This study aimed to assess the protein quality and allergenic potential of chia seeds (Salvia hispanica), introduced in European market as a novel food (Regulation European Union 2015/2283) in 2019. Although chia is increasingly used as a plant-based ingredient, information regarding its protein profile and sequences is still limited. It follows that, without this essential knowledge, information of its allergenic potential is also lacking. Therefore, this work pose itself as a first building block, providing a detailed molecular characterisation of chia proteins and evaluating their allergenic potential and IgE cross-reactivity with other known allergens, such as sesame (Sesamum indicum). Three chia flour samples-partial defatted flour as reference, standardised partial defatted flour, and protein concentrate-were analysed. Protein content was determined by Kjeldahl method. Protein quality was evaluated based on the amino acid profile and the estimation of amino acid score. Proteins were identified by SDS-PAGE through comparison with existing literature and then confirmed by in solution tryptic digestion coupled with high-resolution mass spectrometry analysis. Allergenicity was assessed by in silico sequence homology analysis with characterised sesame linear epitopes and in vitro immunoblotting using sera from sesame-allergic patients. The protein content ranged from 25% to 26% in the raw materials to 56% in the concentrate. Furthermore, SDS-PAGE profiles were comparable between samples, confirming the effectiveness of the extraction method applied. All samples showed balanced amino acid profiles and amino acid scores above one, meeting FAO/WHO requirements for adults and children. The main proteins identified in chia were 11S and 7S globulins, albumins and oleosins. The identified chia peptides were used to obtain a coverage of these with sesame protein sequences, confirming the attended homology. The potential cross-reactivity with sesame proteins, primarily retrieved from the literature, was then confirmed in vitro. IgE-binding was detected for major chia proteins, such as 11S and 7S globulin, and 2S albumin endorsing the attended cross-reactivity with sesame proteins. This study provided insights on the effectiveness of the extraction method applied on chia protein quality, which is essential for their inclusion in balanced food formulations. The approach used for assessing allergenicity also highlighted that the level of molecular and immunological knowledge can differ among novel foods, making it challenging to define a general methodological framework for evaluating their allergenic potential and cross-reactivity risks. These results can be useful both as starting point for the inclusion of protein extracts from chia seeds as safe ingredient, while also highlighting the current lack of comprehensive molecular characterization-including incomplete sequence data and uncharacterized potential epitopes-which limits the full assessment of their allergenic risk.
Enteric infectious diseases claim more than 1 million lives annually and are among the top ten causes of death in children younger than 5 years. Remarkable global investment has been dedicated to enteric infectious disease prevention and control; however, the shifting global health landscape is testing the continuance of progress. To evaluate the current status and guide future interventions, we present the latest epidemiological estimates of enteric infectious diseases from the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2023 and assess progress towards the Global Action Plan for the Prevention and Control of Pneumonia and Diarrhoea (GAPPD) mortality target of fewer than 20 deaths per 100 000 children younger than 5 years by 2025. We quantified the incidence, mortality, and disability-adjusted life-years (DALYs) of enteric infectious diseases by age, sex, and year across 204 countries and territories from 1990 to 2023. In GBD 2023, the following were considered under the category of enteric infectious diseases: diarrhoeal diseases, enteric fever (typhoid and paratyphoid), invasive non-typhoidal Salmonella spp (iNTS) infections, and other intestinal infectious diseases. We also examined 15 aetiologies contributing to diarrhoeal diseases. Incidence and prevalence were estimated with DisMod-MR (version 2.1), a Bayesian meta-regression tool, drawing on data from systematic reviews, population-based surveys, claims data, and hospital sources. Cause-specific mortality was modelled with Cause of Death Ensemble Modelling based on data from sources including vital registration, mortality surveillance, verbal autopsy, and minimally invasive tissue sampling. Years of life lost and years lived with disability were computed and combined to derive DALYs. For aetiology-specific estimation, population-attributable fractions (PAFs) for 15 pathogens were derived with a counterfactual framework. Point estimates and 95% uncertainty intervals (UIs) were generated from 250 draws from the posterior distribution. In 2023, enteric infectious diseases resulted in an estimated 1·27 million (95% UI 0·963-1·68) deaths globally, declining from 3·69 million (3·04-4·56) in 1990. The global age-standardised mortality rate (ASMR) decreased from 74·1 (62·0-92·9) per 100 000 population to 16·4 (12·6-21·3) per 100 000 population during the same period. Diarrhoeal diseases accounted for most deaths in 2023 (1·11 million [0·811-1·54]), followed by enteric fever and iNTS. South Asia and sub-Saharan Africa remained the most affected regions in 2023, with 599 000 (441 000-882 000) and 501 000 (373 000-648 000) deaths due to enteric infectious diseases, respectively, predominantly from diarrhoeal disease. Rotavirus was the leading cause of all-age diarrhoeal disease deaths (PAF 16·3% [12·0-21·5]), followed by norovirus (10·2% [2·4-17·0]) and Shigella spp (9·3% [5·4-15·2]). Among children younger than 5 years, PAFs of deaths due to diarrhoeal diseases were 40·2% (32·5-48·5) for rotavirus, 24·0% (15·1-36·7) for Shigella spp, and 23·4% (13·7-34·3) for adenovirus. Across 204 countries and territories, 141 met the GAPPD mortality target in 2023. The driving aetiologies among countries that did not meet the target in 2023 varied slightly by GBD super-region, but the highest or second-highest number of deaths in children younger than 5 years were consistently attributed to rotavirus. Astrovirus and sapovirus, newly included in GBD 2023, were responsible for 24 600 (6290-49 000) and 18 800 (4650-44 400) deaths, respectively, in 2023, mainly in children younger than 5 years. Our findings show that mortality and ASMRs of enteric infectious diseases declined substantially between 1990 and 2023. This decline is consistent with the expansion of public health measures and broader socioeconomic development. However, the burden in 2023 remains considerably high, with the highest mortality concentrated in sub-Saharan Africa and south Asia. Considering that more than a quarter of all countries had yet to meet the GAPPD mortality target in 2023, sustained efforts are needed to address the persistent burden in affected countries and to adapt to the changing global health landscape. Gates Foundation.
While botulinum neurotoxin type E (BoNT/E) has been shown to have analgesic effects in previous studies, the underlying mechanisms mediating its therapeutic effects remain incompletely understood. This study investigated the involvement of the nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3) inflammasome in the antinociceptive effects of BoNT/E in a rat model of trigeminal neuropathic pain. Neuropathic pain was induced in male Sprague-Dawley rats through inferior alveolar nerve (IAN) injury. IAN injury significantly decreased air-puff thresholds, resulting in mechanical allodynia that persisted for up to 42 days. NLRP3 expression in the ipsilateral trigeminal ganglion (iTG) was significantly increased in the nerve-injured group on postoperative day (POD) 5. In contrast, subcutaneous administration of BoNT/E (6 or 10 U/kg) significantly attenuated mechanical allodynia and suppressed NLRP3 expression in the iTG on POD 5. Moreover, BoNT/E (10 U/kg) markedly reduced the elevated levels of inflammatory cytokines, including interleukin (IL)-1β, IL-18, tumor necrosis factor-alpha (TNF-α), and IL-6 in the iTG by IAN injury. The nerve-injured group also exhibited significant upregulation of hypoxia-inducible factor 1-alpha (HIF-1α) expression in the iTG, which was significantly decreased following BoNT/E (10 U/kg) treatment. Intraganglionic injection of PX-478, a HIF-1α inhibitor, similarly attenuated mechanical allodynia, downregulated NLRP3 expression, and decreased IL-1β, IL-18, TNF-α, and IL-6 levels in the iTG. Collectively, these findings demonstrate that modulation of the HIF-1α-NLRP3 pathway in the iTG plays a critical regulatory role in neuropathic pain development and suggest that BoNT/E may serve as a promising therapeutic strategy for managing chronic neuropathic pain.
Chemical exposure in real-world settings occurs predominantly as mixtures, whereas toxicological assessment has long been shaped by single-substance paradigms. As interest in more human-relevant and mechanistically informative testing strategies has grown, new approach methodologies (NAMs) have been increasingly incorporated into mixture toxicity research. However, the role of advanced in vitro NAMs in chemical mixture toxicity assessment has not yet been examined in a structured manner. This review therefore evaluated how advanced in vitro NAMs are currently being applied in mixture toxicity assessment and considered their potential value for regulatory translation. A PubMed-based literature search covering studies published between 2021 and 2025 identified 353 in vitro NAM-based studies related to chemical mixture toxicity, of which 70 were classified as advanced in vitro NAM-based studies and included for in-depth analysis. Among the advanced in vitro NAM categories, route-of-exposure-relevant models were the most frequently applied, followed by Omics, 3D models, stem cell-based models, and HTS/HCI approaches. The reviewed studies addressed diverse mixtures, including air pollutants, PFAS, phthalates, pesticides/herbicides, consumer product-related mixtures, and other environmentally relevant chemical combinations. Across these studies, advanced in vitro NAMs were used not only to assess overall mixture effects, but also to compare observed and predicted responses, distinguish mixture effects from those of individual chemicals, identify effect-driving components, and interpret non-additive responses under biologically relevant conditions. The reviewed evidence indicates that advanced in vitro NAMs are being used not simply as alternative test systems, but as tools to address key challenges intrinsic to mixture toxicity assessment, including defining relevant mixture entities, identifying toxicity drivers, interpreting non-additivity, and supporting prioritization under combinatorial complexity. These findings suggest that the future value of advanced in vitro NAMs in mixture assessment will depend not only on continued technological development, but also on their integration into question-driven and fit-for-purpose testing strategies. From this perspective, advanced in vitro NAMs may contribute most effectively when used to generate decision-relevant evidence aligned with specific mixture assessment needs and regulatory contexts.
Inhalation is a major route of chemical exposure for both consumers and workers. Physiologically-based kinetic (PBK) modeling is a promising tool to understand the absorption, distribution, metabolism, and excretion (ADME) of inhaled chemicals and to predict systemic concentrations of chemicals in humans. New Approach Methodologies (NAMs) can help generate essential input parameters for PBK models. However, validated NAM-based test methods to assess uptake of inhaled chemicals are currently lacking. Reliable information on respiratory uptake is required to determine relevant exposure concentrations for evaluation of systemic effects using NAMs. This manuscript describes a project that aims to apply robust and reliable in vitro models to study cellular uptake, intracellular accumulation, absorption and systemic exposure of chemicals following inhalation. To evaluate the robustness and predictivity of different NAM-based barrier models, to examine appropriate in vitro to in vivo scaling strategies, and to assess sensitivity and uncertainty in the resulting PBK models, the project will focus on relatively data-rich chemicals, specifically per- and polyfluoroalkyl substances (PFAS). While some have been widely explored and others remain data-poor, the entire chemical family is of interest due to its health hazards. Therefore, the work combines experimental and modeling approaches by generating in vitro data on the respiratory uptake and benchmark this to existing human in vivo data, developing biokinetic models to better understand chemical fate within the test systems, and refining inhalation PBK models to improve estimates of systemic uptake. Read-Across (RAx) will be employed as data gap filling technique to infer on the apparent permeability of non-tested PFAS. Together, the in vitro and in silico results will inform and parameterize PBK models, ultimately enabling more reliable predictions of systemic availability. The project will deliver a workflow to combine in vitro and in silico methods to assess the uptake of inhaled substances, that could be modified and applied to other inhaled substances. Standardized in vitro models for respiratory uptake will improve the evaluation of inhalation as a route of exposure contributing to systemic effects, which is a key requirement for quantitative in vitro to in vivo extrapolation (qIVIVE) and supports the implementation of next-generation risk assessment (NGRA).
Drug-induced liver injury (DILI) is a major cause of drug development failure and market withdrawal. Despite the use of animal and human-derived preclinical models, none reliably predict human hepatotoxicity. Conventional animal studies, based on group-level averages, overlook inter-individual variability critical to idiosyncratic DILI. Although several alternatives to animal testing are available, implementation of alternative methodologies into safety evaluations is very slow and to date, no standalone validated alternative models to assess systemic toxicity exist. To address this, we developed a novel serum-educated rat liver spheroid model that captures individual metabolic diversity, allowing DILI to be assessed both in vitro and in vivo within the same animal. Building on this approach, the present study aimed to validate the model's ability to detect DILI caused by well-characterized hepatotoxic drugs, including diclofenac and bosentan, in rat models. Rats were treated orally with diclofenac or bosentan for 28 days. Blood was collected pre-dosing to generate individualized spheroids containing rat hepatocytes, stellate cells, and macrophages. These spheroids were exposed to each drug across a ten-point concentration range for 3 days and cell viability was quantified using the CellTiter-Glo ATP assay. Clinical chemistry analyses of ALT, AST, ALP, albumin, and bilirubin were performed at in vivo study termination on day 29. In vitro data were analyzed using PredictCan-MIND to derive DILI severity scores and correlate in vitro cytotoxicity with in vivo biomarkers. In vivo, diclofenac caused no significant liver enzyme elevations, although some rats, exclusively females, displayed subtle toxicity revealing sex-dependent susceptibility. Corresponding in vitro spheroids confirmed hepatocellular injury in these individuals. Bosentan produced a mild cholestatic response in vivo without consistent enzyme elevation, while in vitro analysis showed clear hepatotoxicity in 8 of 10 rats. Notably, in vitro DILI severity correlated strongly with in vivo ALP levels, consistent with bosentan's known cholestatic mechanism. The serum-educated rat liver spheroid model captures inter-individual and sex-related differences in hepatotoxicity and demonstrates translational concordance with in vivo cholestatic markers. This approach improves DILI prediction, aligns with the 3Rs principle, and supports a potential 50%-70% reduction in animal use for preclinical liver toxicity testing.
Per- and polyfluoroalkyl substances (PFAS), particularly perfluorooctanoic acid (PFOA), are persistent environmental contaminants known for bioaccumulation and adverse health effects, including neurodevelopmental toxicity. This study investigated the impact of PFOA on primary human neuronal progenitor cells (phNPCs) derived from fetal brain tissue from genetically diverse donors, focusing on lipid metabolism and neuronal differentiation. phNPCs were exposed in vitro to PFOA at high concentrations (10,000-156 μM range) to determine cell viability and cytotoxicity using Alamar blue and lactate dehydrogenase (LDH) assays, respectively. Further experiments were conducted in 300-0.3 μM range where no effects on cell viability or cytotoxicity were observed. phNPCs were treated acutely (2 days) and assessed for changes in lipid droplet accumulation, fatty acid metabolism, lipid peroxidation, mitochondrial damage, and proliferation (EdU, Ki67, pHH3 staining). phNPCs were then exposed to PFOA for 14-days in neuronal differentiation media and assessed for changes in neuronal gene expression using quantitative reverse transcription polymerase chain reaction (RT-qPCR) and MAP2 protein expression and neuronal morphology using high content imaging. To assess differences in cytotoxicity between neuronal progenitors and neurons, fully differentiated neurons and phNPCs were both exposed to high concentrations (10,000-156 μM range) for 14 days and assessed for impacts on cell viability and death using Alamar Blue assays and flow cytometry using Calcein-AM/7-AAD stained cells. Acute PFOA exposure induced dose-dependent lipid droplet accumulation, increased fatty acid uptake, reduced lipid turnover, elevated lipid peroxidation, mitochondrial reactive oxygen species, and fragmented mitochondrial morphology. The PFOA-induced lipid droplet accumulation was attenuated by inhibition of autophagy and lipolysis pathways, suggesting PFOA-induced lipotoxicity. PFOA exposure had minimal effects on phNPC proliferation but 14-day exposure during neuronal differentiation reduced MAP2-positive neurons, neuronal branching and gene expression of neuronal markers (TUBB3, SYN1, MAP2), while increasing the gene expression of progenitor-associated FABP7. Principal component analysis revealed PFOA-exposed cells exhibited intermediate gene expression between progenitors and mature neurons. Treatment of fully differentiated neurons during the same time window resulted in increased death cell and reduced viability compared phNPCs, suggesting neurons are more susceptible to PFOA cytotoxicity. Across donors, greater PFOA-induced lipid accumulation negatively correlated with neuronal differentiation outcomes. These findings indicate that PFOA disrupts human neurodevelopment primarily by impairing neuronal differentiation, potentially through lipotoxicity and mitochondrial stress, highlighting a mechanistic link between dysregulated lipid metabolism and reduced neurogenesis.
Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) extensively prescribed in clinical practice; its use can be associated with toxic effects on multiple organs. These deleterious effects are associated with a combination of oxidative stress, sterile inflammation, and disrupted epithelial barrier integrity. Oridonin has strong antioxidant and anti-inflammatory characteristics, making it a potential candidate for NSAID-induced organ damage. Evaluate the protective effects of oridonin against diclofenac-induced liver, kidney and stomach toxicity, while elucidating molecular mechanisms involved in oxidative stress modulation, endoplasmic reticulum (ER) stress suppression, inflammasome inhibition and barrier preservation of epithelial cells. 30 male Sprague-Dawley rats were divided into five groups: normal control, oridonin control, diclofenac (100 mg/kg, IP), and diclofenac treated with low-dose (10 mg/kg) or high-dose (20 mg/kg) oridonin were the experimental conditions. Renal and hepatic function was assessed by serum and urinary markers. Oxidative stress was assessed by the determination of MDA, GSH, and total antioxidant capacity (TAC) in tissue homogenates. Molecular pathways were studied by ELISA and Western blotting to quantify ER stress markers (IRE1, CHOP, PERK) and the TXNIP/NLRP3/Caspase-1 inflammasome axis. Gastric barrier integrity was confirmed by expression measurement of tight junction proteins (Claudin, ZO-1, Occludin), histopathological, and immunohistochemical markers of NF-κB and IL-1β. Oridonin pretreatment led to the recovery of antioxidant defenses and large decreases in the activation of the TXNIP/NLRP3/Caspase-1 axis, and ER stress signaling. In addition, oridonin maintained the gastric mucosal architecture and the expression of critical tight junction proteins and inhibited NF-κB-induced inflammation. Oridonin ameliorates diclofenac-induced multi-organ toxicity by reducing oxidative stress and ER stress, inhibiting the NLRP3 inflammasome, and preserving epithelial barrier integrity. Thus, oridonin can be considered a suitable drug to ameliorate the unwanted side effects of NSAID treatment.
Colistin is currently reserved as a last-resort antibiotic for multidrug-resistant Gram-negative bacteria such as pan-resistant Escherichia coli. Colistin-resistant E. coli strains have been detected in wastewater, clinical, and agricultural sites, and this resistance is mediated by the plasmid-borne mcr-1 gene, which can spread into other sectors, including the food chain. Herein, we isolated several E. coli strains from mixed wastewater that showed varying resistance to colistin with IC50 values ranging from 6.5 to 27.3 μg/ml as compared to 0.5 μg/mL for the wild-type strain. The strains all contained the mcr-1 gene, and there is no gene duplication to account for the increased resistance to colistin. In addition, the strains displayed resistance to several other antibiotics belonging to different classes, including ciprofloxacin, sulfamethoxazole, and tetracycline, while maintaining wild-type sensitivity to other antibiotics such as tazocin and meropenem. Interestingly, some of the strains showed resistance to the powerful DNA-damaging agents, ultraviolet radiation and hydrogen peroxide, used for eradicating bacteria. Measurement of catalase activity, which decomposes hydrogen peroxide, was not significantly elevated, excluding the possibility that catalase is involved in the resistance of the strains to H2O2. However, we found that H2O2 treatment caused the accumulation of fragmented chromosomal DNA in the wild-type, but not in one of the representative H2O2-resistant strains. Analysis of whole genome sequencing data revealed that the H2O2-resistant strains harbour a high level of missense mutations in several genes, including DNA repair genes that could encode variant proteins with elevated capacity to repair damaged DNA.
Up to 80% of patients with Parkinson's Disease (PD) develop dementia within 20 years of diagnosis. Although the etiology of PD and related neurodegenerative disorders is poorly understood, risk factors including environmental toxicants and viral infections are linked to disease onset and progression. Exposure to high doses of the essential element, manganese (Mn), causes neurotoxicity associated with parkinsonian symptoms and cognitive impairment in humans. Additionally, epidemiologic studies indicate that viral infections increase risk of developing PD. Previously, our lab demonstrated that mice exposed to Mn during juvenile development showed greater neuroinflammatory changes in microglia within the substantia nigra following systemic infection with H1N1 influenza virus (California/04/09 influenza A) than mice infected without prior exposure to Mn. In the present study, this murine dual-hit model was employed to investigate how juvenile Mn exposure alters H1N1-induced neuropathology and glial morphology in the hippocampus. Mice were exposed to Mn in drinking water from post-natal day 21-51 and then intranasally infected with 103 TCID50 A/California/04/2009 H1N1. To assess histopathology following this exposure paradigm, we performed high-content microscopy and machine learning-based image analysis of H&E and IHC-stained sections spanning the hippocampus to quantify pyknotic neurons and reactive microglia. We report a significant increase in the number of pyknotic neurons in the dentate gyrus as well as morphologic changes in microglia that are consistent with inflammatory activation. Our findings highlight the capacity of combined juvenile manganese exposure and adult viral infection to induce substantial microgliosis in the hippocampus.
Non-animal technologies (NATs; also termed new approach methodologies, NAMs) are rapidly reshaping biomedical research, safety testing and drug development by improving human relevance and reducing reliance on animal models. In Australia, substantial expertise exists across organoids, microphysiological systems and in silico approaches; however, national visibility of capabilities, infrastructure access and system-level needs has been limited. We conducted a cross-sectional, online baseline survey (February-October 2025; https://redcap.ohmr.health.nsw.gov.au/surveys/?s=3EWRM48PEXWLFJC7.) integrated into NAT-Net registration and implemented using REDCap. The survey captured organisational characteristics, roles within the NAT ecosystem, research domains, model types, development stage, supporting services and infrastructure access, engagement preferences, and perceived challenges. Quantitative data were summarised descriptively, and free-text responses were analysed thematically. Of 135 submitted records, incomplete entries (n = 2) and duplicates (n = 9) were removed, yielding 124 unique respondents. Respondents were predominantly based in New South Wales (71.0%), followed by Victoria (18.5%) and Queensland (7.3%). Most organisations reported involvement in research and development (79.0%), education and training (41.9%), ethics and the 3Rs (39.5%), and provision of supporting services or facilities (32.3%). Among respondents engaged in NAT R&D (n = 102), commonly reported approaches included in vitro 3D models (64.7%), in vitro 2D culture (56.9%), microphysiological systems/organ-on-chip platforms (35.3%), and in silico methods (30.4%). Among service providers answering the access item (n = 40), 60.0% offered external access. The most frequently identified challenges were the absence of standards and validation processes (75.8%), limited access to resources and infrastructure (74.2%), and difficulty in establishing collaborations or partnerships (71.7%). This NAT-Net baseline survey provides an initial cross-sector ecosystem mapping centred on NAT-Net participants, revealing broad cross-sectoral activity alongside persistent system-level barriers to standardisation, infrastructure access and coordination. These findings provide a critical evidence base to inform coordinated national strategy, investment, policy development and longitudinal monitoring.
Di (2-ethylhexyl) phthalate (DEHP), a ubiquitous environmental plasticizer, is increasingly linked to neurotoxicity and carcinogenesis. However, its role in glioma pathogenesis remains poorly understood. This study integrates network toxicology and machine learning to identify molecular targets of DEHP in glioma. Potential DEHP targets were identified through four databases (CHEMBL, CTD, SwissTargetPrediction, PharmMapper). Glioma-related genes were screened using differential expression analysis and weighted gene co-expression network analysis (WGCNA) on GEO and TCGA datasets. Overlapping genes were subjected to functional enrichment, followed by 127 machine learning models to prioritize core genes. SHAP analysis interpreted model contributions, while COX regression assessed prognostic value. Molecular docking and dynamics simulations evaluated binding stability between DEHP and target proteins. In vitro validation was performed in U87 cells via RT-qPCR and Western blotting. A total of 77 overlapping genes were identified, enriched in neuroactive ligand-receptor interactions, GABAergic synapses, and ion channel activity. Machine learning prioritized 12 key genes (e.g., RELA, ABCA1, HIF1A), forming a parsimonious 12-gene diagnostic model with strong external discrimination (pooled validation AUC = 0.994). A high DEHP-related risk score was associated with poorer survival in the TCGA cohort and showed similar prognostic stratification across the external CGGA_325, CGGA_693, and GSE16011 cohorts. Molecular simulations confirmed stable binding between DEHP and core proteins. Experimental validation demonstrated dose- and time-dependent upregulation of RELA, ABCA1, and HIF1A in DEHP-exposed U87 cells. This integrative approach provides a systems-level framework to prioritize DEHP-associated target genes and molecular signatures in glioma, extending beyond the previously reported PER3-related observation and offering candidate biomarkers for early detection and prognosis under environmental exposure.
The potent synthetic cannabinoid ADB-BUTINACA (also known as ADB-BINACA) was implicated in three fatal intoxications. Post-mortem samples, including femoral and heart blood, urine, gastric content, bile, vitreous humor, cerebrospinal fluid, and various tissues (brain, kidney, liver, lung, muscle), were analyzed. All cases were mixed intoxications with pregabalin, heroin, ketamine, MDPHP, or ethanol. Post-mortem examinations were performed in all three fatalities. Blood and urine were screened by immunoassay, gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for drugs. ADB-BUTINACA was quantified by standard addition in all matrices except vitreous humor and cerebrospinal fluid. For assessing the contribution of ADB-BUTINACA, a Toxicological Significance Score (TSS) was assigned to each case. Metabolite profiles received from analysis of post-mortem matrices were compared with HepG2 cell and human liver microsome data. Additionally, compound stability was assessed over 12 weeks in whole blood. Femoral blood concentrations of ADB-BUTINACA ranged from 4.2-8.2 ng/mL, and in heart blood from 5.7-11 ng/mL. Brain tissue contained 1.0-6.2 ng/g, and vitreous humor 0.33-2.1 ng/mL. TSS for ADB-BUTINACA of 1 - 3 were assigned for all cases. Heart-to-femoral blood ratios (1.3-1.8) indicated relatively low post-mortem redistribution. Metabolite profile assessment indicated that detection of metabolic biomarkers such as the dihydrodiol could be relevant in urine and bile. Matrix storage at -20 °C is highly recommended to avoid stability issues. ADB-BUTINACA was detected in all investigated matrices, with the highest concentrations observed in liver, lung, and kidney tissue. The elevated levels in these organs likely reflect their lipophilicity as well as their involvement in absorption (lungs) and metabolism/excretion processes (liver and kidneys). The results of this study may enhance interpretation of toxicological findings in similar cases.
Thyroid hormone (TH) availability is particularly critical for early brain development. TH transport across the blood-brain barrier is facilitated through two main transmembrane transporters: monocarboxylate transporter 8 (MCT8) and organic anion transporter 1C1 (OATP1C1). Inhibition of MCT8-mediated TH transport has been identified for a number of environmental chemicals using in vitro screening assays. Here we examined the in vivo effects of exposure to a potent in vitro inhibitor of MCT8, the flavonolignan silychristin, on several aspects of the TH system. Adult female rats were daily gavaged with 0, 250, or 500 mg/kg/day (n = 10/group) of silychristin for 7 days and euthanized on day 8. A smaller group (n = 5/group) of rats was administered the related flavonolignan, silybin (900 mg/kg), or the milk-thistle-derived flavonolignan mixture, silymarin (1,500 mg/kg). Serum TH concentrations were not changed in any treatment group. Mct8 and Oatp1c1 expression were upregulated in the choroid plexus upon silymarin exposure, without change in response to silychristin or silybin. Deiodinase 1 and dehalogenase activities, unchanged in the liver, were increased in the thyroid by the high dose of silychristin. These changes may have been triggered by increased thyroidal TH content, consequent to a reduction in MCT8-mediated TH efflux. Pharmacokinetic properties of silychristin and other flavonoids result in their low bioavailability and likely contributed to the largely negative findings. These observations demonstrate the challenges in extrapolating results from in vitro models to studies in intact organisms, showcasing the importance of selecting appropriate animal models and the best experimental design for assessing effects on human health.
Next generation risk assessment (NGRA) demands a fundamental transformation in how toxicological test methods are validated. Traditional validation approaches, designed for animal tests and mainly for simple in vitro methods, are increasingly inadequate for evaluating complex New Approach Methodologies (NAMs), artificial intelligence (AI)-based approaches, and integrated testing strategies (ITS). This paper presents a comprehensive framework for "next-generation validation" that leverages artificial intelligence and modern computational capabilities to create more efficient, thorough, and dynamic validation processes. The proposed framework emphasizes human relevance over simple concordance with animal data and emphasizes key innovations including e-validation, mechanistic validation, and post-validation companion AI agents. Because AI can inherit biases, obscure failure modes, and drift over time, the framework treats AI as both a tool for, and a subject of, validation, requiring transparent performance criteria, uncertainty quantification, and explicit governance for model updates and lifecycle monitoring. To make the framework actionable, we define a method as "NGV-validated for a stated context of use" when it meets pre-specified acceptance criteria across five domains, i.e., technical reliability, biological relevance, predictive performance, uncertainty quantification, and data integrity, supported by defined governance roles, version control, and lifecycle re-review triggers. e-validation employs sophisticated algorithms for reference chemical selection, study simulation, and continuous performance monitoring, while mechanistic validation evaluates whether methods accurately capture relevant biological pathways and mechanisms of toxicity. The paper addresses critical implementation challenges including data quality standardization, regulatory acceptance, and international harmonization, providing specific recommendations for various stakeholders. Looking forward, validation will increasingly embrace dynamic, adaptive approaches that evolve alongside scientific understanding and technological capabilities. The integration of artificial intelligence will enhance analysis of complex data, enable real-time monitoring of method performance, and support more sophisticated uncertainty quantification. Success in this transformation requires coordinated effort across regulatory agencies, industry partners, and academic institutions. In summary, this paper emphasizes a five-pillar framework integrating mechanistic, probabilistic, and AI-driven elements to reform toxicological validation. The proposed framework, exemplified here for tests for developmental neurotoxicants and virtual control groups, represents a crucial step toward more efficient and accurate chemical safety assessment while maintaining necessary standards for public health protection.
Drinking water treatment plants (DWTPs) are designed to protect public health; however, residual contaminants may persist after treatment and elicit biological effects that are not fully covered by routine chemical monitoring. In this study, we combined Polar Organic Chemical Integrative Samplers (POCIS) with in vitro bioassays to evaluate residual biological activity in raw and treated water from six full-scale DWTPs in the Czech Republic. POCIS were deployed at raw- and treated-water points to collect extracts representing mixtures of polar and semi-polar contaminants under realistic exposure conditions. These extracts were evaluated using transcriptional responses in the RTL-W1 rainbow trout cell line and receptor-mediated yeast bioassays specific for estrogenic and progestogenic activity. Among the evaluated biomarkers, gene expression of CYP1A was the most strongly and reliably induced, indicating the presence of AhR-active substances in raw water and, in several cases, incomplete removal of these substances during treatment. In contrast, genes related to phase II detoxification, cell stress, and oxidative stress (GST, HSP, and Nrf2) responded weakly, suggesting a predominantly receptor-mediated mechanism of action rather than generalized cytotoxicity. Estrogenic activity was detected in all raw waters but was below detection limits in all treated waters, indicating efficient removal of estrogenic substances during treatment. Progestogenic activity was not detectable. This study highlights the importance of effect-directed analysis for assessing the efficiency of drinking water treatment processes and confirms the suitability of passive sampling combined with bioassays for identifying treatment-resistant bioactivities.
Heavy metal contamination of coastal waters results in bioaccumulation in fish, posing a significant route of human exposure via seafood consumption. The present study focuses on organ-specific metal distribution and associated health risk indices in three important fish species from the Nagapattinam coast, India. In the current study, three species of fish (Nemipterus japonicus, Oreochromis mossambicus, and Lates calcarifer) were considered with three biological replicates per species obtained from local fish markets of Nagapattinam, Tamil Nadu, India, during January-February 2024. The HM profiling was performed on three organs: liver, gills, and muscle tissues. HMs, including arsenic (As), cadmium (Cd), chromium (Cr), mercury (Hg), lead (Pb), strontium (Sr), and vanadium (V), were analyzed using inductively coupled plasma mass spectrometry (ICP-MS). The concentrations were found to be in the range of 0.025-21.53 μg kg-1. Upon non-carcinogenic risk assessment, the target hazard quotient (THQ) for all species was found to be <1 for both adults and children, indicating low non-carcinogenic risk associated with the consumption of the selected species under the assumed intake scenario. In contrast, the cancer risk (CR) value for chromium in Nemipterus japonicus was found to be elevated (children: 1643.58 × 10-6 and adults: 939.19 × 10-6), assuming a conservative worst-case exposure scenario in which total chromium is considered as Cr(VI), potentially inflating cancer risk estimates. Overall, the findings suggest low non-carcinogenic risk under average consumption conditions; however, the estimated carcinogenic risk for chromium exceeded the commonly referenced acceptable threshold under conservative assumptions and should therefore be interpreted with caution. These results highlight the need for further investigation, particularly chromium speciation analysis, to refine risk estimates for fish consumed from the Nagapattinam marketplace.
Bisphenol S (BPS), a widely used environmental endocrine disruptor, induces multigenerational liver injury, though its underlying mechanisms remain unclear. In this study, pregnant mice were housed separately and randomly divided into control and BPS (0.2, 2, and 20 mg/L) group through drinking water exposure. We observed the pathological changes in the livers of the offspring mice and the indicators of liver lipid metabolism disorder. At the same time, we also measured the levels of liver peroxidation and ferroptosis. Additionally, we established a co-exposure situation of in vitro BPS and ferroptosis inhibitors (Fer-1), analyzed the expression of ferroptosis-related genes to evaluate the hepatotoxicity caused by BPS by disrupting liver lipid metabolism and inducing iron depletion. In this study, we observed that BPS exposure induced liver injury and significantly impaired hepatic function in offspring, as indicated by elevated serum ALT and AST levels. Perinatal BPS exposure altered the expression of genes involved in fatty acid β- oxidation, synthesis, and absorption, and concurrently induced dyslipidemia characterized by elevated triglyceride, total cholesterol, and LDL-cholesterol levels and reduced HDL-cholesterol. We further established that BPS promotes ferroptosis in offspring mice, as evidenced by iron accumulation, mitochondrial damage, and oxidative damage. Mechanistically, BPS increased the expression of genes related to lipid peroxidation while suppressing those involved in the antioxidant system. Notably, Fer-1 cotreatment markedly alleviated both the lipid metabolism disorders and ferroptosis triggered by BPS. this study provides evidence that perinatal BPS exposure leads to hepatic lipid metabolism dysfunction and ferroptosis activation in offspring mice, highlighting BPS as a potential hepatotoxicant.
The small size of nanoplastics (NPs; <1 µm in diameter) facilitates airborne transport, inhalation, and deposition in the lungs, raising significant concerns about potential effects on human health. In occupational settings, such as waste management and recycling facilities, exposure to NPs carrying microbial contaminants may pose an additional health risk to workers. In the present study, we investigated pulmonary cytotoxicity and pro-inflammatory responses after exposure to polyethylene terephthalate nanoplastics (PET-NPs) with or without microbial contaminants. PET-NPs were synthesized from a post-consumer juice bottle (PET b001) and commodity PET pellets (PET c000). The presence of microbial contaminants was assessed via receptor activation in HEK293 Toll-like receptor (TLR) reporter cells expressing TLR2 or TLR4. Co-cultures of human alveolar epithelial cells (A549) and monocyte-derived macrophages (dTHP-1) were exposed to PET-NPs (0, 10 or 100 μg/mL) that tested either negative or positive for TLR2 and TLR4 activation. After 24 h, cell viability was measured, and cytokine responses were quantified at both mRNA and protein levels. PET b001 activated TLR2 and TLR4, indicating the presence of biologically active microbial components, whereas PET c000 showed no activation. In A549/dTHP-1 co-cultures, PET b001 (10 and 100 μg/mL) significantly increased IL-1B, IL-6, IL-8, and TNF mRNA levels and IL-6 and IL-8 protein secretion. In comparison, PET c000 selectively increased IL-8 mRNA levels and protein secretion, and only at the highest tested concentration (100 μg/mL). No changes in cell viability were observed for either particle type. We found that the pro-inflammatory responses to PET-NPs are largely mediated by associated microbial components rather than the polymer itself, highlighting the importance of accounting for environmental context when evaluating their health risks. No evidence of cytotoxicity was observed, as cell viability remained unchanged. Our results further emphasize the need to assess microbial contamination prior to toxicity testing and point to potential occupational health risks in plastic waste and recycling environments.
Current regulatory neurotoxicity guidelines do not include behavioral endpoints that capture stress-related responses. Zebrafish larvae prior to independent feeding offer a promising vertebrate model for developing new approach methodologies (NAMs) because they combine neurobiological relevance with high-throughput potential. In this study, we developed and evaluated a larval thigmotaxis assay to detect behavioral alterations induced by neuroactive substances. Zebrafish larvae at 120 hpf were exposed for 1 h to model compounds and then challenged with visual (light/dark) and acoustic (tapping/silence) stimuli. Thigmotaxis, defined as edge-preference behavior, and locomotor activity were assessed. To increase throughput, we compared the conventional 24-round-well format with a 96-square-well format. Assay performance was evaluated using caffeine and diazepam as reference compounds, followed by additional neuroactive substances (chlorpyrifos, nicotine, dexamethasone, ethylenethiourea) and low-neuroactivity comparators (saccharin, amoxicillin). Benchmark dose modeling was used to compare the sensitivity of thigmotaxis and locomotor endpoints. The 24-well and 96-well formats produced equivalent results, supporting use of the higher-throughput system. Reference compounds confirmed assay performance, with caffeine increasing thigmotaxis and diazepam decreasing it under specific stimulus conditions. Additional neuroactive substances produced stimulus-dependent behavioral responses, whereas saccharin and amoxicillin caused little or no effect. Across compounds, benchmark dose modeling showed that thigmotaxis was generally more sensitive than traditional locomotor activity endpoints. This multiplexed visual-acoustic thigmotaxis assay is reproducible, scalable, and sensitive for detecting neuroactive effects in zebrafish larvae. It can be used either as a stand-alone behavioral NAM or integrated into a broader test battery for neurotoxicity assessment. The method provides a practical and ethical tool to support chemical safety assessment in both ecotoxicology and human toxicology.