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The increasing prevalence of neurodevelopmental disorders has highlighted the need for improved testing methods to determine developmental neurotoxicity (DNT) hazard for thousands of chemicals. This paper proposes the integration of organoid intelligence (OI); leveraging brain organoids to study neuroplasticity in vitro, into the DNT testing paradigm. OI brings a new approach to measure the impacts of xenobiotics on plasticity mechanisms - a critical biological process that is not adequately covered in current DNT in vitro assays. Finally, the integration of artificial intelligence (AI) techniques will further facilitate the analysis of complex brain organoid data to study these plasticity mechanisms. [ABSTRACT FROM AUTHOR]

Under regulations such as REACH, testing of novel and established compounds for their (neuro)toxic potential is a legal requirement in many countries. These are largely based on animal-, cost-, and time-intensive in vivo models, not in line with the 3 Rs' principle of animal experimentation. Thus, the development of alternative test methods has also received increasing attention in neurotoxicology. Such methods focus either on physiological alterations in brain development and neuronal pathways or on behavioral changes. An example of a behavioral developmental neurotoxicity (DNT) assay is the zebrafish (Danio rerio) embryo coiling assay, which quantifies effects of compounds on the development of spontaneous movement of zebrafish embryos. While the importance of embryo-to-embryo contact prior to hatching in response to environmental contaminants or natural threats has been documented for many other clutch-laying fish species, little is known about the relevance of intra-clutch contacts for zebrafish. Here, the model neurotoxin rotenone was used to assess the effect of grouped versus separate rearing of the embryos on the expression of the coiling behavior. Some group-reared embryos reacted with hyperactivity to the exposure, to an extent that could not be recorded effectively with the utilized software. Separately reared embryos showed reduced activity, compared with group-reared individuals when assessing. However, even the control group embryos of the separately reared cohort showed reduced activity, compared with group-reared controls. Rotenone could thus be confirmed to induce neurotoxic effects in zebrafish embryos, yet modifying one parameter in an otherwise well-established neurotoxicity assay such as the coiling assay may lead to changes in behavior influenced by the proximity between individual embryos. This indicates a complex dependence of the outcome of behavior assays on a multitude of environmental parameters. [ABSTRACT FROM AUTHOR]

Arsenic (As) is globally detected in drinking water and food products at levels repeatedly surpassing regulatory thresholds. Several neurological and mental health risks linked to arsenic exposure are proposed; however, the nature of these effects and their association with the chemical forms of arsenic are not fully understood. Gaining a clear understanding of the etiologies and characteristics of these effects is crucial, particularly in association with developmental exposures where the nervous system is most vulnerable. In this study, we investigated the effects of early developmental exposure (6- to 120-h postfertilization [hpf]) of larval zebrafish to environmentally relevant concentrations of arsenic species—trivalent/pentavalent, inorganic/organic forms—on developmental, behavioral, and molecular endpoints to determine their effect on stress response and their potential association with stress-related disorders. At 120 hpf, the developing larvae were assessed for a battery of endpoints including survival, developmental malformities, background activity, and behavioral responses to acute visual and acoustic stimuli. Pooled larval samples were analyzed for alterations in the transcript levels of genes associated with developmental neurotoxicity and stress-related disorders. Developmental exposures at target concentrations did not significantly alter survival, overall development, or background activity, and had minor effects on developmental morphology. Sodium arsenate and monomethylarsonic acid exaggerated the behavioral responses of larval zebrafish, whereas sodium arsenite depressed them. Sodium arsenate induced significant effects on molecular biomarkers. This study highlights the effects of developmental exposure to arsenicals on the behavioral stress response, the role chemical formulation plays in exerting toxicological effects, and the possible association with stress-related disorders. [ABSTRACT FROM AUTHOR]

Sevoflurane induces developmental neurotoxicity in mice; however, the underlying mechanisms remain unclear. Triggering receptor expressed on myeloid cells 2 (TREM2) is essential for microglia-mediated synaptic refinement during the early stages of brain development. We explored the effects of TREM2 on dendritic spine pruning during sevoflurane-induced developmental neurotoxicity in mice. Mice were anaesthetized with sevoflurane on postnatal days 6, 8, and 10. Behavioral performance was assessed using the open field test and Morris water maze test. Genetic knockdown of TREM2 and overexpression of TREM2 by stereotaxic injection were used for mechanistic experiments. Western blotting, immunofluorescence, electron microscopy, three-dimensional reconstruction, Golgi staining, and whole-cell patch-clamp recordings were performed. Sevoflurane exposures upregulated the protein expression of TREM2, increased microglia-mediated pruning of dendritic spines, and reduced synaptic multiplicity and excitability of CA1 neurons. TREM2 genetic knockdown significantly decreased dendritic spine pruning, and partially aggravated neuronal morphological abnormalities and cognitive impairments in sevoflurane-treated mice. In contrast, TREM2 overexpression enhanced microglia-mediated pruning of dendritic spines and rescued neuronal morphological abnormalities and cognitive dysfunction. TREM2 exerts a protective role against neurocognitive impairments in mice after neonatal exposures to sevoflurane by enhancing microglia-mediated pruning of dendritic spines in CA1 neurons. This provides a potential therapeutic target in the prevention of sevoflurane-induced developmental neurotoxicity. [ABSTRACT FROM AUTHOR]

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The adverse outcome pathway (AOP) framework serves as a practical tool for organising scientific knowledge that can be used to infer cause–effect relationships between stressor events and toxicity outcomes in intact organisms. However, a major challenge in the broader application of the AOP concept within regulatory toxicology is the development of a robust AOPs that can withstand peer review and acceptance. This is mainly due to the considerable amount of work required to substantiate the modular units of a complete AOP, which can take years from inception to completion. The methodology used here consisted of an initial assessment of a single chemical hazard using the Integrated Approach to Testing and Assessment (IATA) framework. An evidence‐based approach was then used to gather empirical evidence combining systematic literature review methods with expert knowledge to ensure the effectiveness of the AOP development methodology. The structured framework used assured transparency, objectivity and comprehensiveness, and included expert knowledge elicitation for the evaluation of key event relationships (KERs). This stepwise approach led to the development of an AOP that begins with binding of chemicals to Voltage Gate Sodium Channels (VGSC/Nav) during mammalian development leading to adverse consequences in neurodevelopment evidenced as deficits in cognitive functions. Disruption of the formation of precise neural circuits by alterations in VGSC kinetics during the perinatal stages of brain development may also underlie neurodevelopmental disorders. Gaps in our understanding include the specific critical developmental windows and the quantitative relationship of binding to VGSC and subsequent disruption and cognitive function. Despite the limited quantitative information at all KER levels, regulatory applications of this AOP for DNT assessment have been identified. [ABSTRACT FROM AUTHOR]

Neonicotinoids are synthetic, nicotine-derived insecticides used worldwide to protect crops and domestic animals from pest insects. The reported evidence shows that they are also able to interact with mammalian nicotine receptors (nAChRs), triggering detrimental responses in cultured neurons. Exposure to high neonicotinoid levels during the fetal period induces neurotoxicity in animal models. Considering the persistent exposure to these insecticides and the key role of nAChRs in brain development, their potential neurotoxicity on mammal central nervous system (CNS) needs further investigations. We studied here the neurodevelopmental effects of different generations of neonicotinoids on CNS cells in mouse fetal brain and primary cultures and in neuronal cells and organoids obtained from human induced pluripotent stem cells (iPSC). Neonicotinoids significantly affect neuron viability, with imidacloprid (IMI) inducing relevant alterations in synaptic protein expression, neurofilament structures, and microglia activation in vitro, and in the brain of prenatally exposed mouse fetuses. IMI induces neurotoxic effects also on developing human iPSC-derived neurons and cortical organoids. Collectively, the current findings show that neonicotinoids might induce impairment during neuro/immune-development in mouse and human CNS cells and provide new insights in the characterization of risk for the exposure to this class of pesticides. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Despite the growing epidemiological evidence of an association between toxin exposure and developmental neurotoxicity (DNT), systematic testing of DNT is not mandatory in international regulations for admission of pharmaceuticals or industrial chemicals. However, to date around 200 compounds, ranging from pesticides, pharmaceuticals and industrial chemicals, have been tested for DNT in the current OECD test guidelines (TG-443 or TG-426). There are calls for the development of new approach methodologies (NAMs) for DNT, which has resulted in a DNT testing battery using in vitro human cell-based assays. These assays provide a means to elucidate the molecular mechanisms of toxicity in humans which is lacking in animal-based toxicity tests. However, cell-based assays do not represent all steps of the complex process leading to DNT. Validated models with a multi-organ network of pathways that interact at the molecular, cellular and tissue level at very specific timepoints in a life cycle are currently missing. Consequently, whole model organisms are being developed to screen for, and causally link, new molecular targets of DNT compounds and how they affect whole brain development and neurobehavioral endpoints. Given the practical and ethical restraints associated with vertebrate testing, lower animal models that qualify as 3 R (reduce, refine and replace) models, including the nematode (Caenorhabditis elegans) and the zebrafish (Danio rerio) will prove particularly valuable for unravelling toxicity pathways leading to DNT. Although not as complex as the human brain, these 3 R-models develop a complete functioning brain with numerous neurodevelopmental processes overlapping with human brain development. Importantly, the main signalling pathways relating to (neuro)development, metabolism and growth are highly conserved in these models. We propose the use of whole model organisms specifically zebrafish and C. elegans for DNT relevant endpoints. [ABSTRACT FROM AUTHOR]

Studies on the effects of glyphosate (GlyP) and glyphosate‐based herbicides (GBHs) on cerebellar development are extremely limited. This study examined the effects of maternal exposure to GlyP and GBH on rat cerebellar development in male offspring. From day 6 of gestation until day 21 postpartum at weaning, dams were given GlyP at 1.5% or 3.0% in diet or GBH at 1.0% in drinking water (corresponding to 0.36% GlyP). At weaning, GBH exposure was linked to increased numbers of DCX+ migrating granule cells in the cortex and TUNEL+ apoptotic cells in the internal granular layer (IGL), suggesting the disappearance of mismigrated granule cells via apoptosis. GBH also upregulated Nr4a3 and downregulated Cdk5 in the cerebellar vermis, suggesting a causal relation with the impaired granule cell development at this time. GlyP (3.0%) tended to increase in the number of DCX+ migrating granule cells in the IGL and upregulated Nr4a3 at weaning. Both compounds also upregulated genes related to granule cell migration (Astn1, Astn2, Nfia, and/or Nfix) at weaning and in adulthood, which might be an ameliorative response to delayed granule cell migration. Moreover, GBH induced Purkinje cell misalignment at weaning, which could be the result of delayed granule cell migration. In adulthood, GBH was associated with upregulation of the reelin signaling‐related genes Reln, Dab1, and Efnb1, suggesting a compensatory response to Purkinje cell misalignment. GlyP induced the same gene expression changes. These results suggest that GBH reversibly disrupts cerebellar development, primarily by targeting granule cell migration and differentiation, whereas GlyP exhibited similar toxic potential as GBH. [ABSTRACT FROM AUTHOR]

Adult neurotoxicity (ANT) and developmental neurotoxicity (DNT) assessments aim to understand the adverse effects and underlying mechanisms of toxicants on the human nervous system. In recent years, there has been an increasing focus on the so-called new approach methodologies (NAMs). The Organization for Economic Co-operation and Development (OECD), together with European and American regulatory agencies, promote the use of validated alternative test systems, but to date, guidelines for regulatory DNT and ANT assessment rely primarily on classical animal testing. Alternative methods include both non-animal approaches and test systems on non-vertebrates (e.g., nematodes) or non-mammals (e.g., fish). Therefore, this review summarizes the recent advances of NAMs focusing on ANT and DNT and highlights the potential and current critical issues for the full implementation of these methods in the future. The status of the DNT in vitro battery (DNT IVB) is also reviewed as a first step of NAMs for the assessment of neurotoxicity in the regulatory context. Critical issues such as (i) the need for test batteries and method integration (from in silico and in vitro to in vivo alternatives, e.g., zebrafish, C. elegans) requiring interdisciplinarity to manage complexity, (ii) interlaboratory transferability, and (iii) the urgent need for method validation are discussed. [ABSTRACT FROM AUTHOR]

Acetamiprid is a pesticide active substance with insecticidal action whose approval was renewed by Commission Implementing Regulation (EU) 2018/113. In January 2022, the EFSA PPR Panel published a statement following a request from the European Commission to advise on human health or the environment based on new scientific evidence presented by France during the decision‐making phase. In July 2022, by means of a further mandate received from the European Commission, EFSA was requested to provide advice if new information and any other scientific evidence that has become available since the assessment conducted for the renewal in 2018 warrant re‐evaluation of (i) toxicological parameters used for the risk assessment of acetamiprid during the renewal process, including toxicological endpoints; (ii) the residue definition for acetamiprid in products of plant origin; and (iii) the safety of existing maximum residue levels (MRLs). Meanwhile, the applicant of acetamiprid in the EU submitted new toxicology studies regarding the toxicological profile of the metabolite IM‐2‐1. Furthermore, the European Commission was made aware that several recent publications in scientific literature were made available after the literature searches conducted by EFSA. As the new data could affect the advice that EFSA was expected to deliver through the 2022 mandate, EFSA was further requested to consider this information by means of a revised mandate received in September 2023. As regards re‐evaluation of point (i) in this statement, this was addressed by an EFSA Working Group integrating all the available evidence. The results of the weight of evidence indicated that there are major uncertainties in the body of evidence for the developmental neurotoxicity (DNT) properties of acetamiprid and further data are therefore needed to come to a more robust mechanistic understanding to enable appropriate hazard and risk assessment. In view of these uncertainties, the EFSA WG proposed to lower the acceptable daily intake (ADI) and acute reference dose (ARfD) from 0.025 to 0.005 mg/kg body weight (per day). A revised residue definition for risk assessment was proposed for leafy and fruit crops as sum of acetamiprid and N‐desmethyl‐acetamiprid (IM‐2‐1), expressed as acetamiprid. Regarding pulses/oilseeds, root crops and cereals, the new data received did not indicate a need to modify the existing residue definition for risk assessment, which therefore remains as parent acetamiprid. Regarding the residue definition for enforcement, the available data did not indicate a need to modify the existing definition because acetamiprid is still a sufficient marker of the residues in all crop groups. Considering the new health‐based guidance values derived in the present statement, a risk for consumer has been identified for 38 MRLs currently in place in the EU Regulation. Consequently, EFSA recommended to lower the existing MRLs for 38 commodities based on the assessment of fall‐back Good Agricultural Practices received within an ad hoc data call. Some fall‐back MRLs proposals require further risk management considerations. [ABSTRACT FROM AUTHOR]

Alcohol is toxic to neurons and can trigger alcohol-related brain damage, neuronal loss, and cognitive decline. Neuronal cells may be vulnerable to alcohol toxicity and damage from oxidative stress after differentiation. To consider this further, the toxicity of alcohol to undifferentiated SH-SY5Y cells was compared with that of cells that had been acutely differentiated. Cells were exposed to alcohol over a concentration range of 0–200 mM for up to 24 h and alcohol effects on cell viability were evaluated via MTT and LDH assays. Effects on mitochondrial morphology were examined via transmission electron microscopy, and mitochondrial functionality was examined using measurements of ATP and the production of reactive oxygen species (ROS). Alcohol reduced cell viability and depleted ATP levels in a concentration- and exposure duration-dependent manner, with undifferentiated cells more vulnerable to toxicity. Alcohol exposure resulted in neurite retraction, altered mitochondrial morphology, and increased the levels of ROS in proportion to alcohol concentration; these peaked after 3 and 6 h exposures and were significantly higher in differentiated cells. Protein carbonyl content (PCC) lagged behind ROS production and peaked after 12 and 24 h, increasing in proportion to alcohol concentration, with higher levels in differentiated cells. Carbonylated proteins were characterised by their denatured molecular weights and overlapped with those from adult post-mortem brain tissue, with levels of PCC higher in alcoholic subjects than matched controls. Hence, alcohol can potentially trigger cell and tissue damage from oxidative stress and the accumulation of oxidatively damaged proteins. [ABSTRACT FROM AUTHOR]

Many epidemiological and animal exposure studies have suggested that exposure to environmental substances is a major risk factor for developmental neurotoxicity (DNT), such as in autism, and is related to the increasing relevance of neurodevelopmental disorders. Recent efforts have led to the development of various in vitro approaches that use cell lines and pluripotent stem cells to assess numerous environmental substances. In this study, we developed a method for assessing DNT using a mouse embryonic stem (mES) cell model that focuses on differentiation into neuronal cells (neural cells and astrocytes). Using this model system, we found that six insecticides inhibited the differentiation of neural precursor cells into astrocytes. Our data indicated that the effects of insecticides on glial differentiation were more sensitive than those of several DNT markers reported in previous studies. This mES cell model can make a quick assessment of DNT potential and may be a useful tool for screening substances with potential to induce DNT. [ABSTRACT FROM AUTHOR]

Evaluating the neurodevelopmental effects of thyroid-disrupting chemicals is challenging. Although some standardized developmental and reproductive toxicity studies recommend serum thyroxine (T4) measures in developing rats, extrapolating between a serum T4 reduction and neurodevelopmental outcomes is not straightforward. Previously, we showed that the blood-brain and blood-cerebrospinal fluid barriers may be affected by developmental hypothyroidism in newborn rats. Here, we hypothesized that if the brain barriers were functionally disturbed by abnormal thyroid action, then small molecules may escape from the brain tissue and into general circulation. These small molecules could then be identified in blood samples, serving as a direct readout of thyroid-mediated developmental neurotoxicity. To address these hypotheses, pregnant rats were exposed to propylthiouracil (PTU, 0 or 3 ppm) to induce thyroid hormone insufficiency, and dams were permitted to give birth. PTU significantly reduced serum T4 in postnatal offspring. Consistent with our hypothesis, we show that tight junctions of the brain barriers were abnormal in PTU-exposed pups, and the blood-brain barrier exhibited increased permeability. Next, we performed serum microRNA Sequencing (miRNA-Seq) to identify noncoding RNAs that may reflect these neurodevelopmental disturbances. Of the differentially expressed miRNAs identified, 7 were upregulated in PTU-exposed pups. Validation by qRT-PCR shows that miR-495 and miR-543-3p were similarly upregulated in males and females. Interestingly, these miRNAs have been linked to cell junction dysfunction in other models, paralleling the identified abnormalities in the rat brain. Taken together, these data show that miR-495 and miR-543-3p may be novel in vivo biomarkers of thyroid-mediated developmental neurotoxicity. [ABSTRACT FROM AUTHOR]

With the incidence of neurodevelopmental disorders on the rise, it is imperative to screen and evaluate developmental neurotoxicity (DNT) compounds from a large number of environmental chemicals and understand their mechanisms. In this study, DNT qualitative structure-activity relationship (QSAR) study was carried out for the first time based on DNT data of mammals and structural characterization of DNT compounds was preliminarily illustrated. Five different classification algorithms and two feature selection methods were used to construct prediction models. The best model had good predictive ability on the external test set, but a small application domain (AD). Through combining of three different models, both MCC and AD values were improved. Furthermore, electronical properties, van der Waals volume-related properties and S, Cl or P containing substructure were found to be associated with DNT through modeling descriptors analysis and structure alerts (SAs) identification. This study lays a foundation for further DNT prediction of environmental exposures in human and contributes to the understanding of DNT mechanism. Graphical Abstract [ABSTRACT FROM AUTHOR]

Etomidate (ET) is widely used as an anesthetic in clinical practice. The long-term use of ET can lead to negative effects, such as decreased consciousness and cognitive impairment. However, the developmental neurotoxicity of ET on fish remains unclear. To address this issue, zebrafish embryos were exposed to ET from 2 hpf (hours post-fertilization) to 168 hpf at 0. 010, 0. 091, 0. 501, 9. 400, 84. 31 and 664. 4 μg·L-1. The physiological development, behavior and cell apoptosis, and transcriptional expression of genes related to dopamine (DA) and γ-aminobutyric acid (GABA) pathway were detected to investigate the developmental neurotoxicity of ET. The results showed that 664.4 μg·L-1 ET decreased hatching rate at 48 hpf and 60 hpf. 0.091 μg·L-1 and 0.501 μg· L-1 ET decreased body length in larvae at 168 hpf. At 48 hpf, 0.010, 0.091, 0.501, 9.400, 84.31 and 664.4 μg·L-1 ET increased malformation. 0.501, 9.400, 84.31 and 664.4 μg·L-1 ET increased malformation at 96 hpf. Additionally, 0.501, 9.400 and 664.4 μg·L-1 ET increased malformation at 168 hpf. The 0.010 μg·L-1 ET caused cell apoptosis in the brain, while 664.4 μg·L-1 ET inhibited apoptosis. In addition, behavior analysis indicated that ET inhibited touch behavior at all concentrations. The 0.091 μg·L-1 ET enhanced swimming behavior in zebrafish larvae, while 664.4 μg·L-1 ET weakened swimming behavior. ET increased anxious behavior in a concentration-dependent manner. The qPCR data showed that ET up-regulated the transcriptions of genes related to DA and GABA pathway at low concentration, while ET down-regulated the transcriptions of these genes at high concentration. The above results indicate that ET has significant neurodevelopmental toxicity in early development of zebrafish. [ABSTRACT FROM AUTHOR]

The Health and Environmental Sciences Institute Developmental and Reproductive Toxicology (HESI‐DART) group held a hybrid in‐person and virtual workshop in Washington, DC, in 2022. The workshop was entitled, "Interpretation of DART in Regulatory Contexts and Frameworks." There were 154 participants (37 in person and 117 virtual) across 9 countries. The purpose of the workshop was to capture key consensus approaches used to assess DART risks associated with chemical product exposure when a nonclinical finding is identified. The decision‐making process for determining whether a DART endpoint is considered adverse is critical because the outcome may have downstream implications (e.g., increased animal usage, modifications to reproductive classification and pregnancy labeling, impact on enrollment in clinical trials and value chains). The workshop included a series of webinar modules to train and engage in discussions with federal and international regulators, clinicians, academic investigators, nongovernmental organizations, contract research organization scientists, and private sector scientists on the best practices and principles of interpreting DART and new approach methodologies in the context of regulatory requirements and processes. Despite the differences in regulatory frameworks between the chemical and pharmaceutical sectors, the same foundational principles for data interpretation should be applied. The discussions led to the categorization of principles, which offer guidance for the systematic interpretation of data. Step 1 entails identifying any hazard by closely analyzing the data at the study endpoint level, while Step 2 involves assessing risk using weight of evidence. These guiding principles were derived from the collective outcomes of the workshop deliberations. [ABSTRACT FROM AUTHOR]

Environmental and genetic factors influence synapse formation. Numerous animal experiments have revealed that pesticides, including herbicides, can disturb normal intracellular signals, gene expression, and individual animal behaviors. However, the mechanism underlying the adverse outcomes of pesticide exposure remains elusive. Herein, we investigated the effect of maternal exposure to the herbicide glufosinate ammonium (GLA) on offspring neuronal synapse formation in vitro. Cultured cerebral cortical neurons prepared from mouse embryos with maternal GLA exposure demonstrated impaired synapse formation induced by synaptic organizer neuroligin 1 (NLGN1)-coated beads. Conversely, the direct administration of GLA to the neuronal cultures exhibited negligible effect on the NLGN1-induced synapse formation. The comparison of the transcriptomes of cultured neurons from embryos treated with maternal GLA or vehicle and a subsequent bioinformatics analysis of differentially expressed genes (DEGs) identified "nervous system development," including "synapse," as the top-ranking process for downregulated DEGs in the GLA group. In addition, we detected lower densities of parvalbumin (Pvalb)-positive neurons at the postnatal developmental stage in the medial prefrontal cortex (mPFC) of offspring born to GLA-exposed dams. These results suggest that maternal GLA exposure induces synapse pathology, with alterations in the expression of genes that regulate synaptic development via an indirect pathway distinct from the effect of direct GLA action on neurons. [ABSTRACT FROM AUTHOR]

Exposure to metal mixtures is recognized as a real-life scenario, needing novel studies that can assess their complex effects on brain development. There is still a significant public health concern associated with chronic low levels of metal exposure. In contrast to other metals, these three metals (As, Pb, and Mn) are commonly found in various environmental and industrial contexts. In addition to additive or synergistic interactions, concurrent exposure to this metal mixture may also have neurotoxic effects that differ from those caused by exposure to single components. The NMDA receptor and several important signaling proteins are involved in learning, memory, and synaptic plasticity in the hippocampus, including CaMKII, postsynaptic density protein-95 (PSD-95), synaptic Ras GTPase activating protein (SynGAP), a negative regulator of Ras-MAPK activity, and CREB. We hypothesized that alterations in the above molecular players may contribute to metal mixture developmental neurotoxicity. Thus, the aim of this study was to investigate the effect of these metals and their mixture at low doses (As 4 mg, Pb 4 mg, and Mn 10 mg/kg bw/p.o) on NMDA receptors and their postsynaptic signaling proteins during developing periods (GD6 to PD59) of the rat brain. Rats exposed to As, Pb, and Mn individually or at the same doses in a triple-metal mixture (MM) showed impairments in learning and memory functions in comparison to the control group rats. Declined protein expressions of NR2A, PSD-95, p- CaMKII, and pCREB were observed in the metal mix-exposed rats, while the expression of SynGAP was found to be enhanced in the hippocampus as compared to the controls on PD60. Thereby, our data suggest that alterations in the NMDA receptor complex and postsynaptic signaling proteins could explain the cognitive dysfunctions caused by metal-mixture-induced developmental neurotoxicity in rats. These outcomes indicate that incessant metal mixture exposure may have detrimental consequences on brain development. [ABSTRACT FROM AUTHOR]

Introduction: Flame retardants (FRs) are common bodily and environmental pollutants, creating concern about their potential toxicity. We and others have found that the commercial mixture FireMaster® 550 (FM 550) or its individual brominated (BFR) and organophosphate ester (OPFR) components are potential developmental neurotoxicants. Using Wistar rats, we previously reported that developmental exposure to FM 550 or its component classes produced sex- and compound-specific effects on adult socioemotional behaviors. The underlying mechanisms driving the behavioral phenotypes are unknown. Methods: To further mechanistic understanding, here we conducted transcriptomics in parallel with a novel lipidomics approach using cortical tissues from newborn siblings of the rats in the published behavioral study. Inclusion of lipid composition is significant because it is rarely examined in developmental neurotoxicity studies. Pups were gestationally exposed via oral dosing to the dam to FM 550 or the BFR or OPFR components at environmentally relevant doses. Results: The neonatal cortex was highly sexually dimorphic in lipid and transcriptome composition, and males were more significantly impacted by FR exposure. Multiple adverse modes of action for the BFRs and OPFRs on neurodevelopment were identified, with the OPFRs being more disruptive than the BFRs via multiple mechanisms including dysregulation of mitochondrial function and disruption of cholinergic and glutamatergic systems. Disrupted mitochondrial function by environmental factors has been linked to a higher risk of autism spectrum disorders and neurodegenerative disorders. Impacted lipid classes included ceramides, sphingomyelins, and triacylglycerides. Robust ceramide upregulation in the OPFR females could suggest a heightened risk of brain metabolic disease. Conclusions: This study reveals multiple mechanisms by which the components of a common FR mixture are developmentally neurotoxic and that the OPFRs may be the compounds of greatest concern. [ABSTRACT FROM AUTHOR]

A recent systematic review and dose response meta-analysis, by Iamandii et al., considered the question "Does fluoride exposure affect thyroid function?" The authors concluded that, overall, exposure to high-fluoride drinking water appears to non-linearly affect thyroid function and increase TSH release in children, starting above a threshold of exposure, and to increase the risk of some thyroid diseases. The threshold of exposure for the TSH increase was 2 mg/L, or 2.5 mg/L when the studies with the best quality were considered. Although the lowest-observed-adverse-effect level (LOAEL) observed for TSH was a water fluoride level of approximately 2.5 mg/L, the changes in Total T3 and Total T4, a negative association, were present from very low levels, e.g. a water fluoride level of 0.2 mg/L and did not show a threshold effect. Fluoride in drinking water has been associated, by Hall et al., with an increased risk of hypothyroidism in pregnant women. A 0.5 mg/L increase in drinking water fluoride concentration was associated with a 1.65 (95% confidence interval [CI]: 1.04, 2.60) increased odds of primary hypothyroidism. Children born to women with primary hypothyroidism had lower FSIQ scores compared to children of euthyroid women, especially among boys (B coefficient : -8.42; 95%CI: -15.33, -1.50). The threshold, or point of departure, for fluoride in drinking water varies for different adverse effects such as the development of crippling skeletal fluorosis, dental fluorosis, an elevated TSH, a decreased Total T3 and Total T4, and impaired IQ in children. A recent paper by Grandjean et al. provides evidence that foetal toxicity can occur with a reduction in IQ when the maternal urinary fluoride is above 0.3 mg/L. A BMCL fluoride concentration in maternal urine during pregnancy of about 0.3 mg/L can be estimated to occur with a drinking water fluoride level of 0.24 mg/L, or approximately 0.3 mg/L. These findings suggest that, in order to protect the most vulnerable, a foetus in utero, public health attention should be given to elevated fluoride intakes during pregnancy, whether from drinking water, black tea, or other sources. [ABSTRACT FROM AUTHOR]

Published evidence over the past few decades suggests that general anesthetics could be neurotoxins especially when administered at the extremes of age. The reported pathology is not only at the morphological level when examined in very young and aged brains, given that, importantly, newly developing evidence suggests a variety of behavioral impairments. Since anesthesia is unavoidable in certain clinical settings, we should consider the development of new anesthetics. A promising and safe solution could be a new family of anesthetics referred to as neuroactive steroids. In this review, we summarize the currently available evidence regarding their anesthetic and analgesic properties. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is the fastest-growing neurological disease worldwide, with increases outpacing aging and occurring most rapidly in recently industrialized areas, suggesting a role of environmental factors. Epidemiological, post-mortem, and mechanistic studies suggest that persistent organic pollutants, including the organochlorine pesticide dieldrin, increase PD risk. In mice, developmental dieldrin exposure causes male-specific exacerbation of neuronal susceptibility to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and synucleinopathy. Specifically, in the α-synuclein (α-syn) pre-formed fibril (PFF) model, exposure leads to increased deficits in striatal dopamine (DA) turnover and motor deficits on the challenging beam. Here, we hypothesized that alterations in DA handling contribute to the observed changes and assessed vesicular monoamine transporter 2 (VMAT2) function and DA release in this dieldrin/PFF 2-hit model. Female C57BL/6 mice were exposed to 0.3 mg/kg dieldrin or vehicle every 3 days by feeding, starting at 8 weeks of age and continuing throughout breeding, gestation, and lactation. Male offspring from independent litters underwent unilateral, intrastriatal injections of α-syn PFFs at 12 weeks of age, and vesicular 3H-DA uptake assays and fast-scan cyclic voltammetry were performed 4 months post-PFF injection. Dieldrin-induced an increase in DA release in striatal slices in PFF-injected animals, but no change in VMAT2 activity. These results suggest that developmental dieldrin exposure increases a compensatory response to synucleinopathy-triggered striatal DA loss. These findings are consistent with silent neurotoxicity, where developmental exposure to dieldrin primes the nigrostriatal striatal system to have an exacerbated response to synucleinopathy in the absence of observable changes in typical markers of nigrostriatal dysfunction and degeneration. [ABSTRACT FROM AUTHOR]

Despite the widespread agricultural use of dithianon as an antifungal agent, its neurotoxic implications for humans and wildlife have not been comprehensively explored. Using zebrafish embryonic development as our model, we found that dithianon treatment induced behavioral alterations in zebrafish larvae that appeared normal. Detailed quantitative analyses showed that dithianon at ≥0.0001 µgmL−1 induced cytoplasmic and mitochondrial antioxidant responses sequentially, followed by the disruption of mitochondrial and cellular homeostasis. Additionally, dithianon at 0.01 and 0.1 µgmL−1 downregulated the expressions of glutamatergic (slc17a6b), GABAergic (gad1b), and dopaminergic (th) neuronal markers. Contrarily, dithianon upregulated the expression of the oligodendrocyte marker (olig2) at concentrations of 0.001 and 0.01 µgmL−1, concurrently suppressing the gene expression of the glucose transporter slc2a1a/glut1. Particularly, dithianon-induced increase in reactive oxygen species (ROS) production was reduced by both N-acetylcysteine (NAC) and betaine; however, only NAC prevented dithianon-induced mortality of zebrafish embryos. Moreover, NAC specifically prevented dithianon-induced alterations in glutamatergic and dopaminergic neurons while leaving GABAergic neurons unaffected, demonstrating that the major neurotransmission systems in the central nervous system differentially respond to the protective effects. Our findings contribute to a better understanding of the neurotoxic potential of dithianon and to developing preventive strategies. [ABSTRACT FROM AUTHOR]

Epidemiological and experimental studies indicate that maternal exposure to environmental pollutants impairs the cognitive and motor functions of offspring in humans and laboratory animals. Infant ultrasonic vocalizations (USVs), the communicative behavior of pups toward caregivers, are impaired in rodent models of neurodevelopmental disorders, suggesting a useful method to evaluate the developmental neurotoxicity of environmental pollutants. Therefore, we investigated USVs emitted by mouse pups of dams exposed to 2-chloro-3,7,8-tribromodibenzofuran (TeXDF) and 1,2,3,7,8-pentabromodibenzofuran (PeBDF), which are detected in the actual environment. The USV duration and number in the pups born to dams administered with TeXDF 40 μg/kg body weight (b.w.), but not 8 μg/kg b.w., on gestational day (GD) 12.5, were significantly lower than those in the corresponding pups on postnatal days 3–9. Conversely, there was no statistical change in the USVs emitted by the pups of dams administered with PeBDF 35 or 175 μg/kg b.w. on GD 12.5. To examine whether maternal exposure leads to behavioral impairments in adulthood, we analyzed exploratory behaviors in a novel environment using IntelliCage, a fully automated testing apparatus for group-housed mice. Neither TeXDF nor PeBDF exposure induced significant differences in offspring exploration. Considered together, our findings revealed that TeXDF induces atypical USV emission in infant mice, suggesting the importance of further studies on the risk assessment of mixed brominated/chlorinated dibenzo-p-dioxins and dibenzofurans. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Fluoride can cause developmental neurotoxicity; however, the precise mechanism has yet to be determined. We aimed to explore the possible role and mechanism of fluoride-induced developmental neurotoxicity, specifically the significance of the lysosomal stress response. As an in vivo model, Sprague Dawley rats were exposed to sodium fluoride (NaF) from embryo to 2 months of age. We found that NaF caused autophagic flux blockage and apoptosis in the rat hippocampus. These results were validated in human neuroblastoma (SH-SY5Y) cells in vitro. In addition, in SH-SY5Y cells, NaF hindered autophagosome-lysosome fusion, decreased lysosomal degradation, and elevated lysosomal pH, which is the most prominent hallmark of a lysosomal stress response. Interestingly, rapamycin promoted autophagosome-lysosome fusion, effectively restoring autophagic flux and reducing apoptosis. Notably, bafilomycin A1, a lysosomal lumen alkalizer, unsurprisingly exacerbated the NaF-induced increase in lysosomal pH and decreased lysosomal degradability, as well as enhanced apoptosis of SH-SY5Y cells. In conclusion, our results suggest that NaF exposure initiates excessive lysosomal stress response, resulting in elevated lysosomal pH, decreased lysosomal degradation, and blocked autophagic flux, which leads to neuronal apoptosis. Thus, the lysosomal stress response may be a promising target for the prevention and treatment of fluoride-induced developmental neurotoxicity. Highlights: 1. NaF caused autophagic flux blockage and apoptosis in vivo and in vitro 2. NaF hindered autophagosome-lysosome fusion and elevated lysosomal pH 3. NaF caused excessive lysosomal stress response and impaired lysosomal degradation [ABSTRACT FROM AUTHOR]

Introduction: Acetamiprid (ACE) and imidacloprid (IMI), the neonicotinoid chemicals, are widely used as pesticides because of their rapid insecticidal activity. Although these neonicotinoids exert very low toxicity in mammals, the effects of early, low-level, chronic exposure on the adult central nervous system are largely unclear. This study investigated the effects of low-level, chronic neonicotinoids exposure in early life on the brain functions of adult mice, using environmentally relevant concentrations. Methods: We exposed mice to an acceptable daily intake level of neonicotinoids in drinking water during the prenatal and postnatal periods. Additionally, we also exposed mice to nicotine (NIC) as a positive control. We then examined the effects on the central nervous system in adult male offspring. Results: In the IMI and NIC exposure groups, we detected behavior that displayed impairment in learning and memory. Furthermore, immunohistochemical analysis revealed a decrease in SOX2 (as a neural stem cell marker) and GFAP (as an astrocyte marker) positive cells of the hippocampal dentate gyrus in the IMI and NIC exposure groups compared to the control group. Discussion: These results suggest that exposure to neonicotinoids at low levels in early life affects neural circuit base formation and post-maturation behavior. Therefore, in the central nervous system of male mice, the effects of low-level, chronic neonicotinoids exposure during the perinatal period were different from the expected effects of neonicotinoids exposure in mature animals. [ABSTRACT FROM AUTHOR]

Trichlorfon is an organophosphorus pesticide widely used in aquaculture and has potential neurotoxicity, but the underlying mechanism remains unclear. In the present study, zebrafish embryos were exposed to trichlorfon at concentrations (0, 0.1, 2 and 5 mg/L) used in aquaculture from 2 to 144 h post fertilization. Trichlorfon exposure reduced the survival rate, hatching rate, heartbeat and body length and increased the malformation rate of zebrafish larvae. The locomotor activity of larvae was significantly reduced. The results of molecular docking revealed that trichlorfon could bind to acetylcholinesterase (AChE). Furthermore, trichlorfon significantly inhibited AChE activity, accompanied by decreased acetylcholine, dopamine and serotonin content in larvae. The transcription patterns of genes related to acetylcholine (e.g., ache, chrna7, chata, hact and vacht), dopamine (e.g., drd4a and drd4b) and serotonin systems (e.g., tph1, tph2, tphr, serta, sertb, htrlaa and htrlab) were consistent with the changes in acetylcholine, dopamine, serotonin content and AChE activity. The genes related to the central nervous system (CNS) (e.g., a1-tubulin, mbp, syn2a, shha and gap-43) were downregulated. Our results indicate that the developmental neurotoxicity of trichlorfon might be attributed to disorders of cholinergic, dopaminergic and serotonergic signaling and the development of the CNS. [ABSTRACT FROM AUTHOR]

Per- and polyfluoroalkyl substances (PFASs) are widespread environmental pollutants. There is increasing evidence that PFASs have various adverse health effects, including renal toxicity, metabolic dysfunction, endocrine disruption, and developmental toxicity. PFASs have been found to accumulate in the placenta, and some PFASs can cross the placental barrier and subsequently accumulate in the fetus via the maternal–fetal circulation. An increasing number of studies have shown that early life exposure to PFASs can affect fetal neurodevelopment. This paper reviews the characteristics of indirect exposure to PFASs in early life, the effects on neurodevelopment in offspring, and the possible mechanisms of toxic effects. [ABSTRACT FROM AUTHOR]

Background: In the European Union (EU), the safety assessment of plant protection products relies to a large extent on toxicity studies commissioned by the companies producing them. By law, all performed studies must be included in the dossier submitted to authorities when applying for approval or renewal of the active substance. Methods: For one type of toxicity, i.e. developmental neurotoxicity (DNT), we evaluated if studies submitted to the U.S. Environmental Protection Agency (EPA) had also been disclosed to EU authorities. Results: We identified 35 DNT studies submitted to the U.S. EPA and with the corresponding EU dossiers available. Of these, 9 DNT studies (26%) were not disclosed by the pesticide company to EU authorities. For 7 of these studies, we have identified an actual or potential regulatory impact. Conclusions: We conclude that (1) non-disclosure of DNT studies to EU authorities, in spite of clear legal requirements, seems to be a recurring phenomenon, (2) the non-disclosure may introduce a bias in the regulatory risk assessment, and (3) without full access to all performed toxicity studies, there can be no reliable safety evaluation of pesticides by EU authorities. We suggest that EU authorities should cross-check their data sets with their counterparts in other jurisdictions. In addition, applications for pesticide approval should be cross-checked against lists of studies performed at test facilities operating under Good Laboratory Practice (GLP), to ensure that all studies have been submitted to authorities. Furthermore, rules should be amended so that future studies should be commissioned by authorities rather than companies. This ensures the authorities' knowledge of existing studies and prevents the economic interest of the company from influencing the design, performance, reporting and dissemination of studies. The rules or practices should also be revised to ensure that non-disclosure of toxicity studies carries a significant legal risk for pesticide companies. [ABSTRACT FROM AUTHOR]

Various forms of pesticides have been reported to be among the environmental toxicants, which are detrimental to human health. The active ingredients of these formulations can enter the human body through air, food, or water. Epidemiological studies suggest that these compounds strongly affect the developing brain in fetal and infant stages due to their ability to breach the underdeveloped blood–brain barrier. Since neural progenitor stem cells (NPCs) in the developing brain are the most vulnerable to these compounds, the mechanisms by which NPCs experience toxicity upon exposure to these chemicals must be investigated. Here, we assessed the viability of human fetal NPCs in 2D cultures in the presence of the active ingredients of six widely used pesticides using Live/Dead® and Hoechst staining. The IC50 values ranged from 4.1–201 μM. A significant drop in cell viability with increasing toxicant concentration (p < 0.01) was noted, with the order of toxicity being malathion < 4-aminopyridine < methoprene < prallethrin < temephos < pyriproxyfen. Changes in cellular biomechanical characteristics (Young's modulus, tether force, membrane tension, and tether radius) were quantified using atomic force microscopy, whereas cell migration was elucidated over 48 h using a customized wound-healing assay. The Young's modulus of fetal NPCs exposed to IC50/2 doses of these compounds was reduced by 38–70% and that of those exposed to IC50 doses was reduced by 71–80% (p < 0.001 vs. controls for both; p < 0.01 for IC50 vs. IC50/2 for each compound). Similar patterns were noted for tether forces and membrane tension in fetal NPCs. NPC migration was found to be compound type- and dose-dependent. These results attest to the significant detrimental effects of these compounds on various aspects of the human fetal NPC phenotype, and the utility of cell mechanics as a marker to assess developmental neurotoxicity. [ABSTRACT FROM AUTHOR]

Introduction: The development and maintenance of neural circuits is highly sensitive to neural activity. General anesthetics have profound effects on neural activity and, as such, there is concern that these agents may alter cellular integrity and interfere with brain wiring, such as when exposure occurs during the vulnerable period of brain development. Under those conditions, exposure to anesthetics in clinical use today causes changes in synaptic strength and number, widespread apoptosis, and long-lasting cognitive impairment in a variety of animal models. Remarkably, most anesthetics produce these effects despite having differing receptor mechanisms of action. We hypothesized that anesthetic agents mediate these effects by inducing a shared signaling pathway. Methods: We exposed cultured cortical cells to propofol, etomidate, or dexmedetomidine and assessed the protein levels of dozens of signaling molecules and post-translational modifications using reverse phase protein arrays. To probe the role of neural activity, we performed separate control experiments to alter neural activity with non-anesthetics. Having identified anesthetic-induced changes in vitro, we investigated expression of the target proteins in the cortex of sevoflurane anesthetized postnatal day 7 mice by Western blotting. Results: All the anesthetic agents tested in vitro reduced phosphorylation of the ribosomal protein S6, an important member of the mTOR signaling pathway. We found a comparable decrease in cortical S6 phosphorylation by Western blotting in sevoflurane anesthetized neonatal mice. Using a systems approach, we determined that propofol, etomidate, dexmedetomidine, and APV/TTX all similarly modulate a signaling module that includes pS6 and other cell mediators of the mTOR-signaling pathway. Discussion: Reduction in S6 phosphorylation and subsequent suppression of the mTOR pathway may be a common and novel signaling event that mediates the impact of general anesthetics on neural circuit development. [ABSTRACT FROM AUTHOR]

Organophosphate (OP) and carbamate pesticides are toxic to pests through targeted inhibition of acetylcholinesterase (AChE). However, OPs and carbamates may be harmful to non-target species including humans and could induce developmental neurotoxicity if differentiated or differentiating neurons are particularly vulnerable to neurotoxicant exposures. Hence, this study compared the neurotoxicity of OPs, chlorpyrifos-oxon (CPO), and azamethiphos (AZO) and the carbamate pesticide, aldicarb, to undifferentiated versus differentiated SH-SY5Y neuroblastoma cells. OP and carbamate concentration-response curves for cell viability were undertaken using 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays and cellular bioenergetic capacity assessed via quantitation of cellular ATP levels. Concentration-response curves for inhibition of cellular AChE activity were also generated and the production of reactive oxygen species (ROS) was monitored using a 2′,7′-dichlorofluorescein diacetate (DCFDA) assay. The OPs and aldicarb reduced cell viability, cellular ATP levels, and neurite outgrowth in a concentration-dependent fashion, from a threshold concentration of ≥10 µM. Neurotoxic potency was in the order AZO > CPO > aldicarb for undifferentiated cells but CPO > AZO > aldicarb for differentiated cells and this toxic potency of CPO reflected its more extensive induction of reactive oxygen species (ROS) and generation of carbonylated proteins that were characterized by western blotting. Hence, the relative neurotoxicity of the OPs and aldicarb in part reflects non-cholinergic mechanisms that are likely to contribute to developmental neurotoxicity. [ABSTRACT FROM AUTHOR]

Difenoconazole is a type of triazole fungicide that is widely used in the treatment of plant diseases. Triazole fungicides have been shown in several studies to impair the development of the nervous system in zebrafish embryos. There is still little known about difenoconazole-induced neurotoxicity in fish. In this study, zebrafish embryos were exposed to 0.25, 0.5, and 1 mg/L of difenoconazole solution until 120 h post-fertilization (hpf). The difenoconazole-exposed groups showed concentration-dependent inhibitory tendencies in heart rate and body length. Malformation rate and spontaneous movement of zebrafish embryos increased, and the locomotor activity decreased in the highest exposure group. The content of dopamine and acetylcholine was reduced significantly in difenoconazole treatment groups. The activity of acetylcholinesterase (AChE) was also increased after treatment with difenoconazole. Furthermore, the expression of genes involved in neurodevelopment was remarkably altered, which corresponded with the alterations of neurotransmitter content and AChE activity. These results indicated that difenoconazole might affect the development of the nervous system through influencing neurotransmitter levels, enzyme activity, and the expression of neural-related genes, ultimately leading to abnormal locomotor activity in the early stages of zebrafish. [ABSTRACT FROM AUTHOR]

Building a precise alternative neurotoxicological test is of great importance to respond to societal and ethical requirements. In this study, a new developmental neurotoxicity test (DNT) was established with the human neural progenitor cell line. ReNcell CX cells were exposed to neurotoxic chemicals (aphidicolin, hydroxyurea, cytosine arabinoside, 5-fluorouracil, and ochratoxin A) or non-neurotoxic chemicals (sodium gluconate, sodium bicarbonate, penicillin G, and saccharin). Propidium iodide (PI) was used to evaluate cell viability. BrdU and Ki-76 were employed to determine cell proliferation. Based on the cell viability and proliferation, mathematical models were built by linear discriminant analysis. Furthermore, the neurotoxic-considered chemicals inhibited cell cycle progression at the protein level, supporting the biomolecular rationale for the predictive model. Overall, these results show that the new test method can be used to determine the potential developmental neurotoxicants or new drug candidates. [ABSTRACT FROM AUTHOR]

Exposure to chemicals may pose a greater risk to vulnerable groups, including pregnant women, fetuses, and children, that may lead to diseases linked to the toxicants' target organs. Among chemical contaminants, methylmercury (MeHg), present in aquatic food, is one of the most harmful to the developing nervous system depending on time and level of exposure. Moreover, certain man-made PFAS, such as PFOS and PFOA, used in commercial and industrial products including liquid repellants for paper, packaging, textile, leather, and carpets, are developmental neurotoxicants. There is vast knowledge about the detrimental neurotoxic effects induced by high levels of exposure to these chemicals. Less is known about the consequences that low-level exposures may have on neurodevelopment, although an increasing number of studies link neurotoxic chemical exposures to neurodevelopmental disorders. Still, the mechanisms of toxicity are not identified. Here we review in vitro mechanistic studies using neural stem cells (NSCs) from rodents and humans to dissect the cellular and molecular processes changed by exposure to environmentally relevant levels of MeHg or PFOS/PFOA. All studies show that even low concentrations dysregulate critical neurodevelopmental steps supporting the idea that neurotoxic chemicals may play a role in the onset of neurodevelopmental disorders. [ABSTRACT FROM AUTHOR]

Adverse outcome pathways (AOPs) are organized sequences of key events (KEs) that are triggered by a xenobiotic-induced molecular initiating event (MIE) and summit in an adverse outcome (AO) relevant to human or ecological health. The AOP framework causally connects toxicological mechanistic information with apical endpoints for application in regulatory sciences. AOPs are very useful to link endophenotypic, cellular endpoints in vitro to adverse health effects in vivo. In the field of in vitro developmental neurotoxicity (DNT), such cellular endpoints can be assessed using the human "Neurosphere Assay," which depicts different endophenotypes for a broad variety of neurodevelopmental KEs. Combining this model with large-scale transcriptomics, we evaluated DNT hazards of two selected Chinese herbal medicines (CHMs) Lei Gong Teng (LGT) and Tian Ma (TM), and provided further insight into their modes-of-action (MoA). LGT disrupted hNPC migration eliciting an exceptional migration endophenotype. Time-lapse microscopy and intervention studies indicated that LGT disturbs laminin-dependent cell adhesion. TM impaired oligodendrocyte differentiation in human but not rat NPCs and activated a gene expression network related to oxidative stress. The LGT results supported a previously published AOP on radial glia cell adhesion due to interference with integrin-laminin binding, while the results of TM exposure were incorporated into a novel putative, stressor-based AOP. This study demonstrates that the combination of phenotypic and transcriptomic analyses is a powerful tool to elucidate compounds' MoA and incorporate the results into novel or existing AOPs for a better perception of the DNT hazard in a regulatory context. Highlights: A 3D in vitro model based on human neural progenitor cells was applied for assessing DNT potential of two selected CHMs. Both CHMs caused DNT endophenotypes in vitro, e.g. disturbed radial glia migration or impaired oligodendrocyte differentiation. Combining phenotypic with transcriptomic analyses enlight compounds' modes-of-action. Application and development of AOPs for a better comprehension of DNT hazards. [ABSTRACT FROM AUTHOR]

Two organophosphate esters used as flame retardants and plasticizers, triphenyl phosphate (TPHP) and isopropylated phenyl phosphate (IPP), have been detected in environmental samples around the world. Human exposure primarily occurs via oral ingestion with reported higher concentrations in children. Currently, there are no data to evaluate potential risk from exposure to either TPHP or IPP during fetal development. These short-term perinatal studies in rats provide preliminary toxicity data for TPHP and IPP, including information on transfer to fetus/offspring and across the pup blood-brain barrier. In separate experiments, TPHP or IPP were administered via dosed feed at concentrations 0, 1000, 3000, 10 000, 15 000, or 30 000 ppm to time-mated Hsd:Sprague Dawley SD rats from gestation day (GD) 6 through postnatal day (PND) 28; offspring were provided dosed feed at the same concentration as their dam (PND 28–PND 56). TPHP- and IPP-related toxicity resulted in removal of both 30 000 ppm groups on GD 12 and 15 000 ppm IPP group after parturition. Body weight and organ weights were impacted with exposure in remaining dams. Reproductive performance was perturbed at ≥10 000 ppm TPHP and all IPP exposure groups. In offspring, both TPHP- and IPP-related toxicity was noted in pups at ≥10 000 ppm as well as reduction in bodyweights, delays in pubertal endpoints, and/or reduced cholinesterase enzyme activity starting at 1000 ppm TPHP or IPP. Preliminary internal dose assessment indicated gestational and lactational transfer following exposure to TPHP or IPP. These findings demonstrate that offspring development is sensitive to 1000 ppm TPHP or IPP exposure. [ABSTRACT FROM AUTHOR]

Background: Human exposures to organophosphate flame retardants result from their use as additives in numerous consumer products. These agents are replacements for brominated flame retardants but have not yet faced similar scrutiny for developmental neurotoxicity. We examined a representative organophosphate flame retardant, triphenyl phosphate (TPP) and its potential effects on behavioral development and dopaminergic function. Methods: Female Sprague–Dawley rats were given low doses of TPP (16 or 32 mg kg−1 day−1) via subcutaneous osmotic minipumps, begun preconception and continued into the early postnatal period. Offspring were administered a battery of behavioral tests from adolescence into adulthood, and littermates were used to evaluate dopaminergic synaptic function. Results: Offspring with TPP exposures showed increased latency to begin eating in the novelty‐suppressed feeding test, impaired object recognition memory, impaired choice accuracy in the visual signal detection test, and sex‐selective effects on locomotor activity in adolescence (males) but not adulthood. Male, but not female, offspring showed marked increases in dopamine utilization in the striatum, evidenced by an increase in the ratio of the primary dopamine metabolite (3,4‐dihydroxyphenylacetic acid) relative to dopamine levels. Conclusions: These results indicate that TPP has adverse effects that are similar in some respects to those of organophosphate pesticides, which were restricted because of their developmental neurotoxicity. [ABSTRACT FROM AUTHOR]

D5, a member of the cyclic siloxane family, is widely used in personal care products such as shampoo, cosmetics, and deodorant and as an industrial intermediate. D5 can mainly be absorbed orally or through inhalation. Through these routes, people are exposed to D5 daily. However, the risk of prenatal exposure to D5 has not been fully elucidated. In this study, the effect of D5 on neural development was established through behavioral tests on offspring mice. The result confirmed that the maternal administration of 12 mg/kg of D5 showed depression in tail suspension and decreased performance in the forced swimming test as well as an increase in repetitive activity in both the marble-burying test and grooming test compared to the vehicle group. Furthermore, the 12 mg/kg group showed a decrease in cognitive ability and social behavior in the three-chamber test. In the novel object recognition test, memory impairment and a lack of exploring ability were found in the 12 mg/kg group. In conclusion, it is suggested that maternal D5 exposure has developmental neurotoxicity and can cause behavioral disorders in the offspring of mice. Thus, the usage of D5 needs to be considered carefully. [ABSTRACT FROM AUTHOR]

Purpose: To evaluate the impact of clinical protocol change via active minimisation on the number of general anaesthetic (GA)/sedation episodes for diagnostic ophthalmic purposes at Children's Health Ireland at Temple Street (CHI-TS), Dublin, Ireland, from 2016 to 2019, inclusive. Change was implemented following published cautionary principles in 2016 by the FDA regarding the potential neurotoxic risk from multiple GA exposure in children. Methods: Retrospective analysis of electronic operating theatre records was completed using procedure codes "Ophthalmological examination" and "Examination of fundi". Available records for patients undergoing multiple examination under anaesthesia (EUA) procedures were assessed for demographics, indication. Comparison was made regarding overall EUA numbers and breakdown for each year, before and after the new departmental approach. From 2018 onward, a patient-centred, departmental strategy of active minimisation of EUA was adopted, using strategies of "training, technology and patience". A literature review was conducted using online databases. Results: A total of 450 EUAs were performed over the 4 years investigated. In the former 2 years of the study period, prior to departmental policy change, EUAs represented 32% (304 of 948 total theatre episodes) of the ophthalmic theatre caseload. In the latter 2 years of this study period, this proportion fell to 19% (146 EUAs of 783 theatre episodes). Total theatre case numbers were comparable in both time periods. Eighteen children had multiple EUAs (ie, ≥ 2 EUAs, mean 6.5, SD 2.9) for life/sight threatening indications, totalling 116 EUAs (25.7%). Conclusion: A significant reduction in diagnostic EUA volume was accomplished resulting in reduced individual patient risk and increased capacity for surgical interventions. A detailed description of this methodology is included for the purposes of replication at comparable units. EUA will continue to play a crucial role in the management of life/sight threatening conditions but the application of a cautionary principle to reduce EUA, where possible, is appropriate to reduce potential for neurotoxicity. [ABSTRACT FROM AUTHOR]

The growing production of silver nanoparticles (AgNPs), and their widespread use in medical and consumer products, poses a potential threat to the environment and raises questions about biosafety. Immature organisms are particularly susceptible to various insults during development. The biological characteristics of immature organisms are different from those of adults, and dictate the consequences of exposure to various toxic substances, including AgNPs. Nanoparticles are highly reactive and can easily cross the blood–brain barrier (BBB) to accumulate in brain tissues. It is therefore important to investigate the molecular mechanisms of AgNP-induced neurotoxicity in the developing brain. Immature 2-week-old rats were exposed to a low dose of AgNPs (0.2 mg/kg b.w.) over a long period. Subsequently, brain tissues of the animals were subjected to ultrastructural and molecular analyses to determine endoplasmic reticulum (ER) stress. Ultrastructural markers of ER stress, such as pathological alterations in the ER and elongated forms of mitochondria accompanied by autophagy structures, were confirmed to be present in AgNP-exposed rat brain. Evidence for induction of ER stress in neurons was also provided by molecular markers. Upregulation of genes related to the ER-stress-induced unfolded protein response (UPR) pathway, such as GRP78, PERK, and CHOP ATF-6, was observed at the transcriptional and translational levels. The results show that prolonged exposure of immature rats to a low dose of AgNPs during the developmental period leads to induction of ER stress in the neurons of the developing brain. Simultaneously, in response to AgNP-induced ER stress, neurons promote protective mechanisms that partially compensate for ER stress by regulating the biodynamic processes of mitochondria and autophagy. [ABSTRACT FROM AUTHOR]

Due to their neurodevelopmental toxicity, flame retardants (FRs) like polybrominated diphenyl ethers are banned from the market and replaced by alternative FRs, like organophosphorus FRs, that have mostly unknown toxicological profiles. To study their neurodevelopmental toxicity, we evaluated the hazard of several FRs including phased-out polybrominated FRs and organophosphorus FRs: 2,2′,4,4′-tetrabromodiphenylether (BDE-47), 2,2′,4,4′,5-pentabromodiphenylether (BDE-99), tetrabromobisphenol A, triphenyl phosphate, tris(2-butoxyethyl) phosphate and its metabolite bis-(2-butoxyethyl) phosphate, isodecyl diphenyl phosphate, triphenyl isopropylated phosphate, tricresyl phosphate, tris(1,3-dichloro-2-propyl) phosphate, tert-butylphenyl diphenyl phosphate, 2-ethylhexyl diphenyl phosphate, tris(1-chloroisopropyl) phosphate, and tris(2-chloroethyl) phosphate. Therefore, we used a human cell–based developmental neurotoxicity (DNT) in vitro battery covering a large variety of neurodevelopmental endpoints. Potency according to the respective most sensitive benchmark concentration (BMC) across the battery ranked from <1 μM (5 FRs), 1<10 μM (7 FRs) to the >10 μM range (3 FRs). Evaluation of the data with the ToxPi tool revealed a distinct ranking (a) than with the BMC and (b) compared to the ToxCast data, suggesting that DNT hazard of these FRs is not well predicted by ToxCast assays. Extrapolating the DNT in vitro battery BMCs to human FR exposure via breast milk suggests low risk for individual compounds. However, it raises a potential concern for real-life mixture exposure, especially when different compounds converge through diverse modes-of-action on common endpoints, like oligodendrocyte differentiation in this study. This case study using FRs suggests that human cell–based DNT in vitro battery is a promising approach for neurodevelopmental hazard assessment and compound prioritization in risk assessment. [ABSTRACT FROM AUTHOR]

The safety evaluation of pesticides in the European Union (EU) relies to a large extent on toxicity studies commissioned and funded by the industry. The herbicide glyphosate and four of its salts are currently under evaluation for renewed market approval in the EU. The safety documentation submitted by the applicant companies does not include any animal study regarding developmental neurotoxicity (DNT) that is compliant with test guidelines. For a fifth salt, not included in the present application for re-approval, such a DNT study was sponsored by one of the applicant companies in 2001. That study shows an effect of that form of glyphosate on a neurobehavioural function, motor activity, in rat offspring at a dose previously not known to cause adverse effects. Counter to regulatory requirements, these effects were apparently not communicated to authorities in EU countries where that form of glyphosate was authorised at that time. That DNT study may also be relevant for the ongoing assessment of glyphosate but was not included in the present or previous applications for re-approval.In this commentary, we highlight that it is the responsibility of the industry to evaluate and ensure the safety of their products, taking all available scientific knowledge into account. We argue that the legal obligation for industry to submit all potentially relevant data to EU authorities is clear and far-reaching, but that these obligations were not fulfilled in this case. We claim that authorities cannot reliably pursue a high level of protection of human health, if potentially relevant evidence is withheld from them. We suggest that a retrospective cross-check of lists of studies performed by test laboratories against studies submitted to regulatory authorities should be performed, in order to investigate the completeness of data submitted to authorities. We further suggest that future toxicity studies should be commissioned by authorities rather than by companies, to improve the authorities' oversight over existing data and to prevent that economic conflicts of interest affect the reporting of study results and conclusions. [ABSTRACT FROM AUTHOR]

Neurotoxicants are detrimental to the mammalian nervous system at higher concentrations after exposure, and could result in several neurological abnormalities, especially on the nervous and reproductive systems, and sometimes death. The present study, therefore, aimed to evaluate the research growth on neurotoxicants and their effects on humans and the environment over the last decade from 2011 to 2020. Data on this subject were obtained from the SCI-Expanded of Web of Science, and analyses were performed on the retrieved data in RStudio. The number of published documents fluctuated over the studied years, with an annual growth rate of 4.46%, and the highest number of publications were recorded in 2020 (n = 40). Single authored documents, documents per author, authors per documents, and collaboration index were 24, 0.219, 4.57, and 4.87, respectively. Networks of collaboration in this study were noticeable among authors, institutions, and countries; thus, making efforts to strengthen networking globally would be a good idea. Results from this study also show that the growing trend of research in the field is quite encouraging, thus providing future directions to upcoming researchers, and contributes immensely to reducing the exposure and several disorders linked to these neurotoxicants globally. [ABSTRACT FROM AUTHOR]

Compound toxicity data obtained from independent zebrafish laboratories can vary vastly, complicating the use of zebrafish screening for regulatory decisions. Differences in the assay protocol parameters are the primary source of variability. We investigated this issue by utilizing data from the NTP DNT-DIVER database (https://doi.org/10.22427/NTP-DATA-002-00062-0001-0000-1, last accessed June 2, 2022), which consists of data from zebrafish developmental toxicity (devtox) and locomotor response (designated as "neurotox") screens from 3 independent laboratories, using the same set of 87 compounds. The data were analyzed using the benchmark concentration (BMC) modeling approach, which estimates the concentration of interest based on a predetermined response threshold. We compared the BMC results from 3 laboratories (A, B, C) in 3 toxicity outcome categories: mortality, cumulative devtox, and neurotox, in terms of activity calls and potency values. We found that for devtox screening, laboratories with similar/same protocol parameters (B vs C) had an active call concordance as high as 86% with negligible potency difference. For neurotox screening, active call concordances between paired laboratories are lower than devtox screening (highest 68%). When protocols with different protocol parameters were compared, the concordance dropped, and the potency shift was on average about 3.8-fold for the cumulative devtox outcome and 5.8-fold for the neurotox outcome. The potential contributing protocol parameters for potency shift are listed or ranked. This study provides a quantitative assessment of the source of variability in zebrafish screening protocols and sets the groundwork for the ongoing Systematic Evaluation of the Application of Zebrafish in Toxicology effort at the National Toxicology Program. [ABSTRACT FROM AUTHOR]

Aluminum (Al), a common light metal, affects the developing nervous system. Developmental exposure to Al chloride (AlCl3) induces aberrant neurogenesis by targeting neural stem cells (NSCs) and/or neural progenitor cells (NPCs) in the dentate gyrus (DG) of rats and mice. To investigate whether hippocampal neurogenesis is similarly affected by AlCl3 exposure in a general toxicity study, AlCl3 was orally administered to 5‐week‐old Sprague Dawley rats at dosages of 0, 4000, or 8000 ppm in drinking water for 28 days. AlCl3 downregulated Sox2 transcript level in the DG at the highest dosage and produced a dose‐dependent decrease of SOX2+ cells without altering numbers of GFAP+ or TBR2+ cells in the subgranular zone, suggesting that AlCl3 decreases Type 2a NPCs. High‐dose exposure downregulated Pcna, upregulated Pvalb, and altered expression of genes suggestive of oxidative stress induction (upregulation of Nos2 and downregulation of antioxidant enzyme genes), indicating suppressed proliferation and differentiation of Type 1 NSCs. AlCl3 doses also increased mature granule cells in the DG. Upregulation of Reln may have contributed to an increase of granule cells to compensate for the decrease of Type 2a NPCs. Moreover, upregulation of Calb2, Gria2, Mapk3, and Tgfb3, as well as increased numbers of activated astrocytes in the DG hilus, may represent ameliorating responses against suppressed neurogenesis. These results suggest that 28‐day exposure of young‐adult rats to AlCl3 differentially targeted NPCs and mature granule cells in hippocampal neurogenesis, yielding a different pattern of disrupted neurogenesis from developmental exposure. We previously showed that developmental exposure to aluminum chloride (AlCl3) disrupted hippocampal neurogenesis in rats and mice. The present study investigated whether hippocampal neurogenesis is similarly affected by AlCl3 exposure in a general toxicity study using young‐adult rats. AlCl3 exposure for 28 days decreased Type 2a NPCs but increased mature granule cells through multiple mechanisms, suggesting that AlCl3 differentially targeted NPCs and mature granule cells in hippocampal neurogenesis to generate different patterns of disruption from developmental exposure. [ABSTRACT FROM AUTHOR]

To support a mixture risk assessment with a focus on developmental neurotoxicity we evaluated the strength of evidence for associations of cadmium exposures with declines in IQ by conducting a systematic review and confidence rating. We searched peer-reviewed studies published in English between 2012 and July 2021 and identified 15 eligible studies (11 prospective cohort studies, and 4 cross-sectional studies). Of the 10 studies that observed associations of cadmium exposure with child IQ declines, two achieved an overall "High (H)" confidence rating, five a "Medium to High (M/H)", one a "Medium (M)" and two a "Low (L)" confidence rating. Five studies did not detect significant associations between cadmium exposure and reduced cognitive ability; of these, two received a "High (H)" confidence rating, two an overall rating of "Medium to High (M/H)" and one a "Medium (M)" rating. The null findings reported by the "High (H)" and Medium to High (M/H)" studies could partly be explained by low exposures to cadmium or confounding with high levels of lead. By using a one-compartment toxicokinetic model in a reverse dosimetry approach, we estimated that a daily intake of 0.2 μg/kg body weight/day corresponds to urinary cadmium levels no longer associated with cognitive declines observed in a "High (H)"-confidence study. This estimate is 1.8-fold lower than the current health-based guidance value (HBGV) for kidney toxicity of 0.36 μg/kg bodyweight/day established by the European Food Safety Authority (EFSA). Our value does not have the normative character associated with health-based guidance values and is intended only as a reasonable estimate for the purpose of mixture risk assessments. However, with cadmium exposures in Europe between 0.28 (middle bound) and up to 0.52 μg/kg bodyweight/day (95th percentile), our review suggests that pregnant women and children are poorly protected against neurodevelopmental effects. This warrants a revision of the current HBGV. [ABSTRACT FROM AUTHOR]