Toxicology and Applied Pharmacology

Perfluorooctane sulfonic acid (PFOS), a per- and polyfluoroalkyl substance (PFAS), is a widespread persistent environmental pollutant that has been implicated in various human health conditions, including infertility and cancer. Here, we investigate the effects of acute exposure to PFOS on human fallopian tube epithelial (FNE) cells that are essential for fertility and increasingly recognized as the origin site for high-grade serous ovarian cancer. We show that acute PFOS exposure changes morphology, arrests proliferation, impairs adhesion, and compromises epithelial integrity of FNE cells. Using transcriptomic profiling of FNE cells exposed to PFOS, we found increased expression of genes associated with stress-response signal transduction, including KRAS, and decreased expression of genes related to cholesterol transport and lipid homeostasis. We show that inhibition of MEK/ERK or cholesterol supplementation rescued changes in cell morphology. Further, we performed membrane fluidity measurements of cells exposed to PFOS and found elevated membrane disorder and fluidity. Our results are consistent with a model in which PFOS perturbs plasma membrane, activates stress-response signaling pathways, and impairs epithelial cell function. These studies establish a framework for understanding the effects of PFAS on cell physiology.

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.

SignificanceQuantifying lipid droplet (LD) remodeling in 3D hepatic organoids is often limited to endpoint staining or phototoxic live fluorescence imaging, thereby obscuring droplet-level kinetics. AimWe aimed to develop a label-free method to track LD dynamics in living hepatic organoids under different fatty-acid loads. ApproachTime-lapse 3D refractive-index tomograms were acquired using holotomography and analyzed with a depth-adaptive, multi-threshold segmentation pipeline to quantify LD number, volume, sphericity, and refractive-index-derived concentration and dry mass at single-droplet resolution. ResultsOleic acid and linoleic acid induced LD accumulation while preserving organoid integrity, whereas palmitic acid triggered rapid structural collapse. Despite increases in total LD burden under both oleic acid and linoleic acid, droplet-level dynamics diverged: oleic acid produced volume-dominated accumulation via enlargement of fewer LDs and increased size heterogeneity, whereas linoleic acid produced number-dominated accumulation via sustained increases in LD number, yielding a more uniform population of small droplets. ConclusionsLabel-free holotomography with depth-adaptive analysis enables non-invasive, longitudinal, and multi-scale quantification of LD dynamics in intact organoids and reveals fatty-acid- dependent temporal modes of lipid storage. Statement of DiscoveryWe developed a label-free, longitudinal 3D holotomography framework with depth-adaptive lipid droplet segmentation that quantifies single-droplet dynamics in living mouse hepatic organoids. Using this platform, we found that oleic acid and linoleic acid induce LD accumulation via distinct strategies--oleic acid via droplet enlargement and linoleic acid via sustained increases in droplet number--while palmitic acid rapidly compromises organoid integrity.

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.

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.

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

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.

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

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.

Pregnant women are particularly susceptible to adverse outcomes from environmental heat, yet the physiological effects of acute heat exposure during pregnancy remain poorly understood. Some physiological changes are monitored in humans; however, investigation of underlying molecular mechanisms requires invasive methods that can only be ethically applied in mammalian models. Moreover, research with animal models has largely focused on early and lethal teratogenic effects of heat exposure and lacks longitudinal physiological monitoring, detailed parameterisation of heating regimes and in-depth investigation of underlying mechanisms. Here we used a mouse model to investigate the impact of a controlled acute heat exposure at mid-gestation (E12{middle dot}5), slowly elevating core body temperature (CBT) over 210mins to raise CBT by [~]1{degrees}C. Using high-frequency ultrasound and morphological analyses, we observed delayed alterations in placental and foetal cerebral blood flow indicative of a brain-sparing response, alongside reduced placental labyrinth zone size. Additionally, maternal cardiac function was impaired, accompanied by cardiac and renal fibrosis and elevated circulating soluble Flt-1 levels, an anti-angiogenic biomarker of gestational hypertension. These findings demonstrate that brief heat stress at mid-gestation can induce lasting effects on placental function and maternal cardiovascular health in a mammalian model, highlighting potential risks for pregnancy outcomes under increasing global temperatures. Together this data suggests that an acute exposure to heat elevating core body temperature by 1{middle dot}2{degrees}C can induce a long-term impact on both placenta and maternal health in a mouse model. It will be important to understand the molecular changes which underpin the pathophysiology and whether this is translated to humans.

Ethylene oxide (EtO) is a highly reactive industrial chemical and classified as a known human carcinogen with a putative mutagenic mode of action (MOA). Its genotoxic potential is primarily mediated through alkylation of DNA, resulting in the formation of the mutagenic adduct O6-(2-hydroxyethyl)-2-deoxyguanosine (O6-HE-dG). The N7-(2-hydroxyethyl)guanine (N7-HE-G) adduct is formed in greater abundance and is generally considered to be non-mutagenic. However, dose-response relationships of these DNA adducts, particularly at low inhalation exposure levels (i. e., below 3 ppm), remain unknown. These data are necessary to inform the biological plausibility of different statistical dose-response models that have been applied to human or animal data used for cancer risk assessment. In the present study, male and female B6C3F1 mice were exposed to EtO (0, 0.05, 0.1, 0.5, 1, 50, 100, and 200 ppm) 6 hours/day for 28 consecutive days. Immediately following the last exposure, DNA was extracted from lung, liver, bone marrow, and mammary gland, and further utilized to measure DNA adduct levels using highly sensitive mass spectrometry platforms. N7-HE-G was detected in all tissues and exposure groups, showing linear dose-response relationships in the low-dose range ([&le;]1 ppm) and increased sharply and exposure-disproportionately in the high-dose range ([&ge;]50 ppm). Despite a very low limit of detection, O6-HE-dG, in contrast, was not detected at exposures <50 ppm in any tissue consistent with at most a shallow linear exposure response. At higher exposures ([&ge;]50 ppm), O6-HE-dG exhibited a dose-response pattern of N7-HE-G. Notably the mammary gland, despite being anatomically distant from the site of inhalation, exhibited the second-highest levels of both adducts at higher doses. This study provides the first reliable quantitative dose-response evidence of DNA adducts in tumor target and non-target (liver) tissues across a wide range of EtO exposures. The two DNA adducts differ markedly in their abundance, repairability and mutagenic potential and together provide a molecular MOA dose-response framework to inform both quantitative cancer risk assessment and genotoxic hazard characterization.

ObjectiveChronic obstructive pulmonary disease (COPD) is a leading cause of death in the industrialized world. Although smoking, air pollution, and occupational exposures are well established risk factors, the molecular pathways linking environmental exposures and biological susceptibility to COPD remain incompletely understood. Untargeted metabolomics offers a unique opportunity to simultaneously capture internalized environmental chemicals and endogenous metabolic perturbations. However, large prospective studies integrating broad exposomic and metabolic screening prior to COPD onset are lacking. MethodsWe conducted a nested case-control study within three European population-based cohorts (Doetinchem Cohort Study, KORA, SAPALDIA) and analyzed 1473 prospectively collected plasma samples. COPD was defined by a pre-bronchodilation FEV1/FVC ratio below 0.7 at follow-up (4-16 years after blood sample collection). We applied complementary untargeted liquid- and gas chromatography high-resolution mass spectrometry (LC- and GC-HRMS), enabling extensive coverage of endogenous metabolism and exogenous environmental contaminants, including pesticides, plastic-related chemicals, and polycyclic aromatic hydrocarbons. Controls maintained normal lung function and were matched to cases on age, sex, follow-up time, and sample collection round. We performed separate conditional logistic regression models for each metabolomic feature, and used Mummichog for prediction of biological pathways involved. The false discovery rate (FDR) was controlled using the Benjamini-Hochberg procedure. Long-term measurement reliability was evaluated using intraclass correlation coefficients (ICCs) from repeat samples in the Doetinchem Cohort Study. ResultsIn total, thousands of metabolomic features were screened, including 724 annotated exogenous compounds, 13 endogenous metabolites, and 197 features that could be derived as both. Nicotine and cotinine intensity levels were statistically significantly associated with COPD incidence at an FDR of 10%, validating the analytical and epidemiologic framework. Lower levels of butyrylcarnitine were related to COPD onset in never-smokers. Beyond smoking-related markers, lower levels of butyrylcarnitine were associated with increased COPD risk among never-smokers, implicating altered mitochondrial fatty-acid metabolism as a potential early pathway independent of tobacco exposure. Although most screened environmental contaminants, including PAHs and pesticides, were not associated with COPD at stringent significance thresholds, restricting analyses to temporally stable metabolites identified the insecticide metabolite phenyl N-methylcarbamate as a predictor. ConclusionThis large-scale, prospective untargeted metabolomics study represents one of the most comprehensive assessments to date of both environmental and endogenous metabolic predictors of COPD. Our findings demonstrate the feasibility of exposome-wide molecular screening years before disease onset, identify butyrylcarnitine as a novel metabolic predictor in never-smokers, and highlight the importance of accounting for temporal variability in metabolomic epidemiology.

AbstractInflammation and oxidative stress are key drivers in the pathogenesis of chronic lung diseases, including asthma, pulmonary fibrosis, and chronic obstructive pulmonary disease. Extracellular vesicles derived from the marine microalga Tetraselmis chuii, referred to as nanoalgosomes, have recently gained attention as natural nanocarriers that possess inherent antioxidant and anti-inflammatory properties. In this study, we investigated the biocompatibility and protective effects of aerosolized nanoalgosomes in a bronchial epithelial-macrophage co-culture model at the air-liquid interface. Co-cultures of CALU-3 epithelial cells and differentiated THP-1 macrophages were primed with aerosolised nanoalgosomes and subsequently exposed to either oxidative stress (tert-butyl hydroperoxide) or an inflammatory stimulus (lipopolysaccharide; LPS). Epithelial barrier integrity and cytotoxicity were evaluated using transepithelial electrical resistance and lactate dehydrogenase release assays, respectively, while intracellular reactive oxygen species levels and cytokine secretion were measured to assess antioxidant and immunomodulatory responses. Nanoalgosomes were non-cytotoxic, preserved epithelial barrier integrity, and significantly reduced oxidative stress. In addition, nanoalgosomes priming attenuated LPS-induced secretion of pro-inflammatory cytokines (IL-1{beta}, IL-6, IL-8, IL-18, TNF-) as well as the anti-inflammatory cytokine IL-10, suggesting a balanced immunomodulatory response. Overall, aerosolized nanoalgosomes maintained epithelial homeostasis and mitigated both oxidative and inflammatory stress, underscoring their potential as a safe, sustainable, and effective therapeutic strategy for chronic inflammatory lung diseases. Given their natural origin, excellent biocompatibility, and suitability for aerosol delivery, nanoalgosomes represent a promising class of inhalable biotherapeutics.

Environmental degradation and accumulation of plastics results in micro- and nanoplastics (MNPLs) that are small enough to cross biological barriers, including the blood-brain barrier. Microglia, resident immune cells of brain, are critical regulators of neuroimmune homeostasis and represent a cellular target of nanoplastic exposure. In this study, we assessed the neurotoxic effects of two sizes of polystyrene nanoplastics (PS-NPs; 100 nm and 500 nm) using integrated in vivo and in vitro exposure and washout paradigms. In vivo exposure in mice (60 days; 0.15 or 1.5 mg/day) showed the accumulation of both PS-NP sizes in the cerebral cortex without histopathological damage. However, cortical microglia showed pronounced morphological remodeling, observed as increased expression of Iba1 and GFAP. Transcriptomic profiling of cortical tissue revealed a strong size-dependent response. The 100 nm PS-NP group revealed 18 DEGs (|log2FC| [&ge;] 2, padj < 0.05), whereas the 500 nm PS-NPs showed more than 4,000 DEGs, including upregulation of immune- and microglia-associated genes (CCL5, CXCL10, LCN2, LYZ2) and downregulation of synaptic and neuronal signaling genes (GRIN2B, SYN1, STX1B, MAP1B, ITPR1/2). In vitro assessment, using BV2 microglia cells, showed internalization of PS-NPs via the endolysosomal pathway, with strong co-localization to Rab7- and LAMP2-positive compartments and prolonged intracellular retention following exposure washout. Also, microglial activation markers (Iba1, CD68) exhibited a transient, size- and concentration-dependent increase, correlated with intracellular particle burden rather than cumulative exposure. Overall, these findings demonstrate that PS-NPs accumulate in brain, driving size-dependent microglia activation and transcriptomic reprogramming, even after cessation of exposure to PS-NPs. HighlightsO_LIPS-NPs (100 nm and 500 nm) reach mouse cerebral cortex following 60-day oral exposure. C_LIO_LIPS-NPs were internalized by microglia; accumulated in endolysosomal compartments. C_LIO_LIPS-NP exposure induced transient microglial activation without sustained cytotoxicity. C_LIO_LIMicroglial activation was correlated with intracellular PS-NPs burden. C_LIO_LITranscriptomics revealed disruption of neuroimmune and microglial regulatory pathways. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/712727v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1aba3eaorg.highwire.dtl.DTLVardef@1967641org.highwire.dtl.DTLVardef@12da637org.highwire.dtl.DTLVardef@1fb8441_HPS_FORMAT_FIGEXP M_FIG C_FIG

ObjectiveTo investigate associations between long-term environmental exposures, both external (ambient air pollution and built environment) and internal (circulating anthropogenic chemicals), and the human plasma metabolome, with the aim of generating biologically plausible hypotheses about affected metabolic pathways. MethodsWe analyzed plasma from 989 Estonian Biobank participants using untargeted LC-HRMS (Metabolon HD4). External exposures (PM2.5, PM10, NO2, ozone and built-environment metrics) were assigned using spatiotemporally resolved models developed in the EXPANSE project. Internal exposures were defined as ubiquitous anthropogenic compounds detected in the same metabolomics dataset. Associations between exposures and individual metabolites were quantified using left-censored regression models and then mapped to metabolite classes (Metabolon) and KEGG pathways. For enrichment analyses, one-sided Kolmogorov-Smirnov tests were applied to external exposures and Fishers exact tests to internal exposures. False discovery rate was controlled at 1% per exposure and database. ResultsExternal air pollutants exhibited distinct metabolic patterns: Higher NO2 exposure was associated with enrichment of metabolites involved in tyrosine metabolism; higher ozone with monohydroxy and dicarboxylate fatty acids (consistent with lipid peroxidation); and higher PM2.5 with acyl-carnitine subclasses and carbohydrate metabolism (glycolysis / gluconeogenesis / pyruvate). Built-environment associations were heterogeneous across metabolites and pathways. Internal anthropogenic chemicals showed broader metabolic associations than external exposures, involving a larger number of metabolites and metabolic classes. PFAS (PFOA, PFOS) were associated with long-chain polyunsaturated fatty acids (n3/n6) and lysophospho-lipids. Associations with 4-hydroxychlorothalonil, a fungicide, pointed to androgenic steroid metabolites and alpha-linolenic acid metabolism. The phenolic 2,4-di-tert-butylphenol, a plastic associated chemical, showed widespread associations with lipid classes, suggesting disruption of membrane remodeling and fatty acid handling. ConclusionLong-term environmental exposures, both external and internal, are measurably reflected in the human plasma metabolome. Across exposure domains, recurrent signals involved lipid metabolism, membrane composition, and oxidative stress-related pathways, highlighting these as common biological targets of environmental exposures. The findings generate testable hypotheses, including nitrosative stress-related alterations for NO2, lipid peroxidation for ozone, energy-metabolism perturbations for PM2.5, potential endocrine activity for chlorothalonil metabolites, and possible obesogenic effects of 2,4-di-tert-butylphenol.

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

Silver (Ag+) ions are known to be toxic to bacteria, cells, organisms and living systems; yet its impacts on the locomotion of surface-crawling organisms remain poorly quantified. Here we investigated the short-term (0-6 hours) effects of Ag+ ions on the locomotion of Drosophila melanogaster larvae on flat agarose surfaces containing Ag+ ions at different concentrations (0, 1, 10, and 100 mM). By quantifying their locomotion, we found that Drosophila larvae showed shorter accumulated distances and reduced crawling speed. Additionally, we quantified the go/stop dynamics and peristalsis of the larvae and observed that Ag+ ions disrupted the normal, rhythmic, peristaltic contraction of the larvae and "trapped" them in the stop phase. Such toxic effects were dependent on Ag+ concentration and exposure duration.

Microplastic (MP) pollution, which is present in the ecosystem in vast quantities, adversely affects human health and the environment, making it imperative to develop methods for its mitigation. The challenge of detecting or capturing MPs could potentially be addressed using plastic-binding peptides (PBPs). The ideal PBP for MP remediation would not only bind strongly to plastic, but also have other properties such as high solubility in water or great binding specificity to a certain plastic. However, the scarcity or absence of known PBPs for common plastics along with the lack of methods that can discover PBPs with all of the desired properties precludes the development of peptide-based MP remediation strategies. In this study, we discovered short linear PBPs with high predicted water solubility and binding specificity by employing an in-silico discovery pipeline that combines deep learning and biophysical modeling. First, a long short-term memory (LSTM) network was trained on biophysical modeling data to predict peptide affinity to plastic. High affinity peptides were generated by pairing the trained LSTM with a Monte Carlo tree search (MCTS) algorithm. Molecular dynamics (MD) simulations showed that the PBPs discovered for polyethylene, the most common plastic, had 15% lower binding free energy than PBPs obtained using biophysical modeling alone. PBPs with both high affinity and high predicted solubility in water were found by including the CamSol solubility score in the MCTS peptide scoring function, increasing the average solubility score from 0.2 to 0.9, while only minimally decreasing affinity for polyethylene. The framework also discovered peptides with high binding specificity between polystyrene and polyethylene, two major constituents of MP pollution, using a competitive MCTS approach that optimized the difference in affinity between the two plastics. MD simulations showed that competitive MCTS increased the binding specificity of PBPs for polystyrene and identified peptides with relatively great preference for either of the two plastics. The framework can readily be applied to design PBPs for other types of plastic. Overall, the high-affinity PBPs with desirable properties discovered by marrying artificial intelligence and biophysics can be valuable for remediating MP pollution and protecting the health of humans and the environment.

Background: Cardiovascular diseases (CVD) are more common in lower occupational classes, but the mediating role of health behaviours remains unclear. This study aimed to quantify the extent to which health behaviours mediate the association between occupational class and CVD, evaluate their relative contributions to CVD risk, and assess occupational class differences in the effects of health behaviours. Methods: Municipal employees from Helsinki, aged 40-60 at baseline, were followed from 2000-2002 (response rate 67%) to 2022. CVD events were identified from national registers, including hospitalizations, long-term sickness absence, disability pensions, and mortality. Counterfactual mediation analysis using additive survival regression was used to assess the contribution of health behaviours - excessive alcohol consumption, smoking, unhealthy diet, and insufficient physical activity - to the association of occupational class and CVD. Occupational class differences in the effects of health behaviours were assessed with Cox regression. Results: During follow-up, 50% of participants in the low occupational class and 46% in the high occupational class had a CVD event. All unhealthy behaviours except heavy alcohol use were more common in the low occupational class. Health behaviours explained approximately 40% of the excess risk of CVD when moving from high occupational class to low occupational class. Insufficient physical activity (HR 1.44, 95% CI 1.35-1.54) was the strongest predictor of CVD. Unhealthy diet was more strongly associated with CVD in the high occupational class. Conclusion: Health behaviours explained a part of occupational class inequalities in CVD, but most of the inequality remained unexplained, highlighting broader social determinants.

Establishing whether real-world environmental chemical (EC) exposure can induce heritable epigenetic modifications in large, outbred mammals is key to determining long-term developmental impacts of the human exposome. Using an established biosolids-treated pasture (BS) sheep model, we investigated whether gestational exposure to low-level mixtures of EC induced heritable changes in DNA methylation across three generations of sheep. Reduced-representation bisulfite sequencing of liver, blood, and sperm, combined with a structured, lineage-controlled breeding design, revealed widespread but lineage- and sex-specific differentially methylated loci (DML) in F1 offspring, with detectable alterations evident in F2 and F3 descendants. Although most DML were unique to individual sire lineages, or to a single generation, subsets of loci showed repeated involvement across generations and were associated with altered gene expression in F3 descendants. Sperm from F1 males exhibited reduced methylation at numerous loci and, together with seminal plasma, revealed differential expression of several microRNAs. These effects, however, showed limited persistence in F2 males, indicative of intergenerational rather than fully transgenerational persistence. Collectively, these findings demonstrate that complex, low-level chemical exposures can elicit recurrent, sexually dimorphic epigenetic responses in outbred species, but underscore the challenge of disentangling exposure-induced inheritance from genetically regulated methylation variation. Significance StatementEnvironmental chemical (EC) exposures are ubiquitous, yet their capacity to induce heritable epigenetic changes in large, genetically diverse mammals is poorly understood. Using a real-world exposome-based sheep model, we demonstrate that low-level gestational EC exposure leads to sexually-dimorphic and lineage-dependent alterations in DNA methylation that can extend to unexposed descendants. Although genetic ancestry exerts a dominant influence over these responses, repeated alterations at specific loci suggests that environmentally induced epimutations can reoccur across generations in certain genomic contexts.