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.

While distinct environmental exposures imprint unique mutational signatures on cancer genomes, the specific causal patterns for many known carcinogens remain uncharacterized in relevant human tissues. To address this gap, we developed a novel, physiologically relevant system that uses a combination of airway epithelial cells and whole genome sequencing to characterize mutational patterns induced by genotoxic carcinogens associated with lung cancer. After validating the platforms accuracy by successfully recapturing the known signature for Benzo(a)pyrene (BaP), we used this system to gain detailed insights into the types of mutations that occur with exposure to N-nitrosotris-(2-chloroethyl) urea (NTCU) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), genotoxic compounds that induce lung squamous cell carcinoma and lung adenocarcinoma in mouse models, respectively. Cells exposed to NTCU had significantly more somatic SNVs compared to control samples. An average of 82.3% of mutations in NTCU samples were attributed to a novel mutational signature distinct from those in the COSMIC database but highly correlated with recent in vivo mouse models. In contrast, NNK exposure did not demonstrate a distinct mutational pattern above background at both high and low concentrations. Ultimately, this in vitro system provides a robust platform to define causal links between environmental exposures and mutational patterns in lung cancer mutagenesis. Statement of SignificanceIn vitro exposure of N-nitrosotris-(2-chloroethyl) urea to airway epithelial cells revealed a distinct mutational signature.

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.

Of the cytochrome P450 enzymes, CYP3A4 is the most abundant isoform in the human liver, and this enzyme plays a dominant role in the metabolism of a wide range of clinical drugs and xenobiotics. Previous studies have demonstrated that CYP3A4 participates in the oxidative metabolism of several organophosphorus (OP) pesticides involving both thion (P=S) and oxon (P=O) forms. In the present study, we evaluated the capacity of CYP3A4 to metabolize a structurally diverse set of OP compounds using LC-MS/MS methods and assessed their potential to inhibit CYP3A4 activity using previously developed pFlour50 fluorogenic assay. Our results demonstrate that CYP3A4 preferentially metabolizes thions, as compared to oxons, and several OP compounds were also found to inhibit CYP3A4 activity in a time-dependent manner. To gain further mechanistic structural insight into the CYP3A4-OP interactions, molecular docking studies were performed using a crystal structure of CYP3A4 (PDB ID: 3NXU). Linear correlation analysis between in silico parameters like molecular weight or binding energy correlated with experimental data including inhibition data for 10 or 30 minutes or the LC-MS/MS data showing the degradation at 1 or 2 hours showed moderate but significant correlation. Soman surrogate PiMP, and cyclosarin surrogate CMP, were both effectively metabolized by CYP3A4, while docking of these surrogates and authentic agents with CYP3A4 receptor revealed very similar binding poses and interactions. Collectively, these findings highlight the important role of CYP3A4 in OP metabolism and support the potential of integrating experimental and in silico data to predict CYP3A4-mediated metabolism of existing and emerging OP compounds, including those of toxicological and chemical warfare relevance.

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.

As toxicology shifts towards non-animal testing, quantitative models are essential to predict adverse health effects from molecular or cellular perturbations. Quantitative Adverse Outcome Pathways (qAOPs) represent such models, building on mechanistic knowledge and quantifying the Key Event Relationships (KERs) described in AOPs. Despite the recognized need, the number of qAOPs remains limited. Bayesian-based approaches are often chosen for developing qAOP for their flexibility, but most use discretized variables, limiting their predictive power. In addition, these models are mainly built from newly generated data, underexploiting the large amount of information available. This study successfully leverages data from public literature and presents an innovative framework based on continuous variables to develop a Bayesian-based quantitative model for a central KER towards liver fibrosis. The model predicts the probability of the expression fold change for two key markers of hepatic stellate cell activation (aSMA and COL1A1), given the effects on tissue injury, using in vitro data from 9 chemicals. We propose a newly developed workflow to assist in knowledge identification, organization, and extraction from scientific literature and chemical databases. Based on in vitro data and in vivo information from the Open TG-GATEs (Toxicogenomics Project-Genomics Assisted Toxicity Evaluation System) database, we estimate a biologically relevant range in COL1A1 fold change that indicates an activated state of stellate cells and high liver fibrosis odds ratios. Our study provides a case example of integrating published data and continuous variables to build a Bayesian-based model, which constitutes an essential step for predicting liver fibrosis from in vitro data.

Background & Aims: Per- and polyfluoroalkyl substances (PFAS) are persistent endocrine-disrupting chemicals associated with metabolic dysfunction, including metabolic dysfunction-associated steatotic liver disease (MASLD). While PFAS perturb lipid and bile acid (BA) metabolism in a sex-specific manner, the underlying mechanisms remain unclear. We tested whether steroid hormones mediate PFAS-associated metabolic alterations. Methods: In 104 patients with biopsy-characterized MASLD, we performed sex-stratified analyses applied liquid chromatography coupled to mass spectrometry (LC-MS) for chemical analysis, integrating circulating steroids, PFAS exposure, hepatic lipidomics and BA profiles. Results: Steroid hormones were associated with MASLD severity in a sexually-dimorphic manner. Dihydrotestosterone showed consistent inverse associations with steatosis, fibrosis, necroinflammation and insulin resistance, particularly in females. PFAS exposure was associated with altered steroid profiles, predominantly indicating suppressed steroidogenesis in females. These PFAS-associated hormonal changes were linked to downstream alterations in hepatic lipids and BAs. Mediation analysis supported indirect effects of PFAS on metabolic pathways via steroids, including testosterone/epi-testosterone-mediated effects on ether phospholipids and estradiol-mediated effects on lithocholic acid. Females exhibited stronger PFAS-steroid-BA associations, whereas males showed weaker, lipid-centric effects. Conclusions: PFAS exposure is associated with sex-specific disruption of steroid hormone pathways that may link environmental exposure to lipid and BA dysregulation in MASLD. These findings identify steroid hormones as potential key mediators of PFAS-associated metabolic dysfunction and highlight sex as a critical determinant in environmental liver disease.

Abstract2O_ST_ABSBackgroundC_ST_ABSSustained exposures to high atmospheric levels of PM2.5 at population scale are associated with increased risks for pulmonary inflammatory diseases. These are marked by activation of the TRPC6 (Transient Receptor Potential Canonical type 6) calcium channel, increased reactive oxygen species (ROS) and oxidative stress. Long term exposures are associated with reduced life span, and increased incidences of cardiovascular diseases, dementia, Parkinsons and Alzheimer disease, and increased risk of autism and autism spectrum disorders. It has been proposed that the PM2.5 toxin is benzo[a]pyrene (B[a]P) that is adsorbed to the surface of the PM2.5 particle.. But the mechanism by which B[a]P might drive pulmonary inflammatory diseases, or any other of the indications above, are not known. HypothesisB[a]P was recently reported to bind irreversibly and destructively to the {beta}2 Adrenergic Receptor ({beta}2AR) in the lung. We have therefore hypothesized that B[a]P is the adsorbed PM2.5 toxin, and that {beta}2AR is the B[a]P receptor responsible for TRPC6 activation in lung epithelial cells. ResultsTo test this hypothesis, we exposed a polarized organoid model of normal human lung epithelia, polarized lung epithelial 16HBE14o-cells, and tracheobronchial slice cultures from ferret lung to either PM2.5 or B[a]P. We found that both PM2.5 and B[a]P: (i) irreversibly activated of {beta}2AR signaling via Gi to PI3K/AKT; (ii) increased NF{kappa}B-activated release of proinflammatory cytokines through IKK{beta} activation by PI3K/AKT, which was suppressed by the PI3K inhibitor LY 294002 (iii) desensitized and destroyed the activated {beta}2AR receptor by endocytic recycling; (iv) also destroyed {beta}2ARs signalplex partner CFTR by the same process; (v) activated the CFTR-bound calcium channel protein TRPC6 due to loss of inhibitory CFTR; leading to (vi) increased cytosolic [Ca2+] concentration; (vii) increased ROS due to mitochondrial uncoupling; and (viii) increased expression of oxidative stress. Treatment with the TRPC6 inhibitor BI 749327 blocked steps (vi-viii), and preserved CFTR from endocytic loss. Treatment of tracheobronchial slice cultures of ferret lung with either PM2.5 or B[a]P resulted in increased secretion of IL-6, increased expression of TRPC6, and reduced expression of {beta}2AR and CFTR. Finally, we found that exposure of lung organoids to B[a]P significantly reduced expression of the same five microRNAs (miR-126a-3p, miR-30b-5p, miR-103a-3p, miR-26a-5p, and miR-766-3p) previously identified in sera from service members exposed to PM2.5 from burn pit emissions during deployment to Iraq and Afghanistan. ConclusionPM2.5 and the PM2.5 toxin benzo[a]pyrene (B[a]P) induce inflammation and oxidative stress in the airway by increased expression of TRPC6 and inactivation of {beta}2AR/CFTR signaling. These discoveries mark the first identification of a mechanism by which exposure to PM2.5 or the PM2.5 toxin B[a]P itself can induce inflammation and TRPC6-dependent oxidative stress in lung epithelia.

The composition of culture medium is a major, yet frequently undercontrolled, determinant of hepatic cell state in vitro. For decades, fetal bovine serum (FBS) has been routinely incorporated into liver cell culture. Its undefined and lot-to-lot variable composition can, however, confound cell identity and experimental reproducibility. Serum-free, chemically defined media (CDM) represent an alternative approach that can improve standardization, but the consequences of transitioning from FBS-supplemented media (FBS-SM) to CDM remain insufficiently characterized in hepatic models, particularly with respect to metabolic and detoxification programs that govern xenobiotic handling and hepatotoxicity readouts. Here, we systematically assessed how replacing FBS-SM with CDM remodels transcriptomic profiles in two widely used human hepatic cell lines (HepaRG and HuH7 cells) and compared the results to that obtained from primary human hepatocytes (PHH). Global transcriptomic analyses indicated that cell type was the primary driver of variance, whereas medium induced a model-dependent secondary effect. Functional interpretation showed preferential enhancement of xenobiotic metabolism and transport-associated programs in HepaRG cells, while HuH7 cells response was dominated by lipid/sterol homeostasis and stress-linked processes. Benchmarking against PHH based on hepatic identity and detoxification gene panels further supported improved PHH alignment for HepaRG cells under CDM compared to cultures with FBS-SM, with limited improvement for HuH7 cells. Collectively, these findings address a key knowledge gap by defining how FBS-SM and CDM impact the transcriptomic profiles of HepaRG and HuH7 cells.

BackgroundDespite extensive cataloging of carcinogenic exposures by the International Agency for Research on Cancer (IARC) and pharmacogenomic variation by resources such as PharmVar and CPIC, few platforms unify exposure, metabolic activation and detoxification, DNA damage, and genetic annotation within a single interactive visualization framework. This gap limits systematic evaluation of gene-environment interactions in cancer risk assessment. MethodsWe developed the Carcino-Genomic Knowledge Graph, ExposoGraph, an interactive knowledge-graph platform for carcinogen metabolism and DNA damage pathways. The reference graph integrates curated data and annotations from IARC, KEGG, PharmVar, CPIC, CTD, and supporting literature/resources. The current reference graph contains 96 nodes across 5 entity types (Carcinogens, Enzymes, Metabolites, DNA Adducts, and Pathways) and 102 edges across 6 relationship types (activates, detoxifies, transports, forms adduct, repairs, and pathway). ResultsThe first-generation reference graph captures metabolic activation and detoxification pathways for 9 carcinogen classes spanning 15 index carcinogens. It represents 36 enzymes across Phase I activation (n=14), Phase II conjugation and detoxification (n=14), Phase III transport (n=3), and DNA repair (n=5). Interactive exploration supports carcinogen-class filtering, node- and edge-type filtering, metadata-based search, and detailed hover/detail views with provenance and pharmacogenomic annotations. The androgen branch highlights cross-pathway connectivity by linking androgen metabolism to estrogen quinone formation and DNA adduct generation through CYP19A1-mediated aromatization and downstream catechol estrogen chemistry. In the optional androgen-focused extension, additional receptor, tissue, and variant context further connects this branch to androgen receptor signaling and genotype-specific annotations. ConclusionsExposoGraph provides a first-generation integrated, interactive framework linking carcinogenic exposures to metabolic fates and genetic modulators. The platform supports hypothesis generation for gene-environment interaction studies and may inform future individualized risk modeling, while remaining a research-use framework rather than a clinically validated risk-assessment tool.

Per- and polyfluoroalkyl substances (PFAS) are chemicals linked to obesity and metabolic dysfunction, but their role in bariatric surgery remains poorly understood. This prospective pilot study examined correlations between plasma PFAS concentrations, body composition, and glycemic measures in adults undergoing bariatric surgery. Thirty-two patients (91% female; 66% Black; mean age 43 years) were enrolled preoperatively; twenty-two completed follow-up at a mean 8.6 months post-surgery. Three PFAS (PFHxS, PFNA, and PFOS) were quantified by plasma liquid chromatography-mass spectrometry; body composition and insulin sensitivity were assessed by dual-energy X-ray absorptiometry and intravenous glucose tolerance testing. At baseline, higher plasma PFNA and PFOS concentrations tracked with lower total lean mass ({rho}s = -0.46 and -0.48, respectively) and lean mass index ({rho}s = -0.46 and -0.42), and PFNA was inversely correlated with body weight ({rho}s = -0.40). No baseline associations were observed with adiposity or glycemic indices. Postoperatively, PFHxS concentrations decreased (median = -1.103 ng/mL, p < 0.001), whereas PFNA and PFOS did not change. Average PFNA was positively correlated with postoperative changes in HOMA-IR ({rho}s = 0.51) and total lean mass ({rho}s = 0.49). No significant associations were observed for average PFHxS or PFOS. These findings suggest that PFNA and PFOS may be linked to reduced lean tissue at baseline, and that PFNA burden modestly tracks with attenuated metabolic and body composition recovery. In an ANCOVA, baseline PFNA was not significantly associated with postoperative HOMA-IR or total lean mass. Larger, longitudinal studies are needed to clarify how PFAS influence these associations.

BackgroundRising global temperatures and eutrophication are increasing the intensity and frequency of cyanobacterial harmful algal blooms that release toxins including microcystin-LR (MC-LR). MC-LR inhibits protein phosphatases in the human liver and brain, but its accumulation in the placenta is unclear. Placental transporter expression varies across pregnancy and is influenced by physiological cues, such as low oxygen concentrations which activate HIF1A, and trophoblast cell fusion forming syncytiotrophoblasts that engage CREB-driven transcription. This study examined whether MC-LR accumulates in placental cells, which transporters mediate uptake, and how these transporters are regulated by HIF1A and CREB. MethodsIntracellular accumulation of MC-LR (0.1-10 {micro}M, 3 hour) was measured in human cytotrophoblasts (JAR, BeWo) and extravillous trophoblasts (HTR-8/SVneo) by western blotting for MC-LR-adducted proteins. Organic anion transporting polypeptide (OATP) involvement was tested using cyclosporin A (10 {micro}M), an OATP inhibitor, before exposure to the OATP substrate or MC-LR. Cells were also cultured under 3%, 8%, or 20% O2 to induce hypoxic responses or treated with forskolin (a potent intracellular cAMP inducer) to stimulate cell fusion before MC-LR exposure. ResultsMC-LR accumulated in all three placenta cell lines in a concentration-dependent manner. Cyclosporin A reduced MC-LR uptake by 57% in JAR cells, confirming OATP-mediated transport. Low O2 increased OATP4A1 expression and function but reduced protein phosphatase expression, decreasing MC-LR-bound proteins by 52-72%. Forskolin increased OATP4A1 expression and enhanced MC-LR uptake >2.5-fold. ConclusionMC-LR enters placental trophoblasts via active OATP transport, likely OATP4A1, and uptake increases under hypoxia and trophoblast fusion.

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.

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.

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

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.

Cannabis (marijuana) is the most widely used recreational drug in the USA accounting for about 62 million users in 2024. Among cannabis users, 26% are of prime reproductive age (18-25 years). Delta-9 tetrahydrocannabinol (THC) is the principal psychoactive component of cannabis and has been detected in human seminal fluids. Although abundant evidence indicates adverse effects of THC exposure on spermatogenesis in different species, acute effects of THC on postejaculatory sperm including fertilization potential and subsequent carryover effects on embryo development are largely unknown. The present study was designed to provide missing information on structural and mechanistic effects of THC exposure to postejaculatory sperm function by evaluating sperm indices often overlooked or masked during clinical evaluation. A bovine embryo continuum model was employed to determine effects of THC on sperm structure, kinematics, bioenergetics, and binding mechanisms. Effects of THC on the sperm genomic and epigenomic landscape were determined, complemented by paternal carry over effects on embryo development as a human translational model to elucidate paternal effects on future development, and to mirror sperm exposure during transport within the female reproductive tract. Cryopreserved bovine sperm from three bulls were independently exposed to physiologically relevant concentrations of THC (0 and 32nM, n = 2 individual replicates/bull) for 24 h under non-capacitating conditions at 25{degrees}C followed by quantification of sperm kinematics at 37{degrees}C. Samples of THC-exposed sperm and vehicle-control (0.1% DMSO) were collected in replicate following immediate addition of THC (0 h) and again at 24 h. DNA damage, acrosome integrity, bioenergetics, changes to DNA methylation and embryo development were quantified. Data were analyzed by logistic regression with a generalized linear mixed effect model. Computer-assisted sperm assessment revealed a reduction in progressive motility of THC-exposed sperm after 24 h while other parameters were not affected. Acrosome integrity as determined by flowcytometric analysis with FITC-PSA was severely compromised in THC-exposed sperm (P [&le;] 0.05), despite no detectable difference in capacitation status using merocyanine staining. Similarly, DNA integrity as determined by TUNEL assay was significantly impaired after 24 h of THC exposure (P [&le;] 0.05). Mechanistic effects of THC were explored through characterization of the transmembrane G-protein coupled cannabinoid 1 receptor (CB1). CB1 is expressed in the post-acrosomal region and its abundance decreased as compared to unexposed sperm. Alterations to the methylation landscape of sperm were then determined after 24 h of THC exposure through whole-genome Enzymatic Methyl Sequencing. PCA analysis indicated that sperm from different males formed distinct clusters, implying individual differences among bulls, while the effects of THC exposure produced tighter clusters. Paternal carryover effects on embryos derived by in vitro fertilization from THC exposed sperm had reduced 2-cell cleavage, 8-16 cell morula development, and reduced blastocyst development compared to unexposed sperm (46% vs. 33%). In conclusion, post-ejaculatory mammalian sperm exposure to THC compromises acrosome integrity, induces DNA damage, changes the sperm methylome, and reduces developmental potential. Collectively, these data implicate new considerations for recreational and clinical use of cannabis that impact cellular and molecular mechanisms important for sperm function with detrimental consequences for gamete interaction and embryo development.

Assessing the risk of pesticides to birds requires models that can extrapolate laboratory data to realistic exposure scenarios. In this work, we propose a new modeling framework BIRDkiss (Bird - Impact on Reproduction via Diet, keep it simple and suitable) that accounts for both a simplified Dynamic Energy Budget (DEBkiss) of organisms and the toxicokinetic-toxicodynamic (TKTD) of chemical substances according to a trait-based approach, thereby reducing the number of parameters to identify and strengthening the statistical robustness of the critical endpoints. The BIRDkiss model describes how food intake and toxicant exposure affect growth and egg production in birds over time. The model is fully embedded within an R package, including routines for calibration, validation and prediction under single-compound scenarios performed via Bayesian inference using standard data from the OECD avian reproduction tests. The BIRDkiss model also allows the simulations of scenarios under both varying food availability and multi-compound exposures based on the two classical mixture-toxicity paradigms: Concentration Addition (CA) and Independent Action (IA). The results of calibration for single compounds show good results matching with observed weights and egg counts. From these calibrations, predictions for new exposure scenarios can be readily generated. For mixtures, the IA algorithm is simpler and does not require to scale variables as in CA. Simulations indicate that high food levels do not further increase egg production (saturation), whereas substantial food reductions markedly decrease reproduction because energy is reallocated to maintenance. Exposure to chemicals combined to low food availability amplify the decline in reproductive output. The ready-to-use mechanistic, open-source BIRDkiss tool enables predicting the impact of pesticides on avian reproduction under realistic dietary exposure profiles. The implementation of CA and IA models is a first step toward mechanistic assessment of chemical mixtures, although validation still requires empirical mixture data. The model highlights the importance of food availability and shows that chemical stress can exacerbate the negative effects of nutritional stress. Integrating such models into regulatory frameworks could improve the ecological relevance of risk assessments. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/712277v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@102246dorg.highwire.dtl.DTLVardef@1a58f65org.highwire.dtl.DTLVardef@695cd7org.highwire.dtl.DTLVardef@14e4329_HPS_FORMAT_FIGEXP M_FIG C_FIG