Integrated Approach for Testing and Assessment for Developmental Neurotoxicity (DNT) to Prioritize Aromatic Organophosphorus Flame Retardants.

Organophosphorus flame retardants (OPFRs) are abundant and persistent in the environment but have limited toxicity information. Their similarity in structure to organophosphate pesticides presents great concern for developmental neurotoxicity (DNT). However, current in vivo testing is not suitable to provide DNT information on the amount of OPFRs that lack data. Over the past decade, an in vitro battery was developed to enhance DNT assessment, consisting of assays that evaluate cellular processes in neurodevelopment and function. In this study, behavioral data of small model organisms were also included. To assess if these assays provide sufficient mechanistic coverage to prioritize chemicals for further testing and/or identify hazards, an integrated approach to testing and assessment (IATA) was developed with additional information from the Integrated Chemical Environment (ICE) and the literature. Human biomonitoring and exposure data were identified and physiologically-based toxicokinetic models were applied to relate in vitro toxicity data to human exposure based on maximum plasma concentration. Eight OPFRs were evaluated, including aromatic OPFRs (triphenyl phosphate (TPHP), isopropylated phenyl phosphate (IPP), 2-ethylhexyl diphenyl phosphate (EHDP), tricresyl phosphate (TMPP), isodecyl diphenyl phosphate (IDDP), tert-butylphenyl diphenyl phosphate (BPDP)) and halogenated FRs ((Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP), tris(2-chloroethyl) phosphate (TCEP)). Two representative brominated flame retardants (BFRs) (2,2'4,4'-tetrabromodiphenyl ether (BDE-47) and 3,3',5,5'-tetrabromobisphenol A (TBBPA)) with known DNT potential were selected for toxicity benchmarking. Data from the DNT battery indicate that the aromatic OPFRs have activity at similar concentrations as the BFRs and should therefore be evaluated further. However, these assays provide limited information on the mechanism of the compounds. By integrating information from ICE and the literature, endocrine disruption was identified as a potential mechanism. This IATA case study indicates that human exposure to some OPFRs could lead to a plasma concentration similar to those exerting in vitro activities, indicating potential concern for human health.

Biomarkers of chemotherapy-induced cardiotoxicity: toward precision prevention using extracellular vesicles.

Detrimental side effects of drugs like doxorubicin, which can cause cardiotoxicity, pose barriers for preventing cancer progression, or treating cancer early through molecular interception. Extracellular vesicles (EVs) are valued for their potential as biomarkers of human health, chemical and molecular carcinogenesis, and therapeutics to treat disease at the cellular level. EVs are released both during normal growth and in response to toxicity and cellular death, playing key roles in cellular communication. Consequently, EVs may hold promise as precision biomarkers and therapeutics to prevent or offset damaging off-target effects of chemotherapeutics. EVs have promise as biomarkers of impending cardiotoxicity induced by chemotherapies and as cardioprotective therapeutic agents. However, EVs can also mediate cardiotoxic cues, depending on the identity and past events of their parent cells. Understanding how EVs mediate signaling is critical toward implementing EVs as therapeutic agents to mitigate cardiotoxic effects of chemotherapies. For example, it remains unclear how mixtures of EV populations from cells exposed to toxins or undergoing different stages of cell death contribute to signaling across cardiac tissues. Here, we present our perspective on the outlook of EVs as future clinical tools to mitigate chemotherapy-induced cardiotoxicity, both as biomarkers of impending cardiotoxicity and as cardioprotective agents. Also, we discuss how heterogeneous mixtures of EVs and transient exposures to toxicants may add complexity to predicting outcomes of exogenously applied EVs. Elucidating how EV cargo and signaling properties change during dynamic cellular events may aid precision prevention of cardiotoxicity in anticancer treatments and development of safer chemotherapeutics.

Pluripotent stem cells for target organ developmental toxicity testing.

Prenatal developmental toxicity research focuses on understanding the potential adverse effects of environmental agents, drugs, and chemicals on the development of embryos and fetuses. Traditional methods involve animal testing, but ethical concerns and the need for human-relevant models have prompted the exploration of alternatives. Pluripotent stem cells (PSCs) are versatile cells with the unique ability to differentiate into any cell type, serving as a foundational tool for studying human development. Two-dimensional (2D) PSC models are often chosen for their ease of use and reproducibility for high-throughput screening. However, they lack the complexity of an in vivo environment. Alternatively, three-dimensional (3D) PSC models, such as organoids, offer tissue architecture and intercellular communication more reminiscent of in vivo conditions. However, they are complicated to produce and analyze, usually requiring advanced and expensive techniques. This review discusses recent advances in the use of human PSCs differentiated into brain and heart lineages and emerging tools and methods that can be combined with PSCs to help address important scientific questions in the area of developmental toxicology. These advancements and new approach methods align with the push for more relevant and predictive developmental toxicity assessment, combining innovative techniques with organoid models to advance regulatory decision-making.

Pluripotent Stem Cell-derived Dopaminergic Neurons for Studying Developmental Neurotoxicity.

With the vast number of chemicals in commerce, higher throughput strategies are needed to inform risk assessment. The field of toxicology is therefore moving away from traditional in vivo guideline studies towards in vitro new approach methodologies. There has been a great push for such a shift in the field of developmental neurotoxicity, where there is a particular lack of data. A battery of in vitro new approach methodologies has thus been developed to help fill this gap. Included in this battery are assays for numerous processes critical to neurodevelopment, such as proliferation, migration, and synaptogenesis. The current battery of developmental neurotoxicity new approach methodologies still lacks recapitulation of several critical neurodevelopmental processes, including development of neuronal subtypes. With their pluripotency, alongside other advantages, pluripotent stem cells (PSCs) are uniquely suited to address questions of developmental neurotoxicity, as they can recapitulate the different stages of human in vivo neurodevelopment. Among the various neuronal subtypes, development of dopaminergic neurons (DA) is perhaps the best understood and several approaches exist to differentiate PSCs into DA. Herein we review these approaches and propose utilizing PSCs for screening of the impact of environmental chemicals on development of DA. Related techniques and gaps in knowledge are also addressed.

Category-Based Toxicokinetic Evaluations of Data-Poor Per- and Polyfluoroalkyl Substances (PFAS) using Gas Chromatography Coupled with Mass Spectrometry.

Concern over per- and polyfluoroalkyl substances (PFAS) has increased as more is learned about their environmental presence, persistence, and bioaccumulative potential. The limited monitoring, toxicokinetic (TK), and toxicologic data available are inadequate to inform risk across this diverse domain. Here, 73 PFAS were selected for in vitro TK evaluation to expand knowledge across lesser-studied PFAS alcohols, amides, and acrylates. Targeted methods developed using gas chromatography-tandem mass spectrometry (GC-MS/MS) were used to measure human plasma protein binding and hepatocyte clearance. Forty-three PFAS were successfully evaluated in plasma, with fraction unbound (fup) values ranging from 0.004 to 1. With a median fup of 0.09 (i.e., 91% bound), these PFAS are highly bound but exhibit 10-fold lower binding than legacy perfluoroalkyl acids recently evaluated. Thirty PFAS evaluated in the hepatocyte clearance assay showed abiotic loss, with many exceeding 60% loss within 60 min. Metabolic clearance was noted for 11 of the 13 that were successfully evaluated, with rates up to 49.9 µL/(min × million cells). The chemical transformation simulator revealed potential (bio)transformation products to consider. This effort provides critical information to evaluate PFAS for which volatility, metabolism, and other routes of transformation are likely to modulate their environmental fates.

Integration of toxicodynamic and toxicokinetic new approach methods into a weight-of-evidence analysis for pesticide developmental neurotoxicity assessment: A case-study with DL- and L-glufosinate.

DL-glufosinate ammonium (DL-GLF) is a registered herbicide for which a guideline Developmental Neurotoxicity (DNT) study has been conducted. Offspring effects included altered brain morphometrics, decreased body weight, and increased motor activity. Guideline DNT studies are not available for its enriched isomers L-GLF acid and L-GLF ammonium; conducting one would be time consuming, resource-intensive, and possibly redundant given the existing DL-GLF DNT. To support deciding whether to request a guideline DNT study for the L-GLF isomers, DL-GLF and the L-GLF isomers were screened using in vitro assays for network formation and neurite outgrowth. DL-GLF and L-GLF isomers were without effects in both assays. DL-GLF and L-GLF (1-100 µM) isomers increased mean firing rate of mature networks to 120-140% of baseline. In vitro toxicokinetic assessments were used to derive administered equivalent doses (AEDs) for the in vitro testing concentrations. The AED for L-GLF was ∼3X higher than the NOAEL from the DL-GLF DNT indicating that the available guideline study would be protective of potential DNT due to L-GLF exposure. Based in part on the results of these in vitro studies, EPA is not requiring L-GLF isomer guideline DNT studies, thereby providing a case study for a useful application of DNT screening assays.

Greater Number of Microglia in Telencephalic Proliferative Zones of Human and Nonhuman Primate Compared with Other Vertebrate Species.

Microglial cells, the innate immune cells of the brain, are derived from yolk sac precursor cells, begin to colonize the telencephalon at the onset of cortical neurogenesis, and occupy specific layers including the telencephalic proliferative zones. Microglia are an intrinsic component of cortical germinal zones, establish extensive contacts with neural precursor cells (NPCs) and developing cortical vessels, and regulate the size of the NPC pool through mechanisms that include phagocytosis. Microglia exhibit notable differences in number and distribution in the prenatal neocortex between rat and old world nonhuman primate telencephalon, suggesting that microglia possess distinct properties across vertebrate species. To begin addressing this subject, we quantified the number of microglia and NPCs in proliferative zones of the fetal human, rhesus monkey, ferret, and rat, and the prehatch chick and turtle telencephalon. We show that the ratio of NPCs to microglia varies significantly across species. Few microglia populate the prehatch chick telencephalon, but the number of microglia approaches that of NPCs in fetal human and nonhuman primate telencephalon. These data demonstrate that microglia are in a position to perform important functions in a number of vertebrate species but more heavily colonize proliferative zones of fetal human and rhesus monkey telencephalon.

High-throughput PBTK models for in vitro to in vivo extrapolation.

INTRODUCTION: Toxicity data are unavailable for many thousands of chemicals in commerce and the environment. Therefore, risk assessors need to rapidly screen these chemicals for potential risk to public health. High-throughput screening (HTS) for in vitro bioactivity, when used with high-throughput toxicokinetic (HTTK) data and models, allows characterization of these thousands of chemicals. AREAS COVERED: This review covers generic physiologically based toxicokinetic (PBTK) models and high-throughput PBTK modeling for in vitro-in vivo extrapolation (IVIVE) of HTS data. We focus on 'httk', a public, open-source set of computational modeling tools and in vitro toxicokinetic (TK) data. EXPERT OPINION: HTTK benefits chemical risk assessors with its ability to support rapid chemical screening/prioritization, perform IVIVE, and provide provisional TK modeling for large numbers of chemicals using only limited chemical-specific data. Although generic TK model design can increase prediction uncertainty, these models provide offsetting benefits by increasing model implementation accuracy. Also, public distribution of the models and data enhances reproducibility. For the httk package, the modular and open-source design can enable the tool to be used and continuously improved by a broad user community in support of the critical need for high-throughput chemical prioritization and rapid dose estimation to facilitate rapid hazard assessments.

Fetal Rhesus Monkey First Trimester Zika Virus Infection Impacts Cortical Development in the Second and Third Trimesters.

Zika virus is a teratogen similar to other neurotropic viruses, notably cytomegalovirus and rubella. The goal of these studies was to address the direct impact of Zika virus on fetal development by inoculating early gestation fetal rhesus monkeys using an ultrasound-guided approach (intraperitoneal vs. intraventricular). Growth and development were monitored across gestation, maternal samples collected, and fetal tissues obtained in the second trimester or near term. Although normal growth and anatomical development were observed, significant morphologic changes were noted in the cerebral cortex at 3-weeks post-Zika virus inoculation including massive alterations in the distribution, density, number, and morphology of microglial cells in proliferative regions of the fetal cerebral cortex; an altered distribution of Tbr2+ neural precursor cells; increased diameter and volume of blood vessels in the cortical proliferative zones; and a thinner cortical plate. At 3-months postinoculation, alterations in morphology, distribution, and density of microglial cells were also observed with an increase in blood vessel volume; and a thinner cortical plate. Only transient maternal viremia was observed but sustained maternal immune activation was detected. Overall, these studies suggest persistent changes in cortical structure result from early gestation Zika virus exposure with durable effects on microglial cells.

Microglia: An Intrinsic Component of the Proliferative Zones in the Fetal Rhesus Monkey (Macaca mulatta) Cerebral Cortex.

Microglial cells are increasingly recognized as modulators of brain development. We previously showed that microglia colonize the cortical proliferative zones in the prenatal brain and regulate the number of precursor cells through phagocytosis. To better define cellular interactions between microglia and proliferative cells, we performed lentiviral vector-mediated intraventricular gene transfer to induce enhanced green fluorescent protein expression in fetal cerebrocortical cells. Tissues were collected and counterstained with cell-specific markers to label microglial cells and identify other cortical cell types. We found that microglial cells intimately interact with the radial glial scaffold and make extensive contacts with neural precursor cells throughout the proliferative zones, particularly in the rhesus monkey fetus when compared to rodents. We also identify a subtype of microglia, which we term 'periventricular microglia', that interact closely with mitotic precursor cells in the ventricular zone. Our data suggest that microglia are structural modulators that facilitate remodeling of the proliferative zones as precursor cells migrate away from the ventricle and may facilitate the delamination of precursor cells. Taken together, these results indicate that microglial cells are an integral component of cortical proliferative zones and contribute to the interactive milieu in which cortical precursor cells function.

Maximizing Explanatory Power in Stereological Data Collection: A Protocol for Reliably Integrating Optical Fractionator and Multiple Immunofluorescence Techniques.

With the promise of greater reliability and replicability of estimates, stereological techniques have revolutionized data collection in the neurosciences. At the same time, improvements in immunohistochemistry and fluorescence imaging technologies have facilitated easy application of immunofluorescence protocols, allowing for isolation of multiple target proteins in one tissue sample. Combining multiple immunofluorescence labeling with stereological data collection can provide a powerful tool to maximize explanatory power and efficiency, while minimizing tissue use. Multiple cell classes, subtypes of larger populations, or different cell states can be quantified in one case and even in one sampling run. Here, we present a protocol integrating stereological data collection and multiple immunofluorescence using commonly employed widefield epifluorescence filter sets, optimized for blue (DAPI), green (FITC), and far red (CY5) channels. Our stereological protocol has been designed to accommodate the challenges of fluorescence imaging to overcome limitations like fixed filter sets, photobleaching, and uneven immunolabeling. To enhance fluorescence signal for stereological sampling, our immunolabeling protocol utilizes both high temperature antigen retrieval to improve primary antibody binding and secondary antibodies conjugated to optimally stable fluorophores. To illustrate the utility of this approach, we estimated the number of Ctip2 immunoreactive subcerebral projection neurons and NeuN immunoreactive neurons in rat cerebral cortex at postnatal day 10. We used DAPI (blue) to define the neocortex, anti-NeuN (far red) to identify neurons, and co-labeling of anti-Ctip2 (green) and anti-NeuN (far red) to isolate only subcerebral projection neurons. Our protocol resulted in estimates with low sampling error (CE < 0.05) and high intrarater reliability (ICC > 0.98) that fall within the range of published values, attesting to its efficacy. We show our immunofluorescence techniques can be used to reliably identify other cell types, e.g., different glial cell classes, to highlight the broader applications of our approach. The flexibility of the technique, increasingly reduced costs of fluorescence technologies, and savings in experimental time and tissue use make this approach valuable for neuroscientists interested in incorporating stereology to ask precise neurophysiological and neuroanatomical questions.

The Bat as a New Model of Cortical Development.

The organization of the mammalian cerebral cortex shares fundamental features across species. However, while the radial thickness of grey matter varies within one order of magnitude, the tangential spread of the cortical sheet varies by orders of magnitude across species. A broader sample of model species may provide additional clues for understanding mechanisms that drive cortical expansion. Here, we introduce the bat Carollia perspicillata as a new model species. The brain of C. perspicillata is similar in size to that of mouse but has a cortical neurogenic period at least 5 times longer than mouse, and nearly as long as that of the rhesus macaque, whose brain is 100 times larger. We describe the development of laminar and regional structures, neural precursor cell identity and distribution, immune cell distribution, and a novel population of Tbr2+ cells in the caudal ganglionic eminence of the developing neocortex of C. perspicillata. Our data indicate that unique mechanisms guide bat cortical development, particularly concerning cell cycle length. The bat model provides new perspective on the evolution of developmental programs that regulate neurogenesis in mammalian cerebral cortex, and offers insight into mechanisms that contribute to tangential expansion and gyri formation in the cerebral cortex.