Toxicological Sciences

BackgroundPer- and polyfluoroalkyl substances (PFAS), particularly perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), are persistent organic pollutants ubiquitous in the environment. Epidemiological evidence has closely linked them to an elevated risk of prostate cancer (PCa). However, the precise molecular mechanisms by which combined PFOS/PFOA exposure promotes prostate cancer and their dynamic effects on the tumor microenvironment remain unclear. MethodsThis study constructed a multi-module analytical framework integrating network pharmacology and computational biology: (1) Through ADMET toxicity prediction, multi-database target collection (three-way Venn analysis), panoramic GO/KEGG enrichment, focused androgen receptor (AR) axis analysis, GWAS genetic association validation, protein-protein interaction (PPI) network construction, machine learning-based independent screening, and a relaxed intersection strategy, we systematically identified PFOS/PFOA-prostate cancer core targets. (2) Subsequently, a PFAS-PTS score weighted purely by Cox coefficients was employed to drive gene set variation analysis (GSVA)-based pathway enrichment, tumor microenvironment (TME) deconvolution, ordinary differential equation (ODE)-based kinetic modeling, and drug intervention prediction. ResultsTarget collection identified 100 shared PFOS/PFOA-prostate cancer targets, from which 18 core targets were determined after multi-module screening. These targets were significantly enriched in the AR signaling axis, the PI3K-AKT pathway, and cell cycle regulation. Molecular docking confirmed strong binding affinities of PFOS/PFOA with AR (-9.49/-8.56 kcal/mol), AKT1 (-7.56/-6.93 kcal/mol), and PTEN (-6.36/-6.08 kcal/mol). GSVA revealed that the G2M checkpoint and E2F target gene pathways were significantly upregulated in the high-risk group (padj < 0.001), whereas the androgen response pathway was downregulated (padj = 4.8e-4). TME deconvolution (GSE141445, NNLS) revealed a significantly increased proportion of tumor cells (PCa) (p = 2.4e-4) and markedly reduced CD8+ T cell infiltration (p = 5.7e-4) in the high-risk group, indicating immunosuppressive microenvironment remodeling. ODE-based kinetic modeling confirmed that PFAS promoted tumor cell proliferation and suppressed immune surveillance in a dose-dependent manner. Drug intervention simulation demonstrated that the combination of enzalutamide and Alpelisib achieved optimal tumor cell inhibition (33.9% predicted by the ODE model). ConclusionPFOS/PFOA promote prostate cancer progression primarily through multi-target synergy involving AR axis disruption, PI3K-AKT pathway activation, and cell cycle dysregulation, while reshaping an immunosuppressive tumor microenvironment. The integrative computational framework established in this study provides systematic computational evidence for risk assessment and therapeutic intervention in PFAS-associated prostate cancer.

The early developmental environment plays a critical role in the etiology of cardiovascular diseases (CVDs), but underlying molecular mechanisms are poorly understood. Exposure to per and polyfluoroalkyl substances (PFAS) are linked to various CVDs, but effects of developmental PFAS exposures on the human heart remain unclear. Using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM), the objective of this study was to investigate the effects of PFAS exposure during cardiac differentiation on gene expression and function of cardiomyocytes. We exposed two hiPSC lines (one male and one female donor) to perfluorooctanoic acid (PFOA), a common and ubiquitous PFAS (0.05, 0.5, 5, 50, 100, 150, 200 M), followed by assessment of cellular number and pluripotency marker expression. PFOA exposure for 72 hours had no significant effects on hiPSC pluripotency, and modest inhibition of proliferation was observed only at the highest concentration. hiPSCs were then differentiated into ventricular cardiomyocytes in the continued presence or absence of PFOA (0, 0.5, 5, 50 M) using an established small molecules protocol. Optical mapping studies using voltage and calcium-sensitive dyes revealed dose and cell line-specific effects of PFOA on cardiomyocyte voltage and calcium dynamics that were still present 10 days after cessation of exposure. Patch clamping studies demonstrated small but significant reductions in repolarizing IKr currents with 5{micro}M PFOA exposure in cardiomyocytes from both donors. Using RNA-seq, we found that exposure to PFOA led to significant changes in transcriptional pathways related to lipids and lipoproteins in the female hiPSC-CM. In the male hiPSC-CM, we observed significant effects on developmental pathways and calcium homeostasis. Thus, we found that environmentally relevant PFOA exposure during cardiomyocyte differentiation affects the electrophysiological properties and transcriptome of hiPSC-CM even after cessation of exposure, with effects that differ by donor cell line. These findings provide direct experimental evidence that transient developmental exposure to PFOA can durably reprogram human cardiomyocyte function, supporting a developmental origin of PFAS-associated cardiovascular risk. Impact StatementThese studies demonstrate that exposure to environmentally relevant levels of PFOA during the differentiation of hiPSCs into cardiomyocytes alters cardiac gene expression and function, with effects that persist beyond cessation of exposure.

E-cigarettes have attracted significant attention as a safer substitute for conventional tobacco smoking. However, they have introduced new inhalable toxicants, including benzaldehyde-propylene glycol acetal (BPGA)--a chemical adduct produced by cherry-flavoured e-cigarettes. The health risks associated with such flavour-derived acetals remain insufficiently elucidated at the cellular level. This study investigated the role of BPGA in the progression of epithelial-to-mesenchymal transition (EMT)-like changes in alveolar epithelial cells (A549 cells). A549 cells exposed to various concentrations of BPGA were analysed for cell viability, morphology, mitochondrial function, lysosomal health, and cytoskeletal integrity using viability assays and fluorescence imaging. Intracellular reactive oxygen species (ROS) production was quantified using the 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) assay. Antioxidant enzyme expression, inflammatory responses, and EMT-associated phenotypic alterations were evaluated using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunofluorescence (IF) assays. Exposure of alveolar epithelial cells to BPGA caused a concentration-dependent decrease in cell viability. BPGA exposure resulted in mitochondrial membrane depolarisation, lysosomal damage, cytoskeletal changes, and stress fibre formation, which altered cell morphology. It significantly increased intracellular ROS production. As a result, antioxidant enzyme levels were upregulated as a protective response. However, during severe oxidative stress, this response was overwhelmed. Excess ROS disrupted cellular homeostasis and initiated apoptosis, though not completely. ROS also acted as a signalling molecule, promoting the upregulation of inflammatory mediators. These changes were associated with altered EMT marker expression, suggesting that BPGA might drive EMT-like remodelling. In conclusion, BPGA, a chemical adduct from e-cigarette vapour, induces alveolar injury by promoting oxidative stress, inflammation, and EMT-related changes, which may explain a mechanism by which e-cigarette exposure could lead to lung injury and pulmonary fibrosis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=169 SRC="FIGDIR/small/724520v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@f7739dorg.highwire.dtl.DTLVardef@1c74f11org.highwire.dtl.DTLVardef@180aeeorg.highwire.dtl.DTLVardef@75ae14_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG

Phthalates are pervasive endocrine-disrupting chemicals widely used in consumer products. The wide use of many phthalates results in chronic human exposure to complex mixtures rather than single compounds. Despite extensive studies on individual compounds, the combined effects of phthalate metabolites on oogenesis remain poorly understood. Here, we developed a precise microinjection-based single-oocyte toxicological assay to examine the impact of a defined phthalate metabolite mixture on meiotic progression. Phthalate mixture exposure markedly impaired oocyte maturation, as most oocytes failed to extrude the first polar body. Mechanistic analyses revealed severe meiotic defects, including disrupted spindle morphology, chromosome misalignment, disorganized actin cytoskeleton, and impaired mitochondrial function, accompanied by excessive reactive oxygen species (ROS) accumulation and DNA damage. Single-cell transcriptomic profiling further identified differentially expressed genes enriched in biological processes related to exocytosis, secretory pathway regulation, and cytoskeletal organization, as well as in MAPK, JAK-STAT, cGMP-PKG, and GnRH signaling pathways that are essential for follicular development and oocyte maturation. Together, these findings demonstrate that combined phthalate exposure directly compromises female gamete quality and underscore the importance of evaluating mixture effects when assessing risks to womens reproductive health.

Ethylene oxide is a widely used industrial chemical,yet evidence linking its exposure to Parkinsons disease remains limited.Using data from participants in the United States,we examined whether exposure to ethylene oxide is associated with Parkinson's disease.This cross-sectional study included 8,430 adults from the National Health and Nutrition Examination Survey (NHANES) collected between 2013 and 2020.Information on demographic characteristics,socioeconomic factors,lifestyle behaviors,body mass index,sedentary time and major chronic conditions was analyzed. Levels of hemoglobin ethylene oxide adducts,a biomarker of ethylene oxide exposure, were evaluated in relation to Parkinsons disease using statistical modeling approaches.After accounting for potential confounding factors,higher levels of ethylene oxide exposure were associated with an increased likelihood of Parkinson's disease.The association followed a positive and linear pattern.These findings provide new population-based evidence suggesting that ethylene oxide may be linked to Parkinsons disease and highlight the need for further studies to confirm causality and to better understand the biological mechanisms involved.

Benzo[a]pyrene (BaP), a representative polycyclic aromatic hydrocarbon (PAH), is a widespread environmental toxicant and potent ligand of the aryl hydrocarbon receptor (AHR). Yet, how early developmental exposure to BaP influences human neurodevelopment remains poorly understood. We first examined AHR expression dynamics during human embryonic stem cell (ESC)-derived cerebral organoid development and found that AHR expression was highest at the ESC stage and declined during subsequent differentiation, suggesting a potential window of heightened susceptibility to AHR-mediated environmental perturbations. Based on this observation, ESCs were exposed to BaP (0.1, 1 M) for 7 days prior to organoid generation. BaP exposure did not alter proliferation, cell death, or global transcription of ESCs but increased expression of a subset of AHR target genes. Remarkably, however, organoids derived from BaP-exposed ESCs exhibited profound morphological defects resulting from premature neurogenesis, characterized by disrupted neural rosette organization, reduced EOMES intermediate progenitors, and increased BCL11B neurons. Pharmacological inhibition of AHR with CH-223191 attenuated AHR activation and rescued the progenitor-neuron imbalance. These findings identify AHR signaling as a critical upstream mediator of BaP-induced developmental neurotoxicity and highlight the vulnerability of early pluripotent stages to environmental insults.

The ubiquitously occurring food contaminants altenuene (ALT) and tentoxin (TEN) are recognized as emerging Alternaria mycotoxins, yet substantial data gaps remain when it comes to their toxicological behavior and toxicokinetic characteristics. This study aimed to compare and generate quantitative data on their hepatic metabolism and to obtain semi-quantitative insights into their metabolite profiles. To this end, primary rat and human hepatocytes were incubated with 10 {micro}M ALT or TEN over multiple time points up to 4 h. Both substrate depletion and metabolite identification revealed pronounced interspecies differences. The extent of ALT metabolism was significant, with an 88% and 57% decrease in rat and human hepatocytes after 4 h, respectively. In contrast, TEN showed extensive biotransformation in rats (67%) but only modest turnover in humans (27%) over the same period. Hepatocellular clearances were consistently higher for ALT than TEN, with hepatic extraction ratios indicating intermediate extraction for ALT and low extraction for TEN. High-resolution mass spectrometry combined with targeted analysis of selected metabolites annotated phase II conjugation as the predominant metabolic pathway for ALT and phase I oxidative metabolism for TEN, including mono- and double-metabolized species for the latter. Overall, these results provide a comprehensive characterization of ALT- and TEN-metabolism in hepatocytes, offering a foundation for future studies on their toxicological relevance and impact on human health.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor involved in xenobiotic sensing, as well as development, immunity, and tissue homeostasis. AhR signaling can proceed through a canonical and non-canonical pathway; the present study focuses on the canonical pathway. While ligand-dependent differences in binding affinities and direct ligand degradation kinetics are well known, and subtle differences in ligand binding can shape downstream signaling, it is still unclear which biochemical reaction steps within the canonical pathway are responsible for distinct ligand-specific transcriptional responses. Here, we developed a mechanistic ordinary differential equation model of the canonical AhR pathway. We calibrated the model to time-resolved qPCR measurements of Cyp1a1 and Ahrr mRNA in mouse bone-marrow-derived macrophages exposed to structurally diverse, environmentally relevant ligands with known immunomodulatory activity (3-methylcholanthrene, indolo[3,2-b]carbazole, and bisphenol A) using global optimization under a heteroskedastic likelihood. To dissect ligand specificity, we evaluated 528 candidate models that allow one or two ligand-involving reaction rate constants to vary. Akaike-based model selection reveals a dominant dynamical regime governed by promoter occupancy and target-gene mRNA synthesis, indicating that ligand-specific transcriptional responses are primarily encoded at the level of transcriptional regulation rather than upstream signaling events. The resulting model is made available in SBML and PEtab formats for reproducibility, and to enable further research into whether ligand-specific effects are conserved or rewired across cell types.

The foodborne mycotoxins alternariol (AOH) and alternariol monomethyl ether (AME) have been associated with several adverse effects, including cytotoxicity, genotoxicity, endocrine disruption, and immunomodulation. As these endpoints are typically observed in vitro at micromolar concentrations, the question arises whether such levels are attainable in exposed humans. To address this data gap in chemical risk assessment, a physiologically based kinetic (PBK) model was developed to predict internal exposure doses to AOH and AME in humans. As input parameters, kinetic constants for hepatic glucuronidation were obtained in vitro by incubating Sprague Dawley rat and human liver S9 fractions with 0.5-50 M AOH and 0.5-20 M AME, demonstrating rapid biotransformation in both species. Intestinal absorption of AME and physicochemical parameters were estimated using quantitative structure-activity relationship (QSAR) models. Sensitivity analysis identified parameters describing hepatic glucuronidation and gastrointestinal uptake as among the most influential, confirming the importance of their reliable estimation. The PBK model was evaluated against available rodent toxicokinetic data and subsequently extrapolated to humans. Ultimately, the currently available exposure estimates published by EFSA in 2016 were applied to predict target tissue concentrations, which were compared to points of departure (PoDs) for relevant toxicological endpoints. Even in the most susceptible group of male toddlers, predicted internal concentrations (10-4 M range) were approximately four orders of magnitude below the respective PoDs. Consequently, under the applied exposure assumptions and considering the compounds as isolated chemicals, AOH and AME are not expected to reach systemic or tissue concentrations associated with the investigated effects.

BackgroundWildland firefighters experience repeated occupational exposure to wildfire smoke at high particulate matter (PM) concentrations, leading to elevated cardiovascular disease risk and hypertension prevalence. However, the pathophysiological processes linking cumulative smoke inhalation to vascular damage and blood pressure elevation remain poorly characterized. To evaluate these effects under controlled exposure conditions, we used a preclinical exposure model calibrated to match the cumulative PM burden deposited in wildland firefighter airways over 7-14 years of service. Male apolipoprotein E knockout (Apoe-/-) mice underwent whole-body inhalation of Douglas fir smoke or filtered air for 2 hours/day, 5 days/week, for 8 or 16 weeks at target PM concentrations of 40 mg/m3. ResultsProlonged smoke exposure induced sustained elevation of circulating tumor necrosis factor-alpha (TNF-), interleukin-1 beta (IL-1{beta}), and interleukin-6 (IL-6), coupled with diffused nuclear factor kappa B (NF-{kappa}B) activation throughout the aortic wall. Smoke inhalation disrupted endothelial adherens junctions, upregulated intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and promoted monocyte recruitment to aortic tissues, concurrent with enhanced monocyte chemoattractant protein-1 (MCP-1) expression. Oxidative stress was evidenced by increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit 2 (NOX2) expression, elevated superoxide levels, and endothelial nitric oxide synthase (eNOS) uncoupling in the aorta, leading to lipid peroxidation and accompanied by intimal apoptosis. These inflammatory and oxidative perturbations occurred alongside a pro-fibrotic phenotypic shift characterized by transforming growth factor beta 1 (TGF-{beta}1) upregulation, myofibroblast differentiation, and progressive collagen accumulation in medial and adventitial compartments of the aortic wall. Functionally, smoke exposure progressively impaired aortic cyclic distensibility through combined wall thickening and circumferential tissue stiffening, while severely attenuating endothelium-dependent and nitric oxide (NO)-mediated vasodilation. These functional and structural shifts culminated in elevated systolic and diastolic blood pressures. While endothelial dysfunction reached maximal impairment by 8 weeks, aortic stiffening continued to worsen through 16 weeks of exposure, demonstrating differential temporal progression of vascular damage. ConclusionsThese findings demonstrate that occupationally relevant wildfire smoke exposure produces convergent inflammatory, oxidative, and profibrotic vascular remodeling with progressive loss of arterial compliance and impaired endothelium-dependent vasodilation, underscoring potential vascular targets for cardiovascular health surveillance and risk mitigation in wildland firefighters.

Bioengineers strive to recreate in vivo microenvironments in vitro to reduce our use of animal models and provide insights into human biology. While liver models show promise, sex differences in liver biology remain largely neglected in preclinical studies. Despite the 2014 EU mandate for the inclusion of women in clinical trials, decoupling of research data by sex is historically rare, with only 11% of papers disaggregating data by sex. This gap contributes to women being more susceptible to drug-induced liver injury (DILI) and being underserved in drug development, as well as to costly drug attrition levels. Here we present a novel approach to modelling sex differences in vitro. Human induced pluripotent stem cells (iPSCs) from both male (XY) and female (XX) donors, were differentiated into hepatocyte liver spheroids and exposed to in vivo-mimicking levels of testosterone, progesterone, and oestrogen in high-throughput microwell format. We successfully recapitulated sex-specific metabolic profiles and demonstrated significant differences in CYP1A2 and CYP3A4 drug metabolism and gene expression patterns consistent with reported in vivo observations, without compromising cell viability. These findings validate the utility of sex-differentiated microenvironments in early-stage research, offering a pathway to refine animal and clinical trials and improve therapeutic outcomes for all sexes.

AO_SCPLOWBSTRACTC_SCPLOWAlcohol-associated liver disease (ALD) has been steadily increasing in the United States for many years, as attested by increases in ALD deaths and liver transplant demand. Direct measurement of ALD incidence is challenging as diagnosis often occurs late (or not at all). This study employs a demographically-aware backcalculation method, based on mortality data, to reconstruct latent, age-structured ALD risk and incidence trends in the US population from 2008 to 2022 and uses this information to forecast future ALD trends through 2030. We find that ALD incidence has risen steadily since 2008, with a sharp increase during the 2020 COVID-19 pandemic, and that the average age at onset has also increased over time, with demographic factors playing a substantial role. While our forecasts suggest a continuation of the pre-2020 growth in ALD incidence for most age and sex groups, we also predict marked increases among younger men, a generational shift toward older age cohorts, and substantial rises among older females. Most concerning, between 2022 and 2030, incidence is expected to double among younger men and older females and by 2030 the number of new male ALD cases is projected to be more than twice that of females for all age groups. Our results provide a clearer understanding of evolving ALD trends, highlighting the role of demographic and birth cohort effects. We underscore the urgent need for targeted interventions, particularly among younger men, to reduce ALD-related behaviors and future burden.

O_LIIn this study, we demonstrate that Benzo[a]pyrene (B[a]P) induces keratinocyte senescence and p21Cip1-dependent keratinocyte differentiation. Atmospheric and environmental pollution are known to induce senescence and promote terminal differentiation in human primary keratinocytes, thus driving skin aging. However, much is still unknown about the underlying molecular mechanisms. We observed that B[a]P, a common atmospheric pollutant, induced senescence in primary keratinocytes in both two-dimensional and three-dimensional (reconstructed human epidermis) culture. This was accompanied by signs of DNA damage in B[a]P-treated cells. B[a]P-treated cells also underwent accelerated late-stage terminal differentiation, indicated by increased IVL and FLG expression from 48 to 96 hours post-exposure. While pharmacological and genetic attenuation of p21Cip1 did not rescue cellular senescence, it prevented the expression of IVL and FLG, suggesting that the late-stage terminal differentiation induced by B[a]P exposure was p21-dependent. Our data thus suggest a key role for the p21Cip1 in the keratinocyte response to pollution-induced damage, where p21Cip1 induces terminal differentiation to maintain skin barrier homeostasis. C_LI

Manufacturers are adopting propellants for use in pressurized metered-dose inhalers (pMDIs) that have lower global warming potentials (GWPs) than the propellants traditionally used in pMDIs. Hydrofluoroalkane (HFA)-134a has been used as the propellant in the pMDI used to deliver the fixed-dose triple combination of budesonide, glycopyrrolate and formoterol fumarate (BGF); following successful clinical evaluation, the BGF pMDI is now being transitioned to the next generation propellant hydrofluoroolefin (HFO)-1234ze(E), which has near-zero GWP. We describe formulation development efforts that led to selection of HFO-1234ze(E) over another propellant, HFA-152a, for reformulation. Propellant-specific studies evaluated active pharmaceutical ingredient (API) stability and aerodynamic particle size distribution (aPSD). Those analyses have been complemented by in silico regional lung deposition modeling conducted after the clinical evaluation of the reformulated BGF pMDI. HFO-1234ze(E) supported favorable stability and aPSD characteristics for BGF pMDI reformulation, compared with HFA-152a, and modeling predicted regional deposition consistent with therapeutic intent. Given that each pMDI is a unique combination of APIs, device, propellant, and excipients, propellant substitution requires product-specific evidence and regulatory approval, and typically takes several years. Targeted analyses, such as those described here, helped to identify the most suitable candidate propellant for successful substitution in the BGF pMDI. HighlightsO_LIFormulation development efforts that led to evaluation of a budesonide-glycopyrrolate-formoterol fumarate pressurized metered-dose inhaler (BGF pMDI) reformulated with the next generation propellant HFO-1234ze(E) in a clinical trial program are described; the suitability of another propellant, HFA-152a, was also assessed C_LIO_LIOver 6 months under accelerated storage conditions (40{degrees}C/75% relative humidity [RH]), the HFA-152a formulation approached and, in one replicate, fell below the 90% of formulation label claim threshold of evaluation, whereas the original HFA-134a product and the HFO-1234ze(E) formulation remained above that threshold C_LIO_LIOver 6 months under accelerated storage conditions (40{degrees}C/75% RH) and 18 months under long-term stability storage conditions (25{degrees}C/60% RH), the fine particle mass and fine particle fraction for all active pharmaceutical ingredients (APIs) showed that the HFO-1234ze(E) formulation tracked more closely than the HFA-152a formulation to the original HFA-134a product C_LIO_LILater in silico modeling, conducted after clinical testing, predicted a trend for greater deposition of APIs in early airway generations with HFA-152a, whereas HFO-1234ze(E) was predicted to more closely match HFA-134a, indicating a greater likelihood of achieving equivalence to the original HFA-134a product with HFO-1234ze(E) than with HFA-152a C_LIO_LIBased on these analyses and other formulation development efforts, HFO-1234ze(E) was identified as the most suitable propellant for reformulation of the BGF pMDI; for HFA-152a, analyses raised concerns about storage stability, and differences in aerosol characteristics that can impact API deposition in the lungs and, in turn, efficacy C_LI

Widespread exposure to multiple pesticides might potentially represent a genotoxic risk to humans. However the effects of these mixtures are largely unknown. Genotoxicity is a key characteristic of carcinogens, and its assessment represents an important component of the overall safety assessment of pesticides. In the present study, in vitro micronucleus test on intestinal Caco-2 human cells was performed according to OECD TG 487 in order to ascertain the genotoxicity of ten commonly used pesticides (dose range 0-100 mg L-1), tested as individual pesticides or mixtures. Significant dose-related increases in micronuclei were observed for exposures to lambda-cyhalothrin, tebuconazole, glyphosate, deltamethrin, fluopyram and the synergist piperonyl butoxide. Significant increases of micronuclei were also observed at different doses for cypermethrin, acetamiprid and cyprodinil, however these increases were not dose-dependent. Imazalil genotoxicity could not be analyzed due to confounding of high cytotoxicity even at low doses. Results show that the co-formulant piperonyl butoxide was genotoxic to human cell lines at all tested doses. Moreover, glyphosate, acetamiprid and fluopyram showed genotoxic effects at concentrations of 0.01-1.0 mg L-1. Although previously reported to be not genotoxic cyprodinil and deltamethrin were observed to be genotoxic to Caco-2 cells. A combination of 3 prioritzided pesticides (acetamiprid, glyphosate, tebuconazole) showed genotoxic effects even at the lowest dose. A combination of 8 prioritized pesticides showed genotoxicity at the highest dose. No synergistic interactions in micronuclei formation were evident in either the mixture of 3 or 8 prioritized pesticides. This study provides important information on the genotoxicity of different widely used pesticides and confirms the validity of a component-based approach in genotoxicity assessment of pesticide mixtures. This study was performed as part of the EU SPRINT (Sustainable Plant Protection Transition: A Global Health Approach) project.

Railway catenary sparking generates airborne ultrafine particles (UFPs) that may pose health risks due to their metallic composition and ability to penetrate deep into the alveolar region of the lungs. Copper, widely used in wires and pantographs, is a major component of these emissions, making copper-rich particles common in railway environments such as subways. However, exposure levels and health impacts remain poorly characterized, and localized hotspots may represent an underrecognized risk in densely populated areas. This study investigated the toxicity of copper UFPs under realistic dosimetry and deposition conditions. Copper UFPs were generated using a spark discharge generator and applied to two in vitro lung models: a 3D co-culture of Calu-3 epithelial cells, THP-1-derived macrophages, and EA.hy926 endothelial cells, and a monoculture of A549 alveolar epithelial cells. Cells were exposed at the air-liquid interface (ALI) using an automated platform to mimic inhalation exposure and UFPs deposition. Copper deposition ranged from 6.5 to 41 ng/cm2, within occupationally relevant levels. A549 cells showed cytotoxic responses consistent with previous studies, whereas the 3D co-culture model revealed broader adverse effects, including inflammation, impaired epithelial barrier integrity, oxidative stress, and early DNA damage. Inflammatory activation also differed between models: A549 cells mainly exhibited transcriptional responses, while the 3D model showed significant secretion of IL-6 and IL-8, associated with interferon signaling. These findings highlight the potential health risks of copper UFPs from railway systems and emphasize the need for improved characterization of UFP exposure in environmental and occupational railway settings.

INTRODUCTION: Alzheimers disease (AD) is a multifactorial disorder, yet current research largely focuses on downstream biomarkers with limited attention to environmental contributors. Experimental studies suggest that per and polyfluoroalkyl substances (PFAS) may contribute to neuroimmune and neurodegenerative pathways relevant to AD. OBJECTIVE: To examine associations between PFAS exposure and neuroimmune and AD related plasma biomarkers in cognitively unimpaired rural adults. METHODS: In a cross sectional pilot study (n=48), serum concentrations of 33 PFAS were measured, including four legacy compounds (PFOS, PFHxS, PFOA, PFNA). Plasma neuroimmune related (ITGB2, SMOC1, TREM2, GFAP) and AD related biomarkers (Ab42/40, ptau217) were detected using proteomic analysis. RESULTS: PFOS showed moderate associations with ITGB2, SMOC1, and Ab42/40 in unadjusted analyses, which attenuated after adjustment for age. PFOA and PFNA demonstrated consistent inverse associations with TREM2 before and after adjustment. DISCUSSION: Findings suggest possible compound specific PFAS associations with immune and amyloid related biomarkers, supporting further investigation in longitudinal and PFAS mixture based studies.

Background: Particulate matter (PM) exposure is associated with increased risk and exacerbation of chronic rhinosinusitis (CRS), yet underlying mechanisms remain poorly understood. Methods: Human nasal epithelial cells obtained from ethmoid tissue of CRS (n = 5) and control donors (n = 4) were cultured at an air-liquid interface and exposed to PM. Single-cell RNA sequencing was performed to characterize PM-induced cellular and transcriptional changes. Protein expression, epithelial barrier integrity, cell death, and intracellular PM uptake were evaluated using biochemical, imaging, and ultrastructural approaches. Results: Unsupervised clustering identified seven epithelial cell populations. Gene set analysis revealed baseline enrichment of inflammatory and keratinization pathways and reduced ciliogenesis in CRS compared with controls. Although PM induced inflammation and squamous differentiation in controls, the pathogenic responses were significantly amplified in CRS, including uniquely enhanced IL-1 signaling. Transcriptional changes were validated by ELISA, transepithelial electrical resistance, and immunofluorescence, demonstrating increased inflammation, epithelial barrier disruption, and cell death following PM exposure. Transmission electron microscopy revealed increased intracellular PM within membrane-bound organelles. Pre-treatment with an endocytosis inhibitor rescued PM-induced epithelial barrier dysfunction and inflammation. Conclusion: CRS epithelium exhibits baseline dysfunction that may predispose it to environmental injury. PM exposure both induces CRS-like epithelial changes in controls and exacerbates disease-associated phenotypes.

Until now, there has been no animal-free alternative method for predicting chronic inflammation and delivering the associated dose responses, the timing of onset, and the duration of inflammation, as required by regulatory agencies. We present the results of pre-validation of an in-vitro-learned digital twin (InFiniteLungDT) capable of predicting chronic neutrophilic lung inflammation for regulatory use. The method is based on measuring the dynamics of early biological effects in vitro induced by respirable materials or their mixtures, without the need to know their intrinsic properties. We constructed the digital twin(s) for each of the material, for which we have in vivo exposure data. The instillation data set, comprising 49 different nanomaterials, was used as the primary anchor to calibrate the model. Inhalation data set, comprising 7 different nanomaterials, compliant with OECD TG 412, was used to show the general applicability of the method across species and for different exposure scenaria. In total, about 3094 single mouse exposures and 364 rat exposures (and approx. 775/225 non-exposed mouse/rat controls) were used to predict concentration-dependent time-evolved neutrophil influx into the lung. The accuracy (predictive capacity) of LOAEL determination is 93% for instillation and 84% for inhalation exposure. Taking into account the time-to-deliver-result being less than 1 week, this proves that the effect of inhaled material from acute to chronic conditions can be assessed orders of magnitude faster and cheaper than in a reference animal study.

Mosquito-borne diseases such as dengue, chikungunya, and malaria remain major public health challenges in South Asia, particularly in Bangladesh, where mosquito repellents are widely used as primary preventive tools. This study presents a comparative evaluation of commonly used repellent products, including mosquito coils, liquid vaporizers and aerosols, DEET-based creams, and natural formulations, focusing on their efficacy, protection duration, and potential health risks. Efficacy was assessed using controlled laboratory methods, including chamber-based exposure and arm-in-cage tests against Aedes aegypti (Barnard & Xue, n.d.). Safety was evaluated through in vivo toxicological analysis in a rat model, incorporating clinical observations, hematological and biochemical profiling, and histopathological examination. The results indicated an overall mean effectiveness of 85%, with DEET-based creams demonstrating the highest efficacy (95%), while natural repellents showed the lowest (70%). Mosquito coils provided the longest protection duration ([~]10 hours) but were associated with the highest health risk due to combustion-related emissions. Vaporizers and aerosols offered moderate efficacy with moderate health risk. Toxicological findings revealed that coil exposure induced significant respiratory stress, elevated liver enzymes (ALT, AST), increased leukocyte count, and notable lung tissue damage. Vaporizer and aerosol exposure resulted in mild physiological changes, whereas DEET-based and natural repellents showed minimal systemic toxicity. Overall, the study highlights a clear trade-off between efficacy and safety across repellent types. These findings emphasize the need for informed product selection, stronger regulatory control, and the development of safer, cost-effective, and sustainable mosquito repellent technologies for effective vector control in endemic regions.