NeuroToxicology

Neurodevelopmental disorders, including autism spectrum disorder (ASD), are influenced by both genetic abnormalities and environmental toxicants. Among environmental risk factors, endocrine-disrupting chemicals such as bisphenol A (BPA) and pharmaceutical drugs such as valproic acid (VPA) have been associated with an increased risk of autism. In this study, human induced pluripotent stem cell (iPSC)-derived forebrain organoids were used to model early neurodevelopmental disruptions induced by BPA and VPA exposure. On day 62 of differentiation, forebrain organoids were treated with physiologically relevant concentrations of BPA or VPA for 28 days. Following treatment, morphological, molecular, and electrophysiological changes were assessed across experimental conditions. Both compounds produced distinct alterations in organoid morphology, neurodevelopmental gene expression, and network electrical activity, with VPA inducing markedly stronger effects. Overall, these data suggest forebrain organoids as a robust, physiologically relevant in vitro model system for studying neurodevelopment. This platform enables systematic investigation of environmental and pharmacological risk factors implicated in the pathogenesis of neurodevelopmental disorders.

BackgroundMethylglyoxal (MGO), a highly reactive by-product of glycolysis, has been associated with cognitive decline and Alzheimers disease, though the mechanistic role of MGO remains unclear. Moreover, conflicting findings exist regarding MGOs toxicity on the blood-brain barrier (BBB). This study investigated whether MGO can cross the BBB under physiologically relevant conditions and whether MGO affects BBB permeability. MethodsMice were intravenously injected with highly purified home-made MGO or PBS, and MGO concentration was measured at five timepoints in the cerebral cortex up to 4 hours after injection. MGO toxicity was screened on a human brain endothelial cell line (hCMEC/D3) using a live/dead assay prior to the study of selected MGO concentrations and on hiPSC-derived brain microvascular endothelial cells (EECM-BMECs). EECM-BMECs were cultured on Transwell(R) inserts, and barrier function was assessed by sodium fluorescein permeability and transendothelial passage of 13C3-MGO quantified by UPLC-MS/MS. ResultsMGO levels in the mouse cortex did not increase post-injection. MGO was not toxic to hCMEC/D3 cells, and it had no impact on barrier properties of EECM-BMECs. After 1-hour exposure, [~]13% of total 13C3-MGO was recovered in its free form, and only [~]1% of supplemented MGO was recovered from the abluminal side. ConclusionMGO does not cross the BBB in vivo and does not affect barrier properties of a human in vitro model of the BBB. In vitro MGO passage across the BBB is minimal. These findings suggest that circulating MGO is unlikely to directly affect neuronal function via BBB disruption or enter the brain in its free from.

Background/ObjectivesOccupational exposure to neurotoxicants such as pesticides, metals, and solvents has long been implicated in Parkinsons disease (PD) and Parkinsonism, yet the cumulative impact of multiple occupational exposure families over the working life remains insufficiently characterized. This study evaluated whether long-term cumulative occupational exposures, derived from the ALOHA+ Job-Exposure Matrix (ALOHA+-JEM), were associated with PD and Parkinsonism. MethodsA hospital-based matched case-control study was conducted in the province of Brescia, Italy, including 668 participants (334 PD/Parkinsonism cases and 334 matched controls). Cases and controls were 1:1 matched based on sex, age, and lifetime occupational duration. Lifetime occupational histories were coded using ISCO-08 and harmonized to ISCO-88 for linkage with ALOHA+-JEM. Conditional logistic regression estimated associations between cumulative exposures (none/low/high) and disease status, adjusting for smoking, parental history of PD/tremor, and SNCA rs356219 genotype. Multi-agent occupational exposure burden indexes were evaluated using positively constrained repeated-holdout Weighted Quantile Sum (WQS) regression (100 bootstraps, 100 holdouts) ResultsIn conditional logistic regression, parental history of PD or tremor (OR = 4.55, 95% CI: 2.44-8.48; q < 0.001) and the SNCA rs356219 CC genotype (OR = 2.17, 95% CI: 1.33-3.52; q = 0.013) were significantly associated with disease. High cumulative all pesticide exposure showed positive associations with combined PD + Parkinsonism (OR = 2.98, 95% CI: 1.23-7.25) and PD alone (OR = 3.56, 95% CI: 1.25-10.15). In WQS analyses, the composite occupational exposure burden index was positively associated with disease (combined PD + Parkinsonism: OR = 1.15, 95% CI: 1.00-1.30). All pesticides received the highest mean weight in all models (w = 0.434 for combined PD + Parkinsonism), followed by metals (w = 0.210), identifying them as contributing most strongly to the composite exposure index. ConclusionsLong-term cumulative occupational exposures were associated with increased odds of PD and Parkinsonism. All pesticides and metals were most strongly associated with PD and Parkinsonism, consistent with established neurotoxic mechanisms attributable to occupational environments. These findings underscore the importance of occupational exposure prevention and risk-reduction strategies in occupational settings and highlight workplace exposures as preventable contributors to Parkinsonian disorders.

Developmental exposure to organophosphate flame retardants (OPFRs) is a public health concern due to their endocrine-disrupting potential. We examined perinatal exposure to tris(1,3-dichloro-2-propyl) phosphate, triphenyl phosphate, and tricresyl phosphate in mice. Adult male and female offspring were assessed for memory and anxiety-like behavior. Dopamine and norepinephrine were quantified in the hippocampus and prefrontal cortex (PFC), and bulk RNA sequencing was conducted for the hippocampus. OPFR-treated females in high ovarian hormone states spent less time in the open field test (OFT) center, the Y-maze unknown arm, and with the displaced object in spatial object recognition (SOR) indicating increased anxiety-like behavior and impaired spatial memory. These females also illustrated improved memory on the short-term Barnes maze, and a trending improvement in the novel object recognition test. Females in low ovarian hormone states, demonstrated a trend in center OFT exploration. OPFR-treated males displayed disruption in memory in the SOR and the short- and long-term Barnes maze. Perinatal OPFR reduced hippocampal dopamine in males and altered prefrontal dopamine in females in a hormone-dependent manner. OPFR-treated females in high ovarian hormones states demonstrated a trending decrease in PFC norepinephrine. Perinatal OPFR treatment caused differential gene expression in 121 individual genes and alteration to functional modules related to RNA processing, cellular metabolism, and extracellular organization. Hormone status also affected gene OPFR-induced altered expression, with similarity between males and high ovarian hormone state females. Our findings suggest that perinatal OPFR exposure causes widespread, sex specific, and hormone dependent disruptions in behavior, neurochemistry, and gene expression in adulthood. HighlightsO_LIAnxiety-like behavior in OPFR-treated females varied with ovarian hormone status C_LIO_LIHigh ovarian hormone OPFR females showed task-dependent changes in memory C_LIO_LIMales displayed impaired spatial memory following perinatal OPFR treatment C_LIO_LIPerinatal OPFR modifies hippocampal and prefrontal dopamine and norepinephrine C_LIO_LIOPFR treatment altered individual gene and functional gene module expression C_LI

The nervous system is highly vulnerable to chemical disruption, yet current regulatory guidelines do not include behavioral endpoints that capture changes in stress-related responses. Zebrafish larvae, up to 5 days old, have emerged as a promising model to bridge this gap, offering genetic and neurochemical similarity to humans together with high throughput potential. In this work, we have developed and evaluated a larval thigmotaxis assay as a new approach methodology (NAM) to detect behavioral alterations caused by neuroactive substances. Thigmotaxis, or edge-preference behavior, was studied in zebrafish larvae exposed to a range of model compounds and challenged with both visual (light/dark) and acoustic (sound/silence) stimuli. We compared 24 round well plates, commonly used in behavioral assays, with 96 square well plates to increase throughput. The two formats showed equivalent results, supporting the use of the higher-capacity system. Classical controls confirmed assay performance with caffeine increasing thigmotaxis, while diazepam decreased it. Additional neuroactive substances with diverse modes of action (chlorpyrifos, nicotine, dexamethasone, ethylenethiourea) produced stimulus-dependent responses, whereas negative controls (saccharin, amoxicillin) had little or no effect. Benchmark dose modeling showed that thigmotaxis was generally more sensitive than traditional locomotor activity endpoints. Overall, this multiplexed visual-acoustic thigmotaxis assay proved reproducible, scalable, and sensitive. In neurotoxicity testing this method could be used both as a stand-alone assay or as part of a broader behavioral NAM battery to assess potential effects on the vertebrate nervous system. This method provides a practical and ethical tool to improve chemical safety assessment both in ecotoxicology and human toxicology. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=62 SRC="FIGDIR/small/703464v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1ceea53org.highwire.dtl.DTLVardef@17a2a8borg.highwire.dtl.DTLVardef@17f16b1org.highwire.dtl.DTLVardef@aac24f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Air pollution has been increasingly linked to adverse neurodevelopmental and neurodegenerative outcomes. While experimental and preclinical studies suggest that exposure to particulate matter (PM), particularly during gestation, may disrupt cognitive development, the impact of short-term PM exposure on cognitive and behavioral functioning in healthy young populations remains insufficiently explored in Spain. Moreover, few studies have incorporated individualized dosimetry models to estimate exposure more accurately. This study included 186 healthy young adults (mean age = 20.4 years) recruited from three Spanish cities (Teruel, Almeria, and Talavera) characterized by different pollution levels. Ambient fine and coarse PM concentrations were recorded 8, 15, and 30 days prior to psychological assessment. Instead of relying solely on raw in situ environmental measurements, individualized PM deposition was estimated using the Multiple-Path Particle Dosimetry Model (MPPD), allowing a more biologically meaningful exposure approximation. Psychological outcomes were assessed using validated questionnaires: DASS-21 (depression, anxiety, stress), BIS-11 (impulsivity), UCLA Loneliness Scale, and SWLS (life satisfaction). Behavioral performance was evaluated using computerized versions of the Attentional Network Task (ANT) and the Stroop Task. Blood NRF2 concentrations were analyzed as a biomarker potentially related to oxidative stress mechanisms. In situ data indicated that Talavera presented the highest pollution levels, followed by Almeria and Teruel. Linear regression analyses showed that coarse PM exposure across 8-, 15-, and 30-day windows significantly predicted poorer Executive Control Index performance in the ANT. Additionally, 15-day coarse PM and 30-day fine PM exposure were associated with greater cognitive interference. Oxidative stress markers were significantly associated with PM exposure levels. These findings support emerging evidence that short-term PM exposure may negatively affect executive and attentional processes even in healthy young adults. Further longitudinal research incorporating individualized exposure modeling is warranted to clarify causal pathways and underlying biological mechanisms. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/713644v1_ufig1.gif" ALT="Figure 1"> View larger version (79K): org.highwire.dtl.DTLVardef@1a0ac13org.highwire.dtl.DTLVardef@1812accorg.highwire.dtl.DTLVardef@120bf07org.highwire.dtl.DTLVardef@dd9a7c_HPS_FORMAT_FIGEXP M_FIG C_FIG

1.IntroductionAir pollution (AirPoll) is a major environmental risk factor for age-related cognitive decline and dementia, yet we poorly understood the cellular and molecular mechanisms underlying its effects and their potential attenuation. MethodsWe combined single cell RNA sequencing with immunohistochemistry to determine transcriptional responses in microglia, astrocytes, neurons and neural stem cells in the hippocampus of mice following exposure to chronic diesel exhaust particle (DEP). Differential gene expression profiles were compared between filtered-air and DEP exposed animals. The gamma secretase modulator GSM-15606 (BPN) was used to probe selective rescue of inflammatory signatures across distinct cell populations. ResultsDEP exposure triggered robust inflammatory programs in microglia and astrocytes, including upregulation of cytokine signaling components, innate immune receptors, stress-responsive transcription factors, and markers of reactive glial phenotypes. In neural stem cells, DEP induced activation of gliosis-associated pathways, including Il6st, Stat3, and Txnip, consistent with a pro-inflammatory state that may bias lineage decisions. Immunostaining confirmed a significant reduction in immature neurons in the neurogenic niche after AirPoll exposure. GSM-15606 attenuated many DEP-induced transcriptional alterations in microglia and astrocytes, reducing expression of inflammatory mediators and reactive gliosis markers, but did not modulate the inflammatory profile of neural stem cells. ConclusionsAirPoll activates divergent inflammatory pathways across hippocampal cell populations and suppresses neurogenesis. Targeting inflammation with GSM-15606 selectively reverses glial but not neural stem cell responses, highlighting cell-type-specific mechanisms and potential therapeutic pathways to mitigate pollution-related cognitive vulnerability. These results support GSM-15606 as a protective agent against AirPoll-induced hippocampal dysfunction and amyloidogenic stress.

Exposures to pervasive chemical toxicants such as endocrine disrupting chemicals (EDCs) are associated with adverse neurological and neurodevelopmental deficits. Although EDCs are widespread as sparse mixtures in the environment, most research has focused on single chemicals at high concentrations. Here, we studied the effects of ldEDC: a low-dose mixture of widely prevalent toxicants at doses representative of normal human exposure levels. Primary cultured mouse neurons treated with ldEDC exhibited altered gene expression compared to vehicle controls in genes critical for neuron activity, indicating low doses EDCs can affect neuronal function directly. We next tested persistent exposure through the maternal diet to define perinatal effects on offspring. Exposed offspring exhibited differences in development, tactile sensitivity, and sex-specific changes in motor behavior. Cortical single-nuclei sequencing identified broad transcriptomic changes, particularly in distinct cortical layer subpopulations, excitatory neurons, and astrocytes. Cell-cell signaling between neurons and non-neuronal populations were altered in exposed mice, specifically in pathways associated with cellular adhesion. Transcriptomic differences were also sex-specific. Together, these in vitro and in vivo findings reveal molecular and phenotypic consequences of EDC exposure at a mixture of doses well below commonly studied levels and highlights common functional pathways of susceptibility.

Ethylene oxide (EtO) is primarily used as an intermediate in the manufacture of chemicals, with a minor use as a sterilant for medical equipment and food products. It is a direct-acting alkylating agent that reacts with cellular macromolecules, including proteins and DNA. EtO has been shown to induce tumors in rodents and humans. DNA reactivity has been the postulated mode of action (MOA) for its carcinogenicity. The current study has investigated the dose response for EtO-induced genetic damage to inform the biological plausibility of a dose-response model for cancer risk assessment. Male and female B6C3F1 mice were exposed to 0, 0.05, 0.1, 0.5, 1, 50, 100, or 200 ppm EtO by whole-body inhalation (6 hours/day for 28 days, 7 days/week). Mutagenicity was assessed by determining the frequency of mutant Pig-a phenotype in reticulocytes (RET) and mature red blood cells (RBC) on Day 28. Cytogenetic damage was evaluated by the erythrocyte micronucleus (MN) test in blood samples collected on Days 5 and 28. EtO is a relatively weak genotoxicant with treatment-related increases in Pig-a and MN frequencies being seen primarily at 200 ppm. The hockey-stick shaped dose response for genetic damage may be conservatively interpreted as being no more than a linear response with a single slope. Thus, a cancer risk assessment dose-response model consisting of a single linear slope throughout the exposure range is biologically plausible and consistent if EtO were acting through a mutagenic MoA for its carcinogenicity.

IntroductionAmbient air pollution, especially fine particulate matter 2.5 (PM2.5) has emerged as a critical environmental risk factor for cerebrovascular diseases, contributing to an estimated 7.9 million premature deaths annually. PM2.5 induces cellular toxicity and is hypothesized to disrupt the blood-brain barrier (BBB), a pathological hallmark in cerebrovascular diseases such as ischemic stroke. Despite epidemiological evidence linking PM2.5 to increased stroke incidence, its underlying cellular mechanism driving this association is poorly understood. It remains unclear how environmentally relevant pollution concentrations affects brain endothelial function or influence stroke-related biomarkers such as the lectin-like oxLDL receptor 1 (LOX-1). MethodPrimary adult male human brain microvascular endothelial cells (HBMEC) were exposed to PM2.5 (5, 15, 75, or 300 g/m3) collected from an urban environment in southern Sweden, or control. Thereafter, exposed to normoxia (21% O2) or hypoxia (1% O2) and glucose deprivation, followed by reperfusion as a model for ischemic stroke. Cell viability, oxidative stress, inflammation, BBB integrity (claudin-5, ZO-1) and LOX-1 protein expression were assessed. ResultsPM2.5 exposure induced cellular dysfunction, oxidative stress and inflammation starting at 75 g/m3 PM2.5. Notably, decreased claudin-5 and ZO-1 protein levels and increased LOX-1 expression at concentrations as low as 15 g/m3 PM2.5, levels commonly encountered in urban environments globally. The cellular effects of PM2.5 closely resembled those induced by ischemic-like injury. ConclusionThese findings demonstrate dose-dependent detrimental effects of PM2.5 on HBMEC. The results suggest that ambient urban PM2.5 may act as a predisposing factor for cerebrovascular disease onset, by causing endothelial and barrier dysregulation. HighlightsO_LIUrban PM2.5 dose-dependently disrupts BBB integrity in human brain endothelial cells C_LIO_LIPM2.5 induces endothelial dysfunction resembling ischemic-like injury C_LIO_LIUrban PM2.5 exposure upregulates cardiovascular disease biomarker LOX-1 C_LIO_LIA majority of the global population are exposed to BBB-disrupting PM2.5 levels C_LIO_LIVascular endothelial- and BBB dysfunction enhances risk for cerebrovascular disease C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/702473v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@42cce6org.highwire.dtl.DTLVardef@19a2ff9org.highwire.dtl.DTLVardef@1c38e7corg.highwire.dtl.DTLVardef@1bfa837_HPS_FORMAT_FIGEXP M_FIG C_FIG

Evaluation of unintended immunotoxicity represents an important component of nonclinical safety assessment, as perturbation of immune function may increase susceptibility to infection, impair vaccine responses, and disrupt immune homeostasis. Regulatory guidance, including the ICH S8 Immunotoxicity Guideline, recommends a weight-of-evidence approach in which observations from conventional toxicological endpoints are integrated with functional immune assays to support interpretation of immune system effects. The present study applied an integrated immunotoxicity evaluation framework to examine concordance among structural, functional, and cellular immune endpoints in male Sprague-Dawley rats using a well-characterized immunosuppressive reference compound. Hematological evaluation revealed leukopenia characterized primarily by lymphocyte depletion. Reductions in spleen and thymus weights were accompanied by histopathological evidence of lymphoid depletion in multiple immune tissues, including spleen, thymus, lymph nodes, Peyers patches, and bone marrow. Functional immune competence was assessed through hemagglutination antibody response to sheep red blood cells and delayed-type hypersensitivity assays, both of which demonstrated marked suppression of adaptive immune responses. Flow cytometric immunophenotyping further demonstrated substantial reductions in B-cell populations and decreases in CD4 and CD8 T-cell counts, whereas NK cell populations were comparatively less affected. The concordance of hematological alterations, lymphoid tissue changes, impaired functional immune responses, and lymphocyte subset depletion provides integrated evidence of immune system perturbation. These findings demonstrate that complementary immunotoxicity endpoints collectively support hazard characterization of immune system effects under GLP conditions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/713556v1_ufig1.gif" ALT="Figure 1"> View larger version (72K): org.highwire.dtl.DTLVardef@beaf9dorg.highwire.dtl.DTLVardef@fb9f10org.highwire.dtl.DTLVardef@187ff06org.highwire.dtl.DTLVardef@1780dc2_HPS_FORMAT_FIGEXP M_FIG C_FIG

Exposure of cells or tissues to chemical compounds can be analyzed through transcriptomic signatures, which can be used to classify chemical agents. This information can also enrich Adverse Outcome Pathways (AOP). Transcriptional signatures have generally been obtained using "bulk" analysis, by which the global gene expression pattern of an entire tissue is determined. Although this approach has been useful in toxicology, some information is lost, especially when tissues containing multiple cell types are considered. With the advent of single-cell transcriptomics (scRNA-seq), it is now possible to obtain higher resolution, cell type-specific responses in complex tissues. The aim of the present study was to evaluate the added value of scRNA-seq in analysis of the acute response of human bronchial epithelial cells grown at the air/liquid interface (ALI) to a known toxic compound, CdCl2, with well described transcriptional signatures of exposure. Fully differentiated mucocilliary epithelia obtained from three independent donors were exposed to 10 {micro}M CdCl2 and scRNA-seq analysis was performed on a total of 18255 cells to obtain cell type-specific signatures. Our results show that the contribution of each cell type to the overall transcriptomic bulk response varies. For example, the classical heavy metal detoxification response was only detected in multiciliated and secreting cells, while absent in basal cells. The data demonstrate that scRNA-seq provides high-resolution transcriptional signatures with unexpected features. This added information is likely to have implications for the refinement of AOPs and could serve as a basis for a new generation of tests in predictive toxicology.

Uranium (U) and arsenic (As) are both ubiquitous contaminants in the American southwest, posing risks to humans, animals, and the environment. Depleted uraniums (DU) chronic effects and mechanisms of toxicity are incompletely understood. Differential gene expression of concomitant exposures to identify markers of toxicity have not been undertaken until now. Continuous low-dose, high-dose, and concomitant exposures are investigated using the larval zebrafish (Danio rerio), with exposure paradigms lasting from embryo collection until sampling at 5 days post fertilization (dpf). Herein, we describe overall differential gene expression with counts and pathway enrichment statistics using both gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The raw dataset has been deposited in NCBIs Gene Expression Omnibus (GEO) repository [1] under the accession number GSE319292 [2]. O_TBL View this table: org.highwire.dtl.DTLVardef@9b121aorg.highwire.dtl.DTLVardef@c17073org.highwire.dtl.DTLVardef@1bdc2b9org.highwire.dtl.DTLVardef@13b130aorg.highwire.dtl.DTLVardef@15f1d22_HPS_FORMAT_FIGEXP M_TBL C_TBL VALUE OF THE DATAO_LIUranyl nitrate (UN), a water-soluble depleted uranium species, and sodium arsenite (As) are both ubiquitous contaminants in the American southwest, posing risks to humans, animals, and the environment. The United States Environmental Protection Agency (EPA) has set maximum contaminant limits (MCL) of 30 ppb U atoms and 10 ppb As atoms, respectively. C_LIO_LIThese data show differentially expressed genes (DEGs) from larval zebrafish exposed to 1 or 10 {micro}M As, 30 or 300 {micro}g/L UN, or 1 {micro}M As and 30 {micro}g/L UN in combination. Concentrations were specifically chosen based on environmental relevance. C_LIO_LIGene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of up- and down-regulated DEGs are provided to understand the molecular mechanisms of uranium toxicity and inform future studies. C_LIO_LIThese data should be used for biomarker identification and mechanistic interrogation of single and combinatorial exposures of environmentally relevant compounds at realistic exposure levels. C_LI

Chronic exposure to pesticide mixtures through diet is common, yet their combined metabolic effects and interactions with dietary factors remain unclear. We identified four pesticides prevalent in human exposure (imazalil, thiabendazole, boscalid, lambda-cyhalothrin) and assessed their combined impacts on hepatic metabolism and metabolic homeostasis using human liver cells and male mice fed standard chow or western diets. We found that the pesticide mixture induced metabolic perturbations in human hepatocytes. In addition, the pesticide mixture altered hepatic gene expression in chow-fed mice and exacerbated western diet-induced glucose intolerance, fasting hyperglycemia, and insulin resistance without affecting body weight or liver steatosis. These findings reveal that dietary context influences the metabolic consequences of pesticide mixtures, highlighting the need to consider nutritional status when evaluating environmental contaminant risks. Our results suggest that pesticide mixtures at reference doses may contribute to metabolic dysregulation, particularly under obesogenic dietary conditions. Highlights- Four common pesticides in mixture disrupt metabolism in liver cells - Dietary exposure to this pesticide mixture alters hepatic gene expression in mice - The pesticide mixture exacerbates WD-induced disruptions in glucose homeostasis - Pesticides and diet interact in producing the metabolic effects of a pesticide mixture

Micro and nanoplastics are pollutants which concentration in different biotopes increases continuously over time, which poses the question of their potential effects on health. In animals, these micro and nanoplastics are recognized as particulate materials and thus handled by macrophages, which are therefore a key cell type to study. Most studies have used an experimental scheme in which the cells are exposed to a single dose of plastics, with a readout made immediately after exposure. However, this classical experimental scheme does not take into account the impact of biopersistence, nor the potential cellular adaptation that may take place when cells are exposed repeatedly to a low dose of plastics. We thus used a repeated exposure scheme, in order to better take into account these phenomena. Within this frame, we compared the macrophages responses to a persistent nanoplastic, i.e. polystyrene nanoparticles and to a biodegradable nanoplastic, i.e. polylactide, by a combination of proteomic and targeted experiments. Our results show that under this repeated exposure scheme, the proteome changes were of a lesser (for PS) or similar (for PLA) extent than under the acute exposure mode, indicating cell adaptation. However, PLA particles induced mitochondrial dysfunction and depression of response to bacterial molecules perceived as danger signals, such as lipopolysaccharide. Polystyrene nanoparticles also induced a slight alteration of the immune functions of macrophages. This indicates harmful effects even in the repeated exposure scheme.

Bats are exposed to a variety of pollutants, including cadmium (Cd), that can impair immune function and potentially increase viral shedding and burden. Despite this, little is known about the impacts of heavy metals on bats. This study aimed to determine the impacts of Cd exposure on bat T and B cell immune responses in naive and coronavirus infected bats and determine the impact of Cd on viral replication in Jamaican fruit bat (JFB; Artibeus jamaicensis) cells. To determine the impact of Cd exposure on adaptive immune responses, splenocyte cultures from naive and BANAL-52 coronavirus infected JFB were treated with 0, 1, and 10 {micro}M Cd and stimulated overnight with concanavalin A. RNA was extracted, a SYBR Green qPCR was used to assess gene expression. To determine if Cd exposure increased viral replication, two JFB kidney cell clones were treated with 0, 1, 10, and 50 {micro}M of CdCl2 overnight and then infected with Cedar virus (CedV). Supernatants were collected and viral titers determined. Several transcripts were upregulated in both naive and virus infected JFB splenocytes treated with Cd. B cell transcripts were significantly upregulated in a dose-dependent manner and T cell transcripts were also increased in Cd treated splenocytes. Assessment of transcripts associated with T cell subsets suggest a predominant Th2 response in Cd treated splenocytes. Viral replication was not significantly different in Cd treated kidney clones compared to the non-treated cells. These studies provide evidence that JFB adaptive immune responses are altered when exposed to low Cd concentrations.

Per- and polyfluoroalkyl substances (PFAS) are globally recognised as emerging contaminants of concern due to their persistence, toxicity, endocrine-disrupting and immunosuppressive effects. Because of their extensive industrial use, PFAS are now widespread across ecosystems and accumulate in marine environments. Despite their ubiquity, the extent and drivers of PFAS contamination remain poorly characterised, particularly in marine systems. Odontocetes (toothed whales) are effective bioindicators of marine pollution, integrating contamination across regions, time, and trophic levels. Here, we present the first global assessment of factors influencing PFAS contamination in marine ecosystems by analysing standardised PFAS concentrations of PFNA, PFDA, PFUnDA, PFDoDA and PFOS reported for 713 liver samples across 33 odontocete species spanning 13 countries from 2000 to 2023. Using generalised linear mixed models, we evaluated the effects of genus, location, sex, life stage, and sampling year on PFAS concentrations, combining published datasets with new samples from Australia. Genus and location were the strongest predictors, suggesting that interspecific ecological and physiological traits likely contribute to PFAS accumulation. Concentrations were highest in males and younger individuals, consistent with maternal offloading and possible age-related dilution. Spatio-temporal trends indicate that PFAS contamination is widespread and increasing globally, with highest concentrations reported in the Pacific. This study provides a critical baseline for understanding global PFAS exposure in marine mammals, which underscores the need for coordinated monitoring and further research to address regional data gaps and potential unrecognised biological effects. HighlightsO_LIHigh genus-specific and spatial differences in PFAS contamination across odontocetes globally. C_LIO_LIIncreased contamination in younger/smaller individuals. C_LIO_LISex-specific trends, including higher PFAS levels in male odontocetes. C_LIO_LISpatio-temporal trends suggesting increased PFAS concentration despite global regulatory efforts, with highest concentrations in the Pacific Ocean. C_LI

Nicotinamide riboside chloride (NR-Cl) has been studied predominantly by the oral route, while information regarding its toxicity following parenteral administration is limited. To characterize route-dependent acute toxicity and estimate median lethal doses (LD50), pharmaceutical-grade NR-Cl was evaluated following bolus intravenous (IV), intramuscular (IM), and subcutaneous (SC) administration in female Sprague Dawley rats, in three independent studies. All studies were conducted using an adapted OECD Guideline 425 Up-and-Down procedure, modified for parenteral administration, in the absence of standardized route-specific OECD guidance. Animals received a single dose of NR-Cl via the respective administration route and were monitored for mortality, clinical signs, bodyweight changes, and gross pathological findings over a 14-day observation period. Following IM and SC administration, no mortalities were observed at doses up to 2000 mg/kg, and LD50 values for both routes were determined to be greater than 2000 mg/kg. In contrast, IV administration yielded an estimated LD50 of approximately 2000 mg/kg. These findings demonstrate that the acute toxicity of NR-Cl differs by route of administration and establish foundational safety benchmarks to support future research.

IntroductionWhile longitudinal studies aid in understanding and preventing long-latency disorders like dementia, evidence for cadmiums role in these conditions is still limited. We evaluated associations between cadmium exposure and incident Alzheimers disease (AD) and all-cause dementia in US adults. MethodsNational Health and Nutrition Examination Survey (NHANES) III (1988-1994) and continuous NHANES (1999-2016) data were linked with Medicare claims to identify incident AD and dementia cases through 2018. Urinary and/or blood cadmium were measured during NHANES. We used covariate-adjusted, survey-weighted Cox proportional hazard models to evaluate the associations between cadmium exposure biomarkers and AD/dementia over follow-up. ResultsIn NHANES III (N=6,122), baseline age was 53.9{+/-}0.5 years and urinary cadmium was 0.8{+/-}0.02 ug/L. Over a follow-up of 20.4{+/-}0.3 years, 743 AD and 1,508 all-cause dementia cases occurred. Urinary cadmium was not associated with AD (HR: 1.01, 95% CI: 0.9-1.0) nor all-cause dementia incidence (HR: 1.02, 95% CI: 0.96-1.08). In continuous NHANES (urinary cadmium N=2,833; blood cadmium N=8,038), baseline age was 64.1{+/-}0.2 years, urinary cadmium was 0.5{+/-}0.03 ug/L, and blood cadmium was 0.6{+/-}0.01 ug/L. Over 9.5{+/-}0.1 years, 587 AD and 1,260 all-cause dementia cases occurred. Urinary and blood cadmium showed no associations with AD (HR [95% CI]: 1.09 [0.9, 1.4]; 1.06 [0.9, 1.2]) nor all-cause dementia (HR [95% CI]: 1.07 [0.9, 1.3]; 1.06 [0.95, 1.2]). ConclusionNo association between cadmium exposure and dementia incidence was observed. Our null findings should be interpreted while considering potential methodological issues and verified by subsequent studies.

BackgroundFatal insulin intoxication remains difficult to diagnose because insulin undergoes rapid degradation after death, limiting the reliability of direct biochemical measurements. This creates diagnostic uncertainty when objective molecular confirmation of insulin excess are required. We hypothesised that insulin excess induces systemic metabolic alterations that persist beyond insulin degradation and can be captured using postmortem metabolomics in a forensic setting. MethodsHigh-resolution mass spectrometry (HRMS)-based metabolomics was applied to a national cohort comprising 51 fatal insulin intoxications. Orthogonal partial least squares-discriminant analysis (OPLS-DA) models were trained on cases collected between 2017-2022 to identify insulin-associated metabolite features using a shared-and-unique-structures approach. Performance was evaluated using two temporally distinct test sets (2023-2024): a matched validation cohort and a heterogeneous forensic cohort reflecting biological variability. ResultsHere we show that an insulin-associated metabolomic fingerprint comprising 91 features demonstrated reproducible discrimination across independent cohorts. In the matched cohort (n=59, including 14 insulin cases), insulin intoxication classification achieved 100% sensitivity and 73% specificity within the applicability domain. In the heterogeneous cohort (n=154, including 14 insulin cases), 100% sensitivity was maintained with a 72% specificity despite increased biological variability. Univariate analyses demonstrated significant alterations across multiple metabolite classes, including acylcarnitines, fatty acids/lipids, and purine/nucleoside metabolites, with moderate effect sizes, consistent with systemic effects of insulin-induced hypoglycaemia. ConclusionsFatal insulin intoxication is associated with a reproducible metabolomic fingerprint detectable after death. These findings demonstrate that postmortem metabolomics may serve as a complementary decision-support tool when conventional biomarkers are unreliable.