-dimensional arrangement of the tumor cells and their tissue-specific microenvironments, both of which are important modulators of treatment effects in vivo. Bone cells reside in complex environments that regulate their fate and function. A bioengineered human bone-tumor model has been shown to provide a microphysiological niche for studies of cancer cell behavior. Here, we demonstrate successful transfer between 2 laboratories and utility of this model in efficacy studies using well-established chemotherapeutic agents. The bioengineered human bone-tumor model consisted of Ewing sarcoma (RD-ES) cancer cell aggregates infused into tissue-engineered bone that was grown from human mesenchymal stem cell-derived differentiated into osteoblasts within mineralized bone scaffolds. The tumor model was maintained in culture for over 5 weeks and subjected to clinically relevant doses of linsitinib, doxorubicin, cisplatin, methotrexate, vincristine, dexamethasone, or MAP (methotrexate, doxorubicin, and cisplatin combination). Drug administration cycles were designed to mimic clinical treatment regimens. The bioengineered tumors were evaluated days to weeks after the cessation of treatment to monitor the potential for relapse, using bioengineered bone and ES cell monolayers as controls. Drug binding to the scaffolds and media proteins and gene expression were also evaluated. We show that a bioengineered human bone tumor can be used as a microphysiological model for preclinical studies of anticancer drugs. We found that anticancer efficacy was achieved at concentrations approximating the human Cmax, in contrast to traditional ES cell monolayers. These studies show that the bone-tumor model can be successfully transferred between laboratories and has predictive power in preclinical studies. The effects of drugs on the bone tumors and healthy bone were studied in parallel, in support of the utility of this model for identification of new therapeutic targets.