Proteomic analysis using isobaric tags for relative and absolute quantification technology reveals mechanisms of toxic effects of tris (1,3-dichloro-2-propyl) phosphate on RAW264.7 macrophage cells.

Tris (1,3-dichloro-2-propyl) phosphate (TDCIPP) is one of the most commonly used organophosphorus flame retardants. Immuno-toxicity induced by TDCIPP is becoming of increasing concern. However, effects of TDCIPP on immune cells and mechanisms resulting in those effects are poorly understood. In this study, it was determined, for the first time, by use of isobaric tags for relative and absolute quantification (iTRAQ) based proteomic techniques expression of global proteins in RAW264.7 cells exposed to 10 µM TDCIPP. A total of 180 significantly differentially expressed proteins (DEPs) were identified. Of these, 127 were up-regulated and 53 were down-regulated. The DEPs associated with toxic effects of TDCIPP were then screened by use of Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes for enrichment analysis. Results showed that these DEPs were involved in a number of pathways including apoptosis, DNA damage, cell cycle arrest, immune-toxicity, and signaling pathways, such as the Toll-like receptor, PPAR and p53 signaling pathways. The complex regulatory relationships between different DEPs, which might play an important role in cell death were also observed in the form of a protein-protein interaction network. Meanwhile, mitochondrial membrane potential (MMP) in RAW264.7 cells after TDCIPP treatment was also analyzed, the collapse of the MMP was speculated to play an important role in TDCIPP induced apoptosis. Moreover, some of the important regulator proteins discovered in this study, such as Chk1, Aurora A, would provide novel insight into the molecular mechanisms involved in toxic responses to TDCIPP.

Establishment of an in vitro method of rabbit embryo toxicity with toxicokinetics study.

This report introduces a novel method, rabbit whole embryo culture (WEC) combined with toxicokinetics (TK), for toxicity testing. Rodent WEC has been extensively used for in vitro screening of developmental toxicity. To improve the reliability of in vitro data, it is important to consider TK and species specificity. To test the utility and effectiveness of this method, we investigated the toxic effect of thalidomide on rabbit embryos and its behavior in test systems both in vitro and in vivo under the same experimental condition. The data showed that thalidomide induced embryo malformations such as embryonic brain hypoplasia, short limb buds, and declined embryonic growth both in vitro and in vivo. The toxic effect increased with the increasing exposure of the embryo to thalidomide. In addition, we observed similar toxic effects and exposure-effect relationships in vivo and in vitro. Therefore, we preliminarily conclude that this new method can effectively predict and explain thalidomide toxicity. Furthermore, we investigated the behavior of test compounds in the WEC system for the first time, and this method is expected to be an important technique for in vitro toxicity study after extensive verification.

Development and validation of dual-cardiotoxicity evaluation method based on analysis of field potential and contractile force of human iPSC-derived cardiomyocytes / multielectrode assay platform.

A recent in vitro cardiovascular safety pharmacology test uses cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) to overcome the limitations of the classical test systems, such as species differences and local channel analysis. The Comprehensive in vitro Proarrhythmia Assay (CiPA) is a new proarrhythmia screening paradigm proposed by a CiPA steering expert group, which essentially requires iPSCs derived cardiomyocyte-based electrophysiological evaluation technology. Moreover, the measurement of the contractile force is also emerging as an important parameter to recapitulate non-proarrhythmic cardiotoxicity. Therefore, we constructed an multielectrode assay (MEA) evaluation method that can measure the electrophysiological changes with 6 reference drugs in hiPSC-derived cardiomyocytes. Subsequently, it was confirmed that the electrophysiological were changed in accordance with the mechanism of action of the drugs. Furthermore, based on the multi-probe impedance, we confirmed the decrease in contractile force due to treatment with drugs, and developed a platform to evaluate cardiotoxicity according to drugs along with field potential changes. Our excitation-contraction coupling cardiotoxicity assessment is considered to be more supportive in cardiac safety studies on pharmacologic sensitivity by complementing each assessment parameter.

Improving reliability and reducing costs of cardiotoxicity assessments using laser-induced cell poration on microelectrode arrays.

Drug-induced cardiotoxicity is a major barrier to drug development and a main cause of withdrawal of marketed drugs. Drugs can strongly alter the spontaneous functioning of the heart by interacting with the cardiac membrane ion channels. If these effects only surface during in vivo preclinical tests, clinical trials or worse after commercialization, the societal and economic burden will be significant and seriously hinder the efficient drug development process. Hence, cardiac safety pharmacology requires in vitro electrophysiological screening assays of all drug candidates to predict cardiotoxic effects before clinical trials. In the past 10 years, microelectrode array (MEA) technology began to be considered a valuable approach in pharmaceutical applications. However, an effective tool for high-throughput intracellular measurements, compatible with pharmaceutical standards, is not yet available. Here, we propose laser-induced optoacoustic poration combined with CMOS-MEA technology as a reliable and effective platform to detect cardiotoxicity. This approach enables the acquisition of high-quality action potential recordings from large numbers of cardiomyocytes within the same culture well, providing reliable data using single-well MEA devices and single cardiac syncytia per each drug. Thus, this technology could be applied in drug safety screening platforms reducing times and costs of cardiotoxicity assessments, while simultaneously improving the data reliability.

Dynamic microfluidic bioreactor-Hip simulator (DMBH) system for implant toxicity monitoring.

The generation of degradation products (DPs) like ions and organo-metallic particles from corroding metallic implants is an important healthcare concern. These DPs generate local and systemic toxicity. The impact on local toxicity is well documented, however, little is known about systemic toxicity. This is mainly due to the limited scope of the current microtiter plate-based (static) toxicity assay techniques. These methods do not mimic the systemic (dynamic) conditions. In this study, it is hypothesized that DPs incubated with cells in static conditions might provide improper systemic toxicity results, as there is no movement mimicking the blood circulation around cells. This study reports the development of a three-chambered prototype microfluidic system connected to the operational hip implant simulator to test the cellular response induced by the DPs. This setup is called a dynamic microfluidic bioreactor-hip simulator system. We hypothesize that a dynamic microfluidic system will provide a realistic toxicology response induced by DPs than a static cell culture plate. To prove the hypothesis, Neuro2a (N2a) cells were used as representative cells to study systemic neurotoxicity by the implant DPs. The microfluidic bioreactor system was validated by comparing the cell toxicity against the traditional static system and using COMSOL modeling for media flow with DPs. The hip implant simulator used in this study was a state-of-the-art sliding hip simulator developed in our lab. The results suggested that static toxicity was significantly more compared to dynamic microfluidic-based toxicity. The newly developed DMBH system tested for in situ systemic toxicity on N2a cells and demonstrated very minimum toxicity level (5.23%) compared to static systems (31.16%). Thus, the new DMBH system is an efficient tool for in situ implant metal systemic toxicity testing.

A novel smartphone-based electrochemical cell sensor for evaluating the toxicity of heavy metal ions Cd2+, Hg2+, and Pb2+ in rice.

A novel smartphone-based electrochemical cell sensor was developed to evaluate the toxicity of heavy metal ions, such as cadmium (Cd2+), lead (Pb2+), and mercury (Hg2+) ions on Hep G2 cells. The cell sensor was fabricated with reduced graphene oxide (RGO)/molybdenum sulfide (MoS2) composites to greatly improve the biological adaptability and amplify the electrochemical signals. Differential pulse voltammetry (DPV) was employed to measure the electrical signals induced by the toxicity of heavy metal ions. The results showed that Cd2+, Hg2+, and Pb2+ significantly reduced the viability of Hep G2 cells in a dose-dependent manner. The IC50 values obtained by this method were 49.83, 36.94, and 733.90 µM, respectively. A synergistic effect was observed between Cd2+ and Pb2+ and between Hg2+ and Pb2+, and an antagonistic effect was observed between Cd2+ and Hg2+, and an antagonistic effect at low doses and an additive effect at high doses were found in the ternary mixtures of Cd2+, Hg2+, and Pb2+. These electrochemical results were confirmed via MTT assay, SEM and TEM observation, and flow cytometry. Therefore, this new electrochemical cell sensor provided a more convenient, sensitive, and flexible toxicity assessment strategy than traditional cytotoxicity assessment methods.

Assessing how in vitro assay types predict in vivo toxicology data.

In vivo animal bioassays are increasingly being supplemented with in vitro assays to serve as the new standard for chemical toxicity tests. Despite this shift, investigators face challenges related to increased reliance on in vitro data. The aim of this study was to deploy a streamlined method to assess the ability of in vitro data to predict similar results as in vivo data by correlating chemical toxicity rankings obtained using Benchmark Doses and Benchmark Dose Lower Limits (BMD(L)s) derived from in vivo and in vitro assays. In vitro and in vivo assay characteristics were assessed for their impact on the predictive ability of in vitro data. Minimum best-fit BMD(L)s were calculated for chemicals using Environmental Protection Agency's (EPA's) Benchmark Dose Software (BMDS). Forty-one chemicals met the inclusion criteria of this study. Relative chemical toxicity rankings were assessed through Kappa statistics, Pearson correlations, and/or Ordinary Least Squares (OLS) regressions. Results illustrated likely ability of in vitro data to predict similar results as short-term in vivo data. Further, rankings derived from in vitro cytotoxicity assays, unlike stress response assays, significantly correlated with rankings derived from short-term in vivo assays. These results support the use of in vitro data as a prioritization tool within toxicity testing.

Drug Evaluation Based on a Multi-Channel Cell Chip with a Horizontal Co-Culture.

We developed a multi-channel cell chip containing a three-dimensional (3D) scaffold for horizontal co-culture and drug toxicity screening in multi-organ culture (human glioblastoma, cervical cancer, normal liver cells, and normal lung cells). The polydimethylsiloxane (PDMS) multi-channel cell chip (PMCCC) was based on fused deposition modeling (FDM) technology. The architecture of the PMCCC was an open-type cell chip and did not require a pump or syringe. We investigated cell proliferation and cytotoxicity by conducting 3-(4,5-dimethylthiazol-2-yl)-2,5-dphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays and analysis of oleanolic acid (OA)-treated multi-channel cell chips. The results of the MTT and LDH assays showed that OA treatment in the multi-channel cell chip of four cell lines enhanced chemoresistance of cells compared with that in the 2D culture. Furthermore, we demonstrated the feasibility of the application of our multi-channel cell chip in various analysis methods through Annexin V-fluorescein isothiocyanate/propidium iodide staining, which is not used for conventional cell chips. Taken together, the results demonstrated that the PMCCC may be used as a new 3D platform because it enables simultaneous drug screening in multiple cells by single point injection and allows analysis of various biological processes.

Modulation of Cellular Reactivity for Enhanced Cell-Based Biosensing.

Cell-based sensing platforms provide functional information on cellular effects of bioactive or toxic compounds in a sample. Current challenges concern the rather extended length of the assays as well as their limited reproducibility and sensitivity. We present a biosensing method capable of appraising, on a short time scale and with exquisite sensitivity, the occurrence and the magnitude of cellular alterations induced by low levels of a bioactive/toxic compound. Our method is based on integrating optogenetic control of non-electrogenic human cells, modified to express light sensitive protein channels, into a non-invasive electro-optical analytical platform enabling quantitative assessment of the stimulus dependent, dynamical cellular response. Our system exploits the interplay between optogenetic stimulation and time lapse fast impedance assays in boosting the platform sensitivity when exposing cells to a model exogenous stimulus, under both static and flow conditions. The proposed optogenetically modulated cell-based sensing platform is suitable for in field applications and provides a new paradigm for impedance-based sensing.

Organic Electrochemical Transistor for in Situ Detection of H2O2 Released from Adherent Cells and Its Application in Evaluating the In Vitro Cytotoxicity of Nanomaterial.

Detection of hydrogen peroxide (H2O2) produced by living cells is very significant to fully understand its roles in cellular physiology, as well as providing reliable diagnosis of pathological conditions. However, in situ detection of H2O2 released from adherent cells in cellular culture medium is still insufficiently achieved. Here, we report an electrochemical platform for in situ detection of H2O2 produced by adherent cells in cellular culture medium. It is based on the use of organic electrochemical transistor (OECT) fabricated on a flexible poly(ethylene terephthalate) substrate and Transwell support. A screen-printed carbon paste electrode was modified with carbon nanotubes and platinum nanoparticles and served as the gate of the device. Under optimal conditions, this device exhibits good modulation and sensitivity. It works in the 0.5 µM to 0.1 mM H2O2 concentration range and has a 0.2 µM detection limit. The cells were seeded and grew on the Transwell membrane. Upon being stimulated by N-formylmethionyl-leucyl-phenylalanine peptide, H2O2 produced by the adherent cells diffused into the bottom chamber of the Transwell and was in situ detected by OECT. Moreover, evaluating in vitro cytotoxicity of the nanomaterial using the OECT-Transwell platform was realized. This simple electrochemical platform would be of great interest for in vitro cytotoxicity, cellular physiology study, and diagnosis of pathological conditions.

Multiorgan microfluidic platform with breathable lung chamber for inhalation or intravenous drug screening and development.

Efficient and economical delivery of pharmaceuticals to patients is critical for effective therapy. Here we describe a multiorgan (lung, liver, and breast cancer) microphysiological system ("Body-on-a-Chip") designed to mimic both inhalation therapy and/or intravenous therapy using curcumin as a model drug. This system is "pumpless" and self-contained using a rocker platform for fluid (blood surrogate) bidirectional recirculation. Our lung chamber is constructed to maintain an air-liquid interface and contained a "breathable" component that was designed to mimic breathing by simulating gas exchange, contraction and expansion of the "lung" using a reciprocating pump. Three cell lines were used: A549 for the lung, HepG2 C3A for the liver, and MDA MB231 for breast cancer. All cell lines were maintained with high viability (>85%) in the device for at least 48 hr. Curcumin is used to treat breast cancer and this allowed us to compare inhalation delivery versus intravenous delivery of the drug in terms of effectiveness and potentially toxicity. Inhalation therapy could be potentially applied at home by the patient while intravenous therapy would need to be applied in a clinical setting. Inhalation therapy would be more economical and allow more frequent dosing with a potentially lower level of drug. For 24 hr exposure to 2.5 and 25 µM curcumin in the flow device the effect on lung and liver viability was small to insignificant, while there was a significant decrease in viability of the breast cancer (to 69% at 2.5 µM and 51% at 25 µM). Intravenous delivery also selectively decreased breast cancer viability (to 88% at 2.5 µM and 79% at 25 µM) but was less effective than inhalation therapy. The response in the static device controls was significantly reduced from that with recirculation demonstrating the effect of flow. These results demonstrate for the first time the feasibility of constructing a multiorgan microphysiological system with recirculating flow that incorporates a "breathable" lung module that maintains an air-liquid interface.

Whole body electronic cigarette exposure system for efficient evaluation of diverse inhalation conditions and products.

Objectives: To develop and test a new system for whole body exposure of small animals to support investigation of the biological effects of aerosol generated by electronic cigarette (e-cig) products under diverse inhalation conditions with improved control and monitoring of the e-cig vape exposure and nicotine delivered to the animal's systemic circulation. Methods: A computer-controlled design, with built-in sensors for real time monitoring of O2, CO2, relative humidity, and temperature within the exposure chambers and port for measuring total particulate matter (TPM) was developed, constructed and tested. This design accommodates a variety of commercial vaping devices, offers software flexibility to adjust exposure protocols to mimic different users' puffing patterns, enables variable nicotine delivery to the animal's systemic circulation; minimizes travel time and alterations of aerosol quality or quantity by delivering aerosol directly to the exposure chamber, offers local or remote operation of up to six distinct exposure chambers from a single control unit, and can simultaneously test different exposure conditions or products in diverse animal groups, which reduces inter-run variability, saves time, and increases productivity. Results: The time course pattern of TPM concentration during different phases of the exposure cycle was measured. With increased puffing duration or number of exposure cycles, higher TPM exposure and plasma cotinine levels were observed with plasma cotinine levels in the range reported in light or heavy smokers. Conclusion: Overall, this novel, versatile, and durable exposure system facilitates high-throughput evaluation of the relative safety and potential toxicity of a variety of e-cig devices and liquids.

Cardiotoxicity screening of illicit drugs and new psychoactive substances (NPS) in human iPSC-derived cardiomyocytes using microelectrode array (MEA) recordings.

The use of recreational drugs, including new psychoactive substances (NPS), is paralleled by emergency department visits of drug users with severe cardiotoxicity. Drug-induced cardiotoxicity can be the (secondary) result of increased norepinephrine blood concentrations, but data on potential drug-induced direct effects on cardiomyocyte function are scarce. The presence of hundreds of NPS therefore calls for efficient screening models to assess direct cardiotoxicity. We investigated effects of four reference compounds (3-30 nM dofetilide, nifedipine and isoproterenol, and 1-10 µM mexiletine) and six recreational drugs (0.01-100 µM cocaine, 0.01-1000 µM amphetamine, MDMA, 4-fluoroamphetamine, α-PVP and MDPV) on cardiomyocyte function (beat rate, spike amplitude and field potential duration (FPD ≈ QT interval in ECGs)), using Pluricyte® human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes cultured on ready-to-use CardioPlate™ multi-well microelectrode arrays (mwMEAs). Moreover, the effects of exposure to recreational drugs on cell viability were assessed. Effects of reference compounds were in accordance with the literature, indicating the presence of hERG potassium (dofetilide), sodium (mexiletine) and calcium (nifedipine) channels and α-adrenergic receptors (isoproterenol). All recreational drugs decreased the spike amplitude at 10-100 µM. All amphetamine-type stimulants and α-PVP decreased the beat rate at 300 µM, while cocaine and MDPV did so at 10 µM and 30 µM, respectively. All drugs increased the FPD, however at varying concentrations. MDMA, MDPV and amphetamine affected cardiomyocyte function at concentrations relevant for human exposure, while other drugs affected cardiomyocyte function only at higher concentrations (≥ 10 µM). Cell viability was only mildly affected at concentrations well above the lowest concentrations affecting cardiomyocyte function. We demonstrate that MEA recordings of hiPSC-derived cardiomyocytes enable screening for acute, direct effects on cardiomyocyte function. Our data further indicate that tachycardia in patients exposed to recreational drugs is likely due to indirect drug effects, while prolonged repolarization periods (prolonged QTc interval) could (partly) result from direct drug effects on cardiomyocyte function.

Micropillar-based microfluidic device to regulate neurite networks of uniform-sized neurospheres.

The inability of neurons to undergo mitosis renders damage to the central or peripheral nervous system. Neural stem cell therapy could provide a path for treating the neurodegenerative diseases. However, reliable and simple tools for the developing and testing neural stem cell therapy are still required. Here, we show the development of a micropillar-based microfluidic device to trap the uniform-sized neurospheres. The neurospheres trapped within micropillar arrays were largely differentiated into neuronal cells, and their neurite networks were observed in the microfluidic device. Compared to conventional cultures on glass slides, the neurite networks generated with this method have a higher reproducibility. Furthermore, we demonstrated the effect of thapsigargin on the neurite networks in the microfluidic device, demonstrating that neural networks exposed to thapsigargin were largely diminished and disconnected from each other. Therefore, this micropillar-based microfluidic device could be a potential tool for screening of neurotoxins.

Kidney-based in vitro models for drug-induced toxicity testing.

The kidney is frequently involved in adverse effects caused by exposure to foreign compounds, including drugs. An early prediction of those effects is crucial for allowing novel, safe drugs entering the market. Yet, in current pharmacotherapy, drug-induced nephrotoxicity accounts for up to 25% of the reported serious adverse effects, of which one-third is attributed to antimicrobials use. Adverse drug effects can be due to direct toxicity, for instance as a result of kidney-specific determinants, or indirectly by, e.g., vascular effects or crystals deposition. Currently used in vitro assays do not adequately predict in vivo observed effects, predominantly due to an inadequate preservation of the organs' microenvironment in the models applied. The kidney is highly complex, composed of a filter unit and a tubular segment, together containing over 20 different cell types. The tubular epithelium is highly polarized, and the maintenance of this polarity is critical for optimal functioning and response to environmental signals. Cell polarity is dependent on communication between cells, which includes paracrine and autocrine signals, as well as biomechanic and chemotactic processes. These processes all influence kidney cell proliferation, migration, and differentiation. For drug disposition studies, this microenvironment is essential for prediction of toxic responses. This review provides an overview of drug-induced injuries to the kidney, details on relevant and translational biomarkers, and advances in 3D cultures of human renal cells, including organoids and kidney-on-a-chip platforms.

Characterization of an aerosol generation system to assess inhalation risks of aerosolized nano-enabled consumer products.

Objective: The aerosolization of common nano-enabled consumer products such as cosmetics has significantly increased engineered nanoparticle inhalation risks. While several studies have investigated the impact of cosmetic dermal exposures, inhalation hazards of aerosolized cosmetics are much less known but could pose considerable harm to users due to potential co-exposure of nanoparticles and other product components.Materials and Methods: In this study, we developed a fully automated aerosol generation system to examine the aerosol properties of four aerosolized nano-enabled cosmetics using real-time monitoring and sampling instrumentation. Physicochemical characterization of aerosols was conducted using scanning electron microscopy coupled with energy dispersive x-ray spectroscopy (SEM-EDX). Characterization and calibration of animal exposure pods coupled to the system were also performed by measuring and comparing particle concentrations between pods.Results and Discussion: Results show peak emissions are shade dependent and varied between 12,000-22,000 particles/cm3 with modal diameters ranging from 36 nm-1.3 µm. SEM-EDX analysis determined that the original products and collected aerosols have similar morphological features consisting of micron-sized particles decorated with nanoparticles and crystalline structures. Mean total particle concentration in pods at 5 and 10 mg/m3 target levels were 2.22E + 05 #/cm3 and 4.33E + 05 #/cm3, respectively, with <10% variability between pods.Conclusions: The fully automated exposure platform described herein provides reproducible aerosol generation, conforms to recommended guidelines on chemical testing, and therefore is suitable for future in vivo toxicological assessments to examine potential respiratory hazards of aerosolized nano-enabled consumer products.

Kidney-on-a-chip: untapped opportunities.

The organs-on-a-chip technology has shown strong promise in mimicking the complexity of native tissues in vitro and ex vivo, and recently significant advances have been made in applying this technology to studies of the kidney and its diseases. Individual components of the nephron, including the glomerulus, proximal tubule, and distal tubule/medullary collecting duct, have been successfully mimicked using organs-on-a-chip technology and yielding strong promises in advancing the field of ex vivo drug toxicity testing and augmenting renal replacement therapies. Although these models show promise over 2-dimensional cell systems in recapitulating important nephron features in vitro, nephron functions, such as tubular secretion, intracellular metabolism, and renin and vitamin D production, as well as prostaglandin synthesis are still poorly recapitulated in on-chip models. Moreover, construction of multiple-renal-components-on-a-chip models, in which various structures and cells of the renal system interact with each other, has remained a challenge. Overall, on-chip models show promise in advancing models of normal and pathological renal physiology, in predicting nephrotoxicity, and in advancing treatment of chronic kidney diseases.

Development of Microfluidic Dilution Network-Based System for Lab-on-a-Chip Microalgal Bioassays.

Because of the crucial ecological significance of microalgae, microalgal bioassays have become one of the most demanding tests from all classic aquatic toxicity tests in regulatory frameworks. However, conventional algal tests tend to be lab-intensive and time- and space-consuming, and they have not been utilized to their full potential for routine toxicity assessments. Microfluidics should be a user-friendly alternative. Particularly, dilution to generate gradients that are appropriate for screening experiments can be precisely attained by microfluidic network in a simple and cost-/time-/space-saving way. Here, we demonstrate a microfluidics series toward routine microalgal bioassays, including pretest, single, and joint toxicity test. The chip mainly consists of upstream dilution network (single serial dilution module (logarithmic/linear gradient generator) or multiple (binary/ternary/quaternary) mixing serial dilution module) and downstream diffusible culturing module. It allows the processes of chemical liquid dilution and diffusion, microscale microalgal culture, cell stimulation, and online screening to be integrated into a single device. Electric theorems with the aid of EDA (electronic design automation) simulation were innovatively introduced to minimize design effort for such systems. Using the device, microalgae were successfully cultured and stressed on-chip. The simple assay provides multibiological trait assessments of cell division rate, autofluorescence, esterase activity, and mobile capacity. This work showed promise in developing a high-throughput microfluidic platform for microalgal bioassays as well as lab-on-a-chip screening experiments in the cell-based quantitative assessment of environmental health risks.

Defining toxicological tipping points in neuronal network development.

Measuring electrical activity of neural networks by microelectrode array (MEA) has recently shown promise for screening level assessments of chemical toxicity on network development and function. Important aspects of interneuronal communication can be quantified from a single MEA recording, including individual firing rates, coordinated bursting, and measures of network synchrony, providing rich datasets to evaluate chemical effects. Further, multiple recordings can be made from the same network, including during the formation of these networks in vitro. The ability to perform multiple recording sessions over the in vitro development of network activity may provide further insight into developmental effects of neurotoxicants. In the current study, a recently described MEA-based screen of 86 compounds in primary rat cortical cultures over 12 days in vitro was revisited to establish a framework that integrates all available primary measures of electrical activity from MEA recordings into a composite metric for deviation from normal activity (total scalar perturbation). Examining scalar perturbations over time and increasing concentration of compound allowed for definition of critical concentrations or "tipping points" at which the neural networks switched from recovery to non-recovery trajectories for 42 compounds. These tipping point concentrations occurred at predominantly lower concentrations than those causing overt cell viability loss or disrupting individual network parameters, suggesting tipping points may be a more sensitive measure of network functional loss. Comparing tipping points for six compounds with plasma concentrations known to cause developmental neurotoxicity in vivo demonstrated strong concordance and suggests there is potential for using tipping points for chemical prioritization.

High-throughput metabolism-induced toxicity assays demonstrated on a 384-pillar plate.

The US Environmental Protection Agency (EPA) launched the Transform Tox Testing Challenge in 2016 with the goal of developing practical methods that can be integrated into conventional high-throughput screening (HTS) assays to better predict the toxicity of parent compounds and their metabolites in vivo. In response to this need and to retrofit existing HTS assays for assessing metabolism-induced toxicity of compounds, we have developed a 384-pillar plate that is complementary to traditional 384-well plates and ideally suited for culturing human cells in three dimensions at a microscale. Briefly, human embryonic kidney (HEK) 293 cells in a mixture of alginate and Matrigel were printed on the 384-pillar plates using a microarray spotter, which were coupled with 384-well plates containing nine model compounds provided by the EPA, five representative Phase I and II drug metabolizing enzymes (DMEs), and one no enzyme control. Viability and membrane integrity of HEK 293 cells were measured with the calcein AM and CellTiter-Glo® kit to determine the IC50 values of the nine parent compounds and DME-generated metabolites. The Z' factors and the coefficient of variation measured were above 0.6 and below 14%, respectively, indicating that the assays established on the 384-pillar plate are robust and reproducible. Out of nine compounds tested, six compounds showed augmented toxicity with DMEs and one compound showed detoxification with a Phase II DME. This result indicates that the 384-pillar plate platform can be used to measure metabolism-induced toxicity of compounds in high-throughput with individual DMEs. As xenobiotics metabolism is a complex process with a variety of DMEs involved, the predictivity of our approach could be further improved with mixtures of DMEs.