Chemosphere

Pollution of aquatic environments poses an increasingly severe threat to ecosystems worldwide, and understanding its molecular consequences for aquatic organisms requires extensive research and the development of advanced analytical tools. Phosphoproteomics can be particularly valuable for this purpose, as shifts in phosphorylation states can serve as early molecular indicators of toxic exposure. The cladoceran Daphnia is a keystone species in aquatic ecosystems, linking lower and higher trophic levels, and is therefore widely used as a model organism in ecotoxicology to study biological consequences of pollution. Here, we present a simple and effective strategy to analyse the phosphoproteome of Daphnia magna, a commonly used Daphnia species in ecotoxicology. Following TiO2-based phosphopeptide enrichment and LC-MS/MS analysis, we identified a comprehensive dataset of 3,532 phosphorylation sites across 1,329 phosphoproteins. These proteins were especially involved in signaling pathways and cellular structure and the vast majority have not yet been demonstrated in other Daphnia species. In conclusion, our results demonstrate that a straightforward phosphoproteomic LC-MS/MS workflow in D. magna can serve as a powerful tool for investigating adverse molecular effects caused by anthropogenic pollution, such as microplastics or pharmaceuticals. Statement of significanceThe dataset presented here demonstrates the feasibility of a simple yet effective strategy to perform phosphoprotemics in Daphnia magna, and it will be particularly valuable for future ecotoxicoproteomics research using this model organism.

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.

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.

Alternaria mycotoxins represent an emerging concern due to their frequent occurrence in food and feed. However, available toxicological data remain limited. Under the current EU regulatory framework, and in line with the EFSA/ECHA/JRC guidance for the identification of endocrine disruptors (EDs), assessment of endocrine activity relies on standardized assays performed according to OECD Test Guidelines (TGs) for the estrogen-, androgen- and steroidogenesis- (EAS) modalities. Within the framework of the European Partnership for the Assessment of Risks from Chemicals (PARC), standardized in vitro methods of regulatory relevance were performed for six chemically characterized Alternaria toxins, aiming to address current regulatory gaps on EAS-mediated activity. Alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA), altertoxin-I (ATX-I), tentoxin (TEN) and altenuene (ALT) were assessed over a broad concentration range, from 0.001 up to 60 M, depending on cytotoxicity and solubility profile of each compound. Our findings indicate estrogenic activity for AOH (PC50: 3.9 - 4.6 {micro}M) and AME (PC50: 5.2 - 8.5 {micro}M) in the estrogen receptor transactivation assay (OECD TG 455), as well as an anti-estrogenic activity for ATX-I (IC30: 0.27 - 0.37 {micro}M). Minimal positive responses were observed at high concentrations for AOH (from the dose of 3 {micro}M) and for AME (from the dose of 10 {micro}M) in the agonistic part of the androgen receptor transactivation assay (OECD TG 458), which may also reflect glucocorticoid receptor activation. No effects on estradiol or testosterone production were observed for any of the tested Alternaria compounds in the steroidogenesis assay (OECD TG 456).

O_LIHeavy metals are pervasive environmental contaminants that can impair the health of organisms globally. As the largest anthropogenic source of the potent neurotoxin mercury (Hg), gold mining has amplified these threats throughout the tropics. Consequently, there is a mounting need to monitor Hg contamination of the richest biological communities on Earth. Venous whole blood provides a reliable, nonlethal measurement of recent dietary and site-derived contamination, but collecting and cold-storing samples can be impractical in field conditions. C_LIO_LITo overcome these challenges, we developed and evaluated a method to assay Hg exposure in vascular organisms by measuring the volume of dried blood spots (DBS) in the field, which can be stored at ambient temperatures until analysis. We explored the methods precision and accuracy in estimating whole blood Hg concentrations by collecting paired whole blood and DBS aliquots from birds (n = 527 individuals, 140 species) along a trophic gradient (i.e., granivores to piscivores) in Belize and Peru. C_LIO_LIUsing a Bayesian linear mixed-effects model, we found a highly precise and unbiased relationship between DBS and whole blood total Hg concentrations that was centered at perfect unity (R2 = 0.99; {beta} = 1.00 {+/-} 0.03; 95% CrI: 0.95-1.05). Agreement between individual paired aliquots was more variable, in which approximately 12% of DBS containing at least 1 ng THg differed from whole blood by more than {+/-}20%. However, DBS accuracy increased at higher THg concentrations, suggesting that disagreement at low concentrations is an expected consequence of higher measurement error near the analytical limit of detection of our instruments. C_LIO_LICompared to whole-blood collection and analysis workflows, DBS offer substantial logistical advantages by eliminating cold-chain dependence and reducing transport burden, laboratory handling time, and overall operational costs. Consequently, volume-measured DBS provide a practical and highly reliable alternative for monitoring Hg contamination in both humans and wildlife, particularly for ecological and population-level applications in remote and resource-limited environments. C_LI

Alternaria toxins are naturally occurring food contaminants with limited and often inconsistent genotoxicity and mutagenicity data. Within the European Partnership for the Assessment of Risks from Chemicals (PARC), an OECD-aligned in vitro testing strategy was applied to fill existing data gaps and to characterize the genotoxic potential of major Alternaria toxins using high-purity test materials. Mutagenicity was assessed using bacterial reverse mutation test (OECD TG 471) and SOS/umu assay, while chromosomal damage was assessed using the in vitro micronucleus (MN) assay (OECD TG 487) in TK6 and HepG2 cells, complemented by fluorescence in situ hybridization (FISH) and {gamma}H2AX assay in HepaRG cells. Alternariol (AOH), alternariol monomethyl ether (AME), and altertoxin-I (ATX-I) showed clear mutagenicity in bacteria, whereas altenuene (ALT), tenuazonic acid (TeA), and tentoxin (TEN) were negative under the tested conditions. In mammalian cells, AOH, AME, and ATX-I induced MN formation in TK6 cells at concentrations [≥]5.5 {micro}M, [≥]2.5 {micro}M, and [≥]0.21 {micro}M, respectively, with FISH analysis supporting a clastogenic mode of action. In HepG2 cells, all tested toxins induced chromosomal damage, with effect threshold ranging from [≥]6.25 {micro}M (AOH) to [≥]50 {micro}M (TeA). {gamma}H2AX induction confirmed DNA damage for AOH and ATX-I, and at higher concentrations for TeA (1000 {micro}M). Overall, the data indicate clear in vitro genotoxic potential for AOH, AME, and ATX-I and provide evidence of chromosomal damage for ALT, TEN, and TeA, thereby reducing critical data gaps for hazard assessment.

Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants with poorly understood sublethal effects on insects. Perfluorooctanoic acid (PFOA), one of the most widely distributed legacy PFAS is increasingly recognized for altering organismal physiology beyond traditional toxicity endpoints. Here, we use the fruit fly Drosophila melanogaster as a model to examine how PFOA exposure during larval (juvenile) development reshapes insect life-history progression and metabolic homeostasis. Our studies reveal that at environmentally relevant concentrations (nM to low {micro}M), PFOA induces precocious expression of developmentally-regulated genes and leads to metabolic changes that persist into adulthood. At higher concentrations used to probe mechanism, PFOA accelerates larval development, disrupts mitochondrial membrane potential, and increases whole-organism metabolic heat production - results that suggest altered mitochondrial energetic efficiency. Consistent with this tradeoff, PFOA-exposed larvae that develop faster under permissive conditions exhibit heightened sensitivity to environmental stressors, including elevated temperature and reduced food hydration. Together, these findings demonstrate that PFOA disrupts metabolic and developmental processes in a dose- and context-dependent manner, highlighting sublethal effects that may influence insect resilience under environmental stress. SYNOPSIS STATEMENTHere we describe how PFOA alters the growth, development, and metabolism of the fruit fly Drosophila melanogaster. Specifically, we find that PFOA accelerates Drosophila juvenile growth while also rendering exposed larvae sensitive to environmental stress. These observations suggest that widespread PFOA contamination may impair the developmental fitness of insect populations.

Mancozeb, a widely used fungicide composed of manganese ethylene-bis-dithiocarbamate with zinc salts, has raised concerns due to its potential neurotoxic effects. In this study, we investigated how chronic oral administration of mancozeb affects astrocyte function and neurobehavior in mice, focusing on store-operated Ca{superscript 2} entry (SOCE), mediated by Orai1 and STIM1. Mancozeb treatment at 0.5 {micro}g/kg/day for 4 weeks reduced glial fibrillary acidic protein (GFAP) expression in the hippocampus and corpus callosum of mice, indicating astrocyte atrophy. Further, administration at the human acceptable daily intake (30 {micro}g/kg/day) for 1 week induced hippocampal astrocyte atrophy and hyperlocomotor activity in open field tests. In vitro experiments revealed that mancozeb specifically inhibited SOCE in astrocytes by targeting the Orai1/STIM1 complex, as its inhibitory effect was abolished by short hairpin RNA (shRNA)-mediated knockdown of Orai1 or STIM1, but not by knockdown of TRPA1 or scramble shRNA. This demonstrates that mancozeb-mediated SOCE inhibition critically depends on the presence of Orai1 and STIM1, highlighting the molecular specificity of its action. Furthermore, mancozeb diminished endoplasmic reticulum (ER) Ca{superscript 2} stores and P2Y1 receptor agonist-induced Ca{superscript 2} transients. Electrophysiological analyses revealed that mancozeb selectively decreased the inhibitory postsynaptic current frequency without affecting excitatory currents, suggesting reduced astrocyte-mediated GABA release. Collectively, these findings demonstrate that mancozeb disrupts astrocytic Ca{superscript 2} homeostasis through Orai1/STIM1-dependent SOCE inhibition, leading to astrocyte atrophy and altered inhibitory neurotransmission, which may underlie the observed behavioral changes. These results highlight the potential neurotoxic risk posed by mancozeb via the impairment of astrocyte function and intracellular Ca{superscript 2} regulation. Importantly, these neurotoxic effects occurred at concentrations below current regulatory safety limits (ADI), indicating that mancozeb-induced disruption of astrocytic Ca{superscript 2} signaling provides a mechanistic basis for re-evaluating established human safety exposure standards. Environmental ImplicationsOur findings highlight that the widespread use of mancozeb has a significant impact on brain health. Mancozeb was shown to induce astrocyte atrophy even at low concentrations, amounting to six times the human acceptable daily intake. Mancozeb causes impairment of GABAergic synaptic transmission of neurons by disrupting the Ca{superscript 2} homeostasis via inhibition of Orai1 and STIM1 of astrocytes. These findings indicate that current regulatory standards significantly underestimate the risks of long-term mancozeb exposure to brain health. Therefore, this study underscores the risks of astrocyte-mediated neurotoxicity resulting from pesticide residue ingestion and emphasizes the need to rigorously re-evaluate current exposure limits from the perspective of brain health.

Plastic production has been increasing exponentially. Throughout their lifespan, plastics degrade into smaller particles that accumulate in our bodies and the environment. Recent studies found these plastic particles can cross the placental barrier and reach the fetus. However, the impact of plastic particles on placental function is still unknown. We hypothesized that nanoplastics would disrupt placental growth and function, specifically focusing on transforming growth factor beta (TGF{beta}) signaling. To understand the impact of plastic particles on the placenta, we orally exposed pregnant CD-1 mice to 50 nm or 200 nm polystyrene plastic particles from gestation day 8 to day 15 at a human-relevant concentration of 5 mg/kg/day. After euthanization on day 15, placenta and fetus weights were recorded, and tissues were prepared for histomorphology and gene expression analysis. We observed a statistically significant decrease in the area of the decidua in the placentas for the 200 nm treatment group and a borderline significant decrease in decidua area for the 50 nm treatment group compared to control. However, when we separated by sex, only the male decidua were significantly decreased in the 200 nm group. Gene expression analysis of key signaling factors in the TGF{beta} pathway identified increased expression of Smad2 and Smad3, which may be suppressing prolactin and estrogen receptor signaling. Overall, both particle sizes disrupted placenta structure and signaling in a sex-dependent manner and may be acting as endocrine disruptors.

Depleted uranium (DU) is an environmental contaminant with a 30 g/L (ppb; parts per billion) EPA maximum contaminant level (MCL) for drinking water. The mining of uranium and use of DU in modern weapons underly human exposure that disproportionally impacts military and tribal communities in the United States. Uranium's radiotoxic characteristics are understood, but its chemical hazards much less so. In zebrafish (Danio rerio) and human cell cultures we test the hypothesis that exposure to DU negatively impacts cellular function and development through disruption of mitochondrial metabolism. Using a novel shrapnel model with TEM/SEM+EDS, we showed uranium microparticles caused proximity-dependent mitochondrial disruption. In waterborne exposure paradigms, larval movement was reduced and hatching delayed as a result of reduced movement and not enzyme deficiencies in response to 18 ppb DU, below the MCL. Increased DNA damage accumulation was detected in exposed larva and cells. DNA-damage quantitative PCR of DU-exposed larvae showed increased damage in the ahr1 locus (nuclear gene) and decreased mitochondrial DNA (mtDNA) copy number, but mtDNA damage levels varied across experiments. Mitochondrial function was assessed using a resazurin-based assay in the presence and absence of antioxidants and showed diminished cytoplasmic reductive capacity. DU exposure alone did not enrich antioxidant gene expression, contrasting with arsenic exposure, a known ROS-inducer and Nrf2-activator. Sulforaphane (SFN), a potent Nrf2-activator, did not blunt the effects of DU exposure, despite activation of antioxidant response element (ARE) genes (gstp and gss), but did blunt the effects of arsenic exposure. The most enriched transcript in DU-exposed larvae coded for slingshot protein phosphatase (ssh), further exploration revealed ssh1b as the zebrafish-specific ortholog activated in response to DU, and inhibition using an identified SSH1 inhibitor, Sennoside A, partially rescued the metabolic and hatching defects observed. Our data points to a cytotoxic mechanism in which DU disrupts mitochondrial function through ssh1b enrichment that impairs normal mitophagy, leading to decreased cellular reductive potential independent of either ROS production or ARE-activation. Our results suggest that health impacts from DU exposure may be directly linked to impaired mitochondrial functions.

Cytochrome P450 (CYP)-mediated biotransformation of endogenous and xenobiotic substances can lead to altered exposure, toxicological impact, or adverse drug reactions. CYP induction data are fundamental to regulatory chemical toxicity hazard assessment because they directly affect the in vivo fate of xenobiotics, potentially influencing their safety and efficacy of pharmaceuticals, and impacting the safety assessment of industrial chemicals, and environmental contaminants. Here we report on the third laboratory supplementary validation of an established and previously validated human HepaRGTM in vitro method able to detect CYP1A2, CYP2B6, and CYP3A4 induction, to support the expansion of the chemical applicability domain beyond pharmaceuticals. This study was conducted to support the part 1 study with additional robust data. We established the test method in-house using the 10 previously validated pharmaceutical proficiency chemicals, then tested a further 6 proposed augmentation chemicals, tebuconazole, benfuracarb, chlorpyrifos, N, N-Diethyl-meta-toluamide, fipronil, permethrin, as tested in part 1, and then four additional chemicals: prochloraz, atrazine, pyrimethanil, and chlorpyrifos-methyl. LC-MS/MS was utilised to measure the conversion of a cocktail mixture of prototypical selective CYP probe substrates to their metabolites, in parallel with mRNA measurements. We achieved high concordance with expected classifications for proficiency and additional chemicals. Comparisons with mRNA-based measurements suggested gene expression may serve as a cost-effective pre-screening tool for CYP1A2 and CYP3A4, though with greater uncertainty for CYP2B6. The data support the robustness of the HepaRG method for CYP induction testing and the adoption of the test method in 2026 as an Organisation for Economic Cooperation and Development Test Guideline. Plain language summaryCytochrome P450 (CYP) enzymes metabolize drugs, pesticides, and other chemicals. Chemicals that increase or decrease CYP enzyme activity can change internal exposure levels, potentially leading to unexpected toxicity or impact drug effectiveness. Reliable in vitro methods to assess CYP induction are needed for regulatory chemical safety assessment. This study describes results from a third laboratory applying a previously validated human HepaRG cell-based method to assess induction of CYP1A2, CYP2B6, and CYP3A4. After successful in-house implementation using ten reference pharmaceutical compounds, the method was extended to ten more industrial chemicals. CYP induction was evaluated by measuring enzyme activity and changes in gene expression. The test method showed a high level of agreement with expected induction outcomes. Gene expression data supported enzyme activity results, particularly for CYP1A2 and CYP3A4. These results strengthen confidence in the robustness and wider applicability of the method for Organisation for Economic Cooperation and Development Test Guideline adoption.

High concentrations of steroidal hormone compounds are a growing source of concern for environmental pollution in aquatic ecosystems. In this study, we examine the effects of two estrogenic compounds (estriol and 17-ethinylestradiol) on fitness traits in the aquatic microcrustacean, Daphnia magna, a key bioindicator species for toxicology studies. The impacts were compared of two forms representing a natural and synthetic estrogenic compound. Growth and reproduction traits were assayed by exposing Daphnia to each estrogen type at four concentrations reflecting potential environmental exposure conditions up to acute toxicity levels (ranging from 0.1 - 50 {micro}g/L). Assaying the effects at a variety of concentrations is important given that it is known that hormone exposures can often result in non-monotonic responses. Both forms of estrogen impact a subset of the traits assessed, in some cases leading to beneficial changes and others causing harm. Estriol, the naturally-occurring estrogen, and EE2, the synthetic version, at high doses shift fitness traits in opposite directions such as adult growth rate as do at low doses for fecundity. In conclusion, our results support the need to assay a wide array of traits using multiple forms of steroidal hormones at a range of doses in order to assess non-monotonic patterns and their impact on an organismal fitness. In particular, assays that extend beyond the conventional measurements of lethality during acute exposure windows will be essential for understanding the impact of increased levels of hormone pollution on aquatic organisms and ecosystem health.

Background The placenta serves a critical role in nutrient uptake and waste elimination for the developing fetus. The placenta is also responsible for the uptake and/or exchange of xenobiotics, including medications, between the maternal and fetal bloodstreams. An estimated 40-80% of women take medications or drugs during pregnancy for a variety of conditions. Very little is understood about fetal drug and nutrient exposure during pregnancy and how it may change over the course of fetal development. Objective This study aimed to characterize the abundance of transport proteins in placental tissue, which are important in modulating fetal nutrient and drug exposure, over the duration of pregnancy. Mass spectrometry-based global proteomic analysis revealed trends in the expression of thousands of proteins throughout gestation. Focusing on the membrane-associated proteome enabled an increased emphasis on the solute carrier and ATP-binding cassette families of transporter proteins that are critical for nutrient and xenobiotic transport across the maternal-fetal barrier. Study Design Using data-independent acquisition proteomics, relative abundance of proteins in placental tissue samples was profiled across all three trimesters of pregnancy (Trimester 1 = 16, Trimester 2 = 9, and Term = 9). Membrane fractions were generated to enrich membrane-associated proteins for proteomic analysis. Placental samples were grouped into randomized batches for membrane fraction generation and mass spectrometry analysis. Proteomic search results from each batch were imported into the R programming environment from Skyline, concatenated, and normalized as one data set for downstream analysis. Results A total of 6,331 proteins were detected across all samples with 4,210 proteins identified in every sample. Pathway analysis revealed that as gestational age increases, membrane-associated proteins involved in more complex metabolic pathways increase in relative abundance while those involved in extracellular remodeling events and simple organic ion transport tended to decrease. A total of 139 solute carrier and ATP-binding cassette transport proteins were identified in all samples, and 80 were identified in every sample. In general, membrane-associated proteins, including solute carrier and ATP-binding cassette transport proteins, were significantly enriched in placental tissue collected during early gestation compared to term placental tissue. Conclusion This study presents a comprehensive profiling of membrane-associated proteomic changes during gestation and identifies significant gestational age associated abundance changes at the protein level in several transport protein families. The application of data-independent acquisition global proteomic techniques enabled in-depth analysis of thousands of proteomic changes across pregnancy in a single experiment. These data provide critical information to support future studies into the understanding of fetal exposure to xenobiotics and nutrients circulating in the maternal bloodstream.

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.

Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants associated with developmental abnormalities and adverse health outcomes, yet it remains unclear whether PFAS exposure imposes novel transcriptional programs during development or perturbs endogenous developmental processes. Here, we continuously exposed African killifish (Nothobranchius furzeri) to an environmentally relevant concentration of perfluorooctanoic acid (PFOA) from egg laying through juvenile development to mimic prenatal-to-adolescent exposure and performed whole-transcriptome sequencing at two developmental stages. Despite four weeks of embryonic exposure, newly hatched juveniles, approximately equivalent to human infants, exhibited remarkably limited transcriptional responses, with only a few differentially expressed genes identified. In contrast, older juveniles, equivalent to human adolescents, exposed for eight weeks displayed a dramatic expansion of transcriptional perturbation, with approximately 30-fold more differentially expressed genes spanning pathways involved in cell-cycle regulation, endocrine signaling, immune function, oxidative stress, and lipid metabolism. Unexpectedly, more than half of the PFOA-induced genes were the same genes that normally increase during juvenile maturation, representing a highly significant enrichment of the endogenous developmental program. These findings indicate that the embryonic transcriptome is largely buffered against chronic PFOA exposure, whereas post-hatch stages exhibit heightened vulnerability. Rather than inducing a distinct toxicological state, PFOA predominantly acted by amplifying existing developmental gene regulatory programs. Our results identify the juvenile stage following hatching, equivalent to human neonatal and adolescent developmental stages, as a critical window of PFAS susceptibility and suggest that environmental contaminants may exert their effects by exaggerating normal developmental trajectories, with potential consequences for growth, maturation, and long-term health.

BackgroundThe cardiovascular system is significantly affected by exogenous factors, but understanding the risks posed by pharmaceuticals and environmental chemicals is restricted due to limited data availability. New approach methodologies (NAMs) apply in vitro, in chemico, and in silico methods to characterize hazard and risk, thus offering rapid, multiscale human biology-based strategies to overcome regulatory challenges and the potential to complement or replace animal testing for understanding chemical cardiovascular effects. MethodsIn the present study, we applied a systems-based workflow using physiologically based pharmacokinetic (PBPK) models to convert bioactive concentrations from >300 high-throughput screening (HTS) assays with cardiovascular-relevant molecular and cellular targets to human equivalent administered doses (EADs) for >800 substances with widespread human exposure potential. To derive human-relevant risk predictions, the in vitro activity-derived EADs were compared with human exposure estimates and in vivo points of departure (PODs) from toxicological animal studies. For a subset of chemicals, we applied a geospatial analysis to assess the combined risks for populations across regions of the US. ResultsThe combined HTS assay data, human exposure predictions, animal study-based PODs, geospatial exposure data, and PBPK modeling identified compounds with potential cardiovascular toxicity at relevant exposure levels. Personal care product ingredients, flame retardants, herbicides, pesticides, pharmaceuticals, and byproducts of various industrial processes were noted as agents of concern preferentially targeting endothelial cell signaling, nuclear hormone receptors, and other critical cardiovascular targets. Of the 859 chemicals assessed, in vitro CV-relevant assays were more risk protective than animal studies for 96.4% of the chemicals. A set of 17 chemicals had a log10 bioactivity exposure ratio (BER) below -2, indicating estimated human exposure more than 100-fold above the in vitro-derived bioactive dose. ConclusionsThis study establishes an integrative, multiscale framework linking molecular perturbations to population-level cardiovascular risk, enabling systematic identification of potentially cardiotoxic chemicals and the communities most vulnerable to their effects. By bridging mechanistic toxicology with pharmacokinetic modeling and epidemiologic context, this approach enhances the biological relevance and translational impact of human health risk assessment. This scalable, adaptable framework supports timely, evidence-based decision-making and aligns with the growing adoption of NAMs to advance cardiovascular research and disease prevention. Novelty and SignificanceO_ST_ABSWhat is known?C_ST_ABSHigh-throughput screening (HTS) assays can identify chemicals with activity at cardiovascular (CV) relevant molecular targets, but translating in vitro bioactivity concentrations into biologically meaningful human equivalent doses requires physiologically based pharmacokinetic (PBPK) modelling. The bioactivity exposure ratio (BER) provides a data-driven metric for comparing in vitro-derived equivalent administered doses against population exposure estimates, but its application to CV endpoints across a large and chemically diverse environmental chemical landscape has not been demonstrated. Geospatial mapping of CV chemical exposure risk has been demonstrated for a limited set of air pollutants but has not been extended to a broad environmental chemical landscape using human-relevant in vitro bioactivity data. What new information does this article contribute?Integrated in vitro to in vivo extrapolation (IVIVE) across 859 environmental chemicals demonstrates that cardiovascular-relevant in vitro endpoints are sensitive indicators of broader systemic toxicity, 96.4% of chemicals showed positive POD ratios, meaning in vitro CV assays flagged hazard at lower doses than non-specific animal toxicity studies despite the absence of endpoint matching. Seventeen chemicals including PFAS, brominated flame retardants, endocrine disruptors, and agricultural herbicides, had a BER below -2, indicating estimated human exposure more than 100-fold above the in vitro-derived cardiovascular bioactive dose, with convergent evidence from both in vitro and in vivo data supporting regulatory priority. County-level geospatial mapping reveals that cardiovascular chemical exposure risk is geographically heterogeneous across the United States, concentrated in industrially active regions already associated with elevated cardiovascular disease mortality, identifying specific populations for targeted environmental monitoring. SummaryThis study presents a scalable, systems-based IVIVE framework that integrates cardiovascular-relevant in vitro HTS bioactivity data with reverse dosimetry, population exposure predictions, and in vivo animal toxicity data to prioritize environmental chemicals for cardiovascular risk assessment. Applied to 859 chemicals spanning personal care products, flame retardants, pesticides, pharmaceuticals, and industrial compounds, the framework demonstrates that CV-relevant in vitro endpoints are sensitive indicators of systemic toxicity even in the absence of direct endpoint matching with in vivo studies. The BER emerges as a flexible and resource-adaptable prioritization metric, identifying 92 chemicals where estimated human exposure falls within the CV bioactive range, of which 17 represent the highest regulatory priority based on convergent evidence from both data streams. Geospatial mapping further reveals regional heterogeneity in cardiovascular chemical exposure risk concentrated in industrial areas of the central and southeastern United States. This work advances the application of new approach methodologies for cardiovascular chemical risk assessment at a time of accelerating regulatory transition toward human-relevant in vitro-based safety evaluation, providing a reproducible computational workflow directly applicable to chemical prioritization under evolving EPA and FDA regulatory frameworks.

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.

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.

Pesticides are ubiquitous in agroecosystems and pose substantial risks to non-target organisms. Traditional ecotoxicological assessments focus on survival, reproduction, or overt behavior, yet these endpoints may fail to detect subtle, molecular-level stress. Here, we investigated the effects of sublethal deltamethrin exposure on the head proteome of field-collected European earwig (Forficula auricularia) females, sampled at two life stages (pre-oviposition and post-family life) to account for physiological context. Our results reveal that deltamethrin induces a robust proteomic response shared across developmental stages, including the regulation of key detoxification enzymes (NADPH- cytochrome P450 reductase, arginine kinase). In parallel, stage-specific responses were observed, involving proteins related to metabolism, stress response, and cellular organization. Strikingly, these molecular perturbations occurred without detectable changes in reproductive traits, highlighting a disconnect between cellular stress and organismal phenotypes. Several uncharacterized proteins were consistently regulated, representing promising targets for future studies on pesticide adaptation and potential detoxification pathways. Overall, these findings suggest that classical phenotypic assays may underestimate sublethal pesticide effects, and that proteomic profiling provides a sensitive framework to uncover underlying molecular responses. By integrating natural variability, realistic exposure, and reproductive physiology, our study emphasizes the need for molecular approaches in environmental risk assessment and offers a new perspective on the subtle, cryptic effects of agrochemicals.

Nanoplastics (NPs) are emerging environmental contaminants able to cross biological barriers, disrupt cellular and organelle homeostasis, and alter the brain microenvironment. This study investigated whether NPs affect microglia to neuron communication, a key mechanism underlying neuronal resilience, via the nuclear factor erythroid 2 like 2 (NFE2L2) pathway. Using an in vitro model, we evaluated the effects of polystyrene nanoplastics on microglial metabolic fitness and microglia-mediated neuronal stress responses. Increasing NP concentrations induced a dose dependent biphasic effect. Low to intermediate concentrations increased intracellular adenosine triphosphate (ATP) levels in microglia and enhanced microglia-mediated activation of neuronal NFE2L2. In contrast, high NP concentration impaired microglial metabolism, reduced ATP availability, and decreased microglia to neuron communication. These findings indicate that NPs alter microglial energetic status and modulate neuroprotective signalling, potentially contributing to impaired neuron to microglia interactions and increased susceptibility to neurotoxicity.