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Per- and polyfluoroalkyl substances (PFAS) are widely used, and their fluorinated state contributes to unique uses and stability but also long half-lives in the environment and humans. PFAS have been shown to be toxic, leading to immunosuppression, cancer, and other adverse health outcomes. Only a small fraction of the PFAS in commerce have been evaluated for toxicity using in vivo tests, which leads to a need to prioritize which compounds to examine further. Here, we demonstrate a prioritization approach that combines human biomonitoring data (blood concentrations) with bioactivity data (concentrations at which bioactivity is observed in vitro) for 31 PFAS. The in vitro data are taken from a battery of cell-based assays, mostly run on human cells. The result is a Bioactive Concentration to Blood Concentration Ratio (BCBCR), similar to a margin of exposure (MoE). Chemicals with low BCBCR values could then be prioritized for further risk assessment. Using this method, two of the PFAS, PFOA (Perfluorooctanoic Acid) and PFOS (Perfluorooctane Sulfonic Acid), have BCBCR values < 1 for some populations. An additional 9 PFAS have BCBCR values < 100 for some populations. This study shows a promising approach to screening level risk assessments of compounds such as PFAS that are long-lived in humans and other species. more...

To date, approximately 200 chemicals have been tested in US Environmental Protection Agency (EPA) or Organization for Economic Co-operation and Development (OECD) developmental neurotoxicity (DNT) guideline studies, leaving thousands of chemicals without traditional animal information on DNT hazard potential. To address this data gap, a battery of in vitro DNT new approach methodologies (NAMs) has been proposed. Evaluation of the performance of this battery will increase the confidence in its use to determine DNT chemical hazards. One approach to evaluate DNT NAM performance is to use a set of chemicals to evaluate sensitivity and specificity. Since a list of chemicals with potential evidence of in vivo DNT has been established, this study aims to develop a curated list of "negative" chemicals for inclusion in a "DNT NAM evaluation set". A workflow, including a literature search followed by an expert-driven literature review, was used to systematically screen 39 chemicals for lack of DNT effect. Expert panel members evaluated the scientific robustness of relevant studies to inform chemical categorizations. Following review, the panel discussed each chemical and made categorical determinations of "Favorable", "Not Favorable", or "Indeterminate" reflecting acceptance, lack of suitability, or uncertainty given specific limitations and considerations, respectively. The panel determined that 10, 22, and 7 chemicals met the criteria for "Favorable", "Not Favorable", and "Indeterminate", for use as negatives in a DNT NAM evaluation set. Ultimately, this approach not only supports DNT NAM performance evaluation but also highlights challenges in identifying large numbers of negative DNT chemicals., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Published by Elsevier Inc.) more...

Studies in in vivo rodent models have been the accepted approach by regulatory agencies to evaluate potential developmental neurotoxicity (DNT) of chemicals for decades. These studies, however, are inefficient and cannot meet the demand for the thousands of chemicals that need to be assessed for DNT hazard. As such, several in vitro new approach methods (NAMs) have been developed to circumvent limitations of these traditional studies. The DNT NAMs, some of which utilize human-derived cell models, are intended to be employed in a testing battery approach, each focused on a specific neurodevelopmental process. The need for multiple assays, however, to evaluate each process can prolong testing and prioritization of chemicals for more in depth assessments. Therefore, a multi-endpoint higher-throughput approach to assess DNT hazard potential would be of value. Accordingly, we have adapted a high-throughput phenotypic profiling (HTPP) approach for use with human-derived neural progenitor (hNP1) cells. HTPP is a fluorescence-based assay that quantitatively measures alterations in cellular morphology. This approach, however, required optimization of several laboratory procedures prior to chemical screening. First, we had to determine an appropriate cell plating density in 384-well plates. We then had to identify the minimum laminin concentration required for optimal cell growth and attachment. And finally, we had to evaluate whether addition of antibiotics to the culture medium would alter cellular morphology. We selected 6,000 cells/well as an appropriate plating density, 20 µg/ml laminin for optimal cell growth and attachment, and antibiotic addition in the culture medium. After optimizing hNP1 cell culture conditions for HTPP, it was then necessary to select appropriate in-plate assay controls from a reference chemical set. These reference chemicals were previously demonstrated to elicit unique phenotypic profiles in various other cell types. Aphidicolin, bafilomycin A1, berberine chloride, and cucurbitacin I induced robust phenotypic profiles as compared to dimethyl sulfoxide vehicle control in the hNP1 cells, and thus can be employed as in-plate assay controls for subsequent chemical screens. We have optimized HTPP for hNP1 cells, and consequently this approach can now be assessed as a potential NAM for DNT hazard evaluation and results compared to previously developed DNT assays., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The U.S. Environmental Protection Agency has provided administrative review and approved this manuscript for publication. The views expressed in this manuscript, however, are those of the authors and do not necessarily reflect U.S. EPA policies. Reference to commercial products does not constitute endorsement., (Copyright © 2022 Culbreth, Nyffeler, Willis and Harrill.) more...

Phenotypic profiling assays are untargeted screening assays that measure a large number (hundreds to thousands) of cellular features in response to a stimulus and often yield diverse and unanticipated profiles of phenotypic effects, leading to challenges in distinguishing active from inactive treatments. Here, we compare a variety of different strategies for hit identification in imaging-based phenotypic profiling assays using a previously published Cell Painting data set. Hit identification strategies based on multiconcentration analysis involve curve fitting at several levels of data aggregation (e.g., individual feature level, aggregation of similarly derived features into categories, and global modeling of all features) and on computed metrics (e.g., Euclidean and Mahalanobis distance metrics and eigenfeatures). Hit identification strategies based on single-concentration analysis included measurement of signal strength (e.g., total effect magnitude) and correlation of profiles among biological replicates. Modeling parameters for each approach were optimized to retain the ability to detect a reference chemical with subtle phenotypic effects while limiting the false-positive rate to 10%. The percentage of test chemicals identified as hits was highest for feature-level and category-based approaches, followed by global fitting, whereas signal strength and profile correlation approaches detected the fewest number of active hits at the fixed false-positive rate. Approaches involving fitting of distance metrics had the lowest likelihood for identifying high-potency false-positive hits that may be associated with assay noise. Most of the methods achieved a 100% hit rate for the reference chemical and high concordance for 82% of test chemicals, indicating that hit calls are robust across different analysis approaches. more...

Background: Synaptogenesis is a critical neurodevelopmental process whereby pre- and postsynaptic neurons form apposed sites of contact specialized for chemical neurotransmission. Many neurodevelopmental disorders are thought to reflect altered patterns of synaptic connectivity, including imbalances between excitatory and inhibitory synapses. Developing rapid throughput approaches for assessing synaptogenesis will facilitate toxicologic and drug screening studies of neurodevelopmental disorders. The current study describes the use of high-content imaging to quantify the ontogeny of excitatory and inhibitory synapses using in vitro models of neurodevelopment. These data are compared to biochemical and functional measures of synaptogenesis., Results: The ontogenetic patterns of synapse formation were compared between primary rodent hippocampal and cortical neurons over 28 days in vitro (DIV). As determined by ELISA, the increase in synaptophysin expression levels as cultures matured was similar between hippocampal and cortical cultures. High-content imaging of immunoreactivity of excitatory and inhibitory synaptic biomarkers demonstrated an overall greater number of synapses in hippocampal relative to cortical neurons with marked differences in the pattern of inhibitory synapse development between these two neuronal cell types. Functional assays revealed that both the mean firing rates and mean bursting rates were significantly increased in cortical cultures relative to hippocampal cultures. This difference may reflect decreased inhibitory synaptic tone in cortical versus hippocampal cultures., Conclusions: These data demonstrate differences and similarities in the ontogeny of synaptogenesis between hippocampal and cortical neurons, depending on the biological level examined. Assessment of synaptophysin protein levels by ELISA showed a general increase in synapse formation in both cell types with increasing time in culture, while high-content imaging was able to delineate cell type-dependent differences in formation of excitatory versus inhibitory synapses. The functional significance of differences in the balance of excitatory to inhibitory synapses was confirmed by the assessment of network activity using microelectrode arrays. These results suggest that high-content imaging and microelectrode arrays provide complementary approaches for quantitative assessment of synaptogenesis, which should provide a robust readout of toxicologic and pharmacologic effects on this critical neurodevelopmental event. more...

Various growth factor cocktails have been used to proliferate and then differentiate human neural progenitor (NP) cells derived from embryonic stem cells (ESC) for in vitro and in vivo studies. However, the cytokine leukemia inhibitory factor (LIF) has been largely overlooked. Here, we demonstrate that LIF significantly enhanced in vitro survival and promoted differentiation of human ESC-derived NP cells. In NP cells, as well as NP-derived neurons, LIF reduced caspase-mediated apoptosis and reduced both spontaneous and H2O2-induced reactive oxygen species in culture. In vitro, NP cell proliferation and the yield of differentiated neurons were significantly higher in the presence of LIF. In NP cells, LIF enhanced cMyc phosphorylation, commonly associated with self-renewal/proliferation. Also, in differentiating NP cells LIF activated the phosphoinositide 3-kinase and signal transducer and activator of transcription 3 pathways, associated with cell survival and reduced apoptosis. When differentiated in LIF+ media, neurite outgrowth and ERK1/2 phosphorylation were potentiated together with increased expression of gp130, a component of the LIF receptor complex. NP cells, pretreated in vitro with LIF, were effective in reducing infarct volume in a model of focal ischemic stroke but LIF did not lead to significantly improved initial NP cell survival over nontreated NP cells. Our results show that LIF signaling significantly promotes human NP cell proliferation, survival, and differentiation in vitro. Activated LIF signaling should be considered in cell culture expansion systems for future human NP cell-based therapeutic transplant studies., (Copyright © 2012 AlphaMed Press.) more...

Because the Developmental Neurotoxicity Testing Guidelines require large numbers of animals and is expensive, development of in vitro approaches to screen chemicals for potential developmental neurotoxicity is a high priority. Many proposed approaches for screening are biochemical or morphological, and do not assess function of neuronal networks. In this study, microelectrode arrays (MEAs) were used to determine if chemical-induced changes in function could be detected by assessing the development of spontaneous network activity. MEAs record individual action potential spikes as well as groups of spikes (bursts) in neuronal networks, and activity can be assessed repeatedly over days in vitro (DIV). Primary cultures of rat cortical neurons were prepared on MEAs and spontaneous activity was assessed on DIV 2, 6, 9, 13, and 20 to determine the in vitro developmental profile of spontaneous spiking and bursting in cortical networks. In addition, 5 μM of the protein kinase C inhibitor bisindolylmaleamide-1 (Bis-1) was added to MEAs (n = 9-18) on DIV 5 to determine if changes in spontaneous activity could be detected in response to inhibition of neurite outgrowth. A clear profile of in vitro activity development occurred in control MEAs, with the number of active channels increasing from 0/MEA on DIV 2 to 37 ± 5/MEA by DIV 13; the rate of increase was most rapid between DIV 6 and 9, and activity declined by DIV 20. A similar pattern was observed for the number of bursting channels, as well as the total number of bursts. Bis-1 decreased the number of active channels/MEA and the number of bursting channels/MEA. Burst characteristics, such as burst duration and the number of spikes in a burst, were unchanged by Bis-1. These results demonstrate that MEAs can be used to assess the development of functional neuronal networks in vitro, as well as chemical-induced dysfunction. more...

Background: Pyrethroids are neurotoxic pesticides that interact with membrane bound ion channels in neurons and disrupt nerve function. The purpose of this study was to characterize and explore changes in gene expression that occur in the rat frontal cortex, an area of CNS affected by pyrethroids, following an acute low-dose exposure., Results: Rats were acutely exposed to either deltamethrin (0.3 - 3 mg/kg) or permethrin (1 - 100 mg/kg) followed by collection of cortical tissue at 6 hours. The doses used range from those that cause minimal signs of intoxication at the behavioral level to doses well below apparent no effect levels in the whole animal. A statistical framework based on parallel linear (SAM) and isotonic regression (PIR) methods identified 95 and 53 probe sets as dose-responsive. The PIR analysis was most sensitive for detecting transcripts with changes in expression at the NOAEL dose. A sub-set of genes (Camk1g, Ddc, Gpd3, c-fos and Egr1) was then confirmed by qRT-PCR and examined in a time course study. Changes in mRNA levels were typically less than 3-fold in magnitude across all components of the study. The responses observed are consistent with pyrethroids producing increased neuronal excitation in the cortex following a low-dose in vivo exposure. In addition, Significance Analysis of Function and Expression (SAFE) identified significantly enriched gene categories common for both pyrethroids, including some relating to branching morphogenesis. Exposure of primary cortical cell cultures to both compounds resulted in an increase (approximately 25%) in the number of neurite branch points, supporting the results of the SAFE analysis., Conclusion: In the present study, pyrethroids induced changes in gene expression in the frontal cortex near the threshold for decreases in ambulatory motor activity in vivo. The penalized regression methods performed similarly in detecting dose-dependent changes in gene transcription. Finally, SAFE analysis of gene expression data identified branching morphogenesis as a biological process sensitive to pyrethroids and subsequent in vitro experiments confirmed this predicted effect. The novel findings regarding pyrethroid effects on branching morphogenesis indicate these compounds may act as developmental neurotoxicants that affect normal neuronal morphology. more...

Pyrethroids are pesticides with high selectivity for insects. In order to identify strengths and gaps in the database for pyrethroid neurobehavioral toxicology, we have critically analyzed the data from peer-reviewed literature. This review includes dose-response data that have been recently generated demonstrating consistent findings for low-dose, acute, oral exposure to pyrethroids in small rodents. All pyrethroids tested (i.e., about twenty compounds), regardless of structure, produce a decrease in motor activity in a variety of test protocols. The range of relative potencies varies more than two orders of magnitude, and thresholds for motor activity were found well below doses that produce overt signs of poisoning. Six compounds (allethrin, permethrin, cis-permethrin, deltamethrin, cypermethrin, and fenvalerate) impair schedule-controlled operant responding, seven compounds (pyrethrum, bifenthrin, S-bioallethrin, permethrin, beta-cyfluthrin, cypermethrin, and deltamethrin) decrease grip strength, and two compounds (deltamethrin and alpha-cypermethrin) produce incoordination using the rotarod. In addition, while compounds lacking an alpha-cyano group (e.g., cismethrin, permethrin, bifenthrin) induce an increase in acoustic-evoked startle response amplitude, cyano compounds (e.g., deltamethrin, cypermethrin, cyfluthrin) produce the opposite outcome. Other endpoints (e.g., tremor intensity, sensory response) have been only occasionally explored. A synthesis of the neurobehavioral evidence relating to the action of pyrethroids indicates that some differences in the experimental findings across compounds are also present in the low-effective dose range. For risk assessment purposes, a strategy that takes into account data from an array of neurobehavioral endpoints is needed to capture the heterogeneity of pyrethroid-induced adverse effects and accurately inform policy decisions. more...