Chemosphere

Chemical exposures during pregnancy are linked to an increased risk of pregnancy complications that contribute significantly to maternal and infant morbidity and mortality and can lead to long term health consequences for both the mother and the offspring. The placenta, a central regulator of pregnancy health, is a direct target of environmental toxicants. Epidermal growth factor receptor (EGFR), highly expressed in the placenta, regulates proliferation, migration, invasion, fusion, and cellular bioenergetics. To identify compounds of environmental concern with potential for EGFR-disrupting activity, we optimized a high-throughput virtual screening protocol for the identification of EGFR inhibitors and achieved enrichment factors of EF1% = 10.09, EF5% = 3.86, and EF10% = 3.0 in a benchmarking dataset. We applied this protocol to screen the Collaborative Estrogen Receptor Activity Prediction Project database, finding that top-scoring compounds were enriched for aromatic and fused-ring chemical classes, including dyes. Kinase activity assays revealed that two out of thirteen selected compounds, Vat Red 32 and Reactive Red 136, inhibited EGFR kinase activity with micromolar IC50 values. Additionally, pose refinement with molecular dynamics simulations characterized the binding interactions of Reactive Red 136 within the EGFR kinase domain, and functional assays in HTR-8/SVneo placental trophoblast cells showed that Reactive Red 136, but not Vat Red 32, partially attenuated EGF-mediated cell migration despite both compounds inhibiting EGFR kinase activity. Together, this study has generated an enriched dataset of candidate environmental EGFR modulators, with experimental validation confirming enrichment for EGFR-disrupting activity among the selected compounds. These results provide a valuable resource for toxicological studies.

Pesticide use is ubiquitous. While pesticide resistance in target species such as insects is a well-known phenomenon, it is less known how pesticide exposures affect the genetics of non-target species. Here, we demonstrate the research value of integrating existing datasets to study the relationship between chemicals and biodiversity from the genetics perspective by analyzing spatial pesticide concentration data and genetic data from Europe that were not initially generated for toxicological purposes. Specifically, we found associations between environmental pesticide concentrations and genetic variation (i.e., single nucleotide polymorphisms, SNPs) in a non-target species, the wild boar. Gene Ontology (GO) terms and biological signaling pathways related to nervous system function were overrepresented for the impacted genes, especially for insecticides and fungicides. Further, we found that external studies characterizing the molecular mechanisms of these pesticides revealed the same pesticide - GO relationships as those observed in our analysis. Our results show that pesticide-induced genetic changes may not be constrained to target species but may also have an impact on the long-term viability of other wildlife populations. We therefore encourage further research into this area and collaboration across disciplines to develop more comprehensive designated datasets for such analyses. This finding also urges the consideration and inclusion of chemical exposure into conservation protection strategies and greater consideration of genetic impacts from chemicals in environmental risk assessment.

Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants associated with adverse female reproductive outcomes. However, most prior studies examined individual PFAS, despite that humans are exposed to complex PFAS mixtures. Here, we investigated the ovarian effects of a mixture of five commonly detected long-chain PFAS, including PFOS, PFOA, PFNA, PFDA, and PFHxS, using complementary in vivo mouse and in vitro 3D ovarian follicle culture models. Young adult female mice were exposed to a range of concentrations of the PFAS mixture (PFASmix) through daily drinking water for 8 weeks. PFASmix at high concentrations (PFASmix-high) disrupted mouse estrous cyclicity, decreased circulating estradiol, increased testosterone, and promoted follicular atresia. Antral follicles from exposed mice showed decreased Lhcgr and increased Amh expression. Although the number of ovulated oocytes was unchanged following superovulation, PFASmix-high reduced oocyte size and polar body extrusion and altered oocyte cytoskeletal characteristics. In the 3D follicle culture model, direct PFASmix exposure impaired follicle growth, ovulation, and oocyte maturation in a concentration-dependent manner. PFASmix-high suppressed the periovulatory expression of Areg, Ereg, Tnfaip6, and Adamts1 and impaired corpus luteal (CL) development and function, as evidenced by reduced CL spheroid growth and cell survival, decreased expression of Star, Cyp11a1, Hsd3b1, and Lhcgr, and reduced progesterone secretion. PFASmix-high exposure additionally altered the expression of multiple PPAR{gamma}-associated genes in cultured ovarian follicles. Benchmark dose modeling identified estrous cyclicity and follicle rupture as sensitive in vivo and in vitro endpoints, respectively. Together, these findings demonstrated that exposure to the mixture of long-chain PFAS disrupts multiple stages of ovarian function, from follicular development, oocyte maturation to ovulation and luteal development and function and provided mechanistic and quantitative evidence for the reproductive toxicity of PFAS mixtures.

Environmental exposure involves pollutant mixtures. Building on evidence that individual bisphenols alter zebrafish larval physiology and transcriptomic profiles at ng/L concentrations, we assessed the effects of specific mixtures of BPS, BPF, and BPAF without (M1) or with (M2) BPA. Experimentally, M1 and M2 induced 4.16% and 6.54% mortality in zebrafish larvae, despite the concentration addition model predicting no toxicity. Both mixtures reduced swimming-locomotor activities. These behaviors were accompanied by organ-level functional defects, as in vivo optical mapping revealed bradycardia and increased high-frequency (30-80 Hz) brain oscillations for both mixtures, while M2 additionally altered low-frequency (0.2-4 Hz) brain activity. The neurophysiological changes were accompanied by neurovascular remodeling, with in vivo two-photon microscopy revealing decreased vascular length and branching. Finally, transcriptomic analysis revealed common M1- and M2-induced alterations linked to pathways of vascular function, neuronal communication, and metabolism. M2 altered additional pathways of cardiac and neuronal signaling, consistent with the reported organ-physiology effects. We discuss the environmental relevance of mixture exposures and assess whether BPA intensifies mixture-induced responses.

Typical environmental exposures to the toxic class of chemicals known as polycyclic aromatic hydrocarbons (PAHs) involve complex mixtures; however, relatively few mechanistic toxicity studies have evaluated them as environmental mixtures, instead focusing on individual compounds or simple mixtures. In this study, we first derived Republic Sediment Extract (REPSE), a complex PAH mixture extracted from sediment at the Republic Creosoting site of the Elizabeth River in Norfolk, Virginia. After characterizing the PAH contents of REPSE, we evaluated its mechanisms of developmental neurotoxicity in three evolutionarily distinct taxa, leveraging the unique strengths of Atlantic killifish, zebrafish, and Caenorhabditis elegans as model species, with a focus on the Aryl hydrocarbon Receptor (AhR) pathway. Embryonic REPSE exposure caused induction of CYP1A in both fish species at sub-teratogenic concentrations, consistent with activation of the canonical AhR pathway. These sub-teratogenic exposures nevertheless induced neurotoxicity across both fish species, altering neurobehavioral phenotypes in fish, and induced dopaminergic neuronal damage in worms, again at non-teratogenic concentrations. To determine whether these effects were linked to canonical AhR response pathways, we examined killifish offspring from the pollution-adapted Republic Creosoting population, which exhibited characteristic recalcitrance to CYP1A induction, but remained susceptible to the neurobehavioral effects of REPSE. The induction of neuronal damage in worms provides orthogonal evidence for a non-AhR mechanism, because C. elegans AhR is not transcriptionally activated by PAHs as in vertebrates. Further probing of potential mechanisms underlying REPSE-induced neurotoxicity in worms revealed altered neuronal redox status (roGFP) and energy availability (ATP:ADP ratio). Collectively, our multispecies approach reveals conserved mechanisms of PAH mixture neurotoxicity, including effects that extend beyond canonical AhR signaling.

Benzyl paraben (BzP) and butyl paraben (BuP) are used as preservatives and antimicrobial agents in cosmetics, pharmaceuticals, and foods. BzP and BuP have been detected in urine, placental tissue, and ovarian follicular fluid and are understudied with regards to their endocrine activity, raising concerns on their impact to the reproductive system. We tested the hypothesis that BzP and BuP are toxic to the ovary and behave similarly to other structurally similar endocrine disrupting chemicals, such as phthalates and terephthalates, by altering hormone levels and impacting cell growth and gene expression in vitro. Untreated mouse antral follicles were exposed to vehicle, 0.1, 1, 10, or 100 g/mL BzP, BuP, or the BuP analog monobutyl terephthalate (MBuTP) for 96 hours. The highest dose of BzP and BuP were cytotoxic to follicles, suggesting that medium chain length hydroxybenzoates are more cytotoxic to the ovary than corresponding terephthalic acids. BzP significantly increased levels of estradiol and testosterone compared to control. BuP decreased follicle growth and disrupted expression of cell cycle regulators, similar to phthalates. The peroxisome proliferator-activated receptor gamma (PPARg)-transcriptionally controlled gene Fabp4 was significantly upregulated by both BzP and BuP, whereas MBuTP upregulated all four PPAR target genes and decreased follicle growth. Overall, BzP, BuP, and MBuTP disrupted hormone levels, follicle growth, and cell cycle progression, suggesting similarities in toxicity to phthalates and potential endocrine disruption. Further study should be done on the reproductive toxicity and endocrine disrupting effects of chemicals similar in structure to phthalates to reduce exposure to endocrine disruptors and inform design of safer replacements.

Omics methods have been envisioned to complement traditional toxicological testing for chemical risk assessment, in which identifying adverse effects is a critical step. However, the high dimensionality of transcriptomic data has historically led to reliance on context-dependent analysis. Liberality is a quantitative index that reduces genome-scale data dimensionality, with its changes reflecting underlying biological phenomena. In this study, we measured liberality in mouse liver RNA sequencing (RNA-Seq) datasets from studies in which mice were exposed to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) every 4 days for 28 or 92 days, comparing dose-liberality relationships. Both 28- and 92-day TCDD treatments increased liberality but exhibited different dose-liberality relationships. Analysis of genes contributing to liberality revealed that longer exposure duration induced more extensive alterations in transcriptomic architecture. These findings suggest that liberality may serve as an unbiased metric to assess the extent of treatment-induced transcriptome perturbation.

Large-scale chemical screens are important tools for hazard identification and chemical prioritization, but they less commonly progress from identifying a phenotype to identifying its mechanism. Here, we used zebrafish embryos to screen 4,657 chemicals from the U.S. EPA ToxCast Phase III library for disruption of embryonic development and advanced selected hits through sequential validation using original library stocks, independently sourced chemicals, concentration-response analysis, transcriptomics, and functional experiments. Of 61 primary hits subjected to repeat testing, 33 reproduced the original phenotype, and four of eight compounds subsequently tested using independently sourced chemicals exhibited reproducible concentration-dependent developmental toxicity. We then investigated purpurin, an understudied anthraquinone pigment that caused pericardial edema, circulation defects, and body-axis abnormalities. Transcriptomic analysis of purpurin-exposed embryos revealed coordinated suppression of pathways involved in calcium regulation, ion transport, and neuronal signaling. Increasing extracellular calcium produced a concentration-dependent rescue of purpurin-induced developmental abnormalities, whereas equivalent magnesium supplementation did not, supporting a role for calcium availability or homeostasis in purpurin developmental toxicity. These results demonstrate that large-scale in vivo toxicity screening can be integrated with independent chemical validation and functional follow-up to move beyond hazard identification toward mechanistic understanding of how environmental chemicals disrupt embryonic development.

Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants with the potential to disrupt metabolic processes and contribute to carcinogenesis; however, molecular evidence linking alternative and emerging PFAS to ovarian cancer (OC) remains limited. We conducted a biomarker-based case-control study integrating exposureomics with multi-omics profiling. Plasma samples from 128 patients with OC and 128 controls were analyzed for 26 PFAS, untargeted metabolomics, lipidomics, and clinical phenotypes, while urine samples from an additional 100 patients and 100 controls underwent untargeted metabolomic and clinical profiling. Single-compound, burden-based, and mixture models were integrated with PFAS-omics association scans, cross-matrix metabolite prioritization, observational exposure-omics-clinical association-chain analyses, and repeated internal cross-validation. Higher PFAS burdens were consistently associated with OC case status, and DONA, GenX, PFNA, PFPeS, PFUdA, and PFTrDA constituted an OC-prioritized six-PFAS mixture. DONA and GenX exhibited the broadest lipid-related bridging signals, particularly across phospholipids, lysophospholipids, sphingolipids, neutral lipids, and acylcarnitines. Integrated plasma and urine analyses further identified five shared metabolites-elaidic acid, hippuric acid, betaine, corticosterone, and taurocholic acid-that provided incremental and complementary discrimination beyond reference models based on clinical variables and tumor markers in internal cross-validation. Collectively, these findings demonstrate that both alternative and emerging PFAS are associated with a distinct OC-related molecular phenotype involving coordinated lipidomic, metabolomic, and hematologic alterations, while highlighting DONA and GenX as the most prominent exposure-related signals.

Tetrabromobisphenol A (TBBPA), is one the most widely produced brominated flame retardant, detected in human matrices including cord plasma, raising concern over its impact on embryonic development. Our previous work demonstrated that TBBPA disrupts craniofacial cartilage development in zebrafish; however, inhibition of bone morphogenetic protein (BMP) signaling did not rescue these defects, suggesting the involvement of alternative molecular mechanisms. Transcriptomic profiling revealed significant upregulation of aryl hydrocarbon receptor (Ahr) signaling genes, including cyp1a and cyp1c, as well as reactive oxygen species (ROS) responsive genes, such as nfe and gstp. Consistent with these findings, KEGG pathway enrichment analysis identified significant enrichment of pathways involved in xenobiotic metabolism, cytochrome P450-mediated metabolism, and molecular docking predicted stronger binding of TBBPA to Ahr2 than the Ahr2 agonist TCDD. Then we investigated the role of Ahr signaling and ROS in TBBPA-induced craniofacial cartilage defects using microinjection of Ahr2 translation-blocking morpholino and the antioxidant N-acetylcysteine (NAC), respectively. Immunohistochemistry confirmed concentration-dependent induction of Cyp1a and ROS at environmentally relevant concentrations (0.05;0.005 uM). Ahr2 knockdown using a translation-blocking morpholino rescued TBBPA-induced alterations in Cyp1a expression, ROS levels, DNA damage, the expression of chondrogenesis-related markers (Sox10 and Sox2), and epithelial to mesenchymal transition (EMT) markers (E-cadherin, N-cadherin, and Snail2). Furthermore, Ahr2 knockdown rescued several TBBPA-induced alterations in craniofacial cartilage parameters. Co-exposure of TBBPA with the antioxidant NAC rescued the ROS and DNA damage and rescued only ceratohyal cartilage length, whereas other cartilage parameters remained disrupted. These findings establish a causal role for the Ahr-Cyp1a axis in TBBPA-induced craniofacial cartilage developmental toxicity and identify ROS as an important downstream contributor, indicating that both ROS-dependent and ROS-independent Ahr mechanisms underlined the observed TBBPA induced craniofacial defects.

Endocrine-disrupting chemicals can target ovaries and interfere with key milestones of reproduction. Previously, we found that mixtures of the chemical exposome measured in follicular fluid (FF) were cumulatively associated with lower oocyte yield. Because ovaries age faster than many other organs, our current aim was to evaluate associations of FF chemical mixtures with epigenetic age acceleration and epigenetic pathways in FF cells. FF was collected during oocyte retrieval from 76 patients undergoing assisted reproduction in Atlanta. The exposome was measured using untargeted high-resolution mass spectrometry with gas (GC) and liquid (LC) chromatography. Weighted quantile sum (WQS-RS) indices were constructed for three mixtures of chemicals in association with oocyte yield, separated by instrument configuration (GC, LC-HILIC, LC-C18). DNA methylation was measured from the cellular component of FF using Illumina MethylationEPIC BeadChip, with age acceleration based on the GrimAge clock. Regression and pathway enrichment analyses elucidated relationships between chemical exposures or mixture indices and epigenetic markers, adjusted for age and technical covariates. All three chemical mixture indices were associated with epigenetic age acceleration in FF (p<0.05). For example, a standard-deviation increase in the GC-detected mixture was associated with 0.23 standard-deviations higher accelerated aging (95% CI: 0.0058-0.45; p=0.048). Twenty-seven frequently detected chemicals, including benzo[a]pyrene, plasticizers, flame retardants, forever chemicals, and pesticides, were associated with epigenetic pathways related to ovarian follicle growth and hormone signaling (p<0.05; six under false discovery rate<5%). In summary, environmental chemicals may accumulate in ovaries, contribute to accelerated epigenetic aging of ovarian somatic cells, and potentially affect follicle development.

BACKGROUND: Pesticides are extensively used in agriculture, raising concerns about their potential impact on human health through dietary and environmental exposure. OBJECTIVES: This study evaluated the in vitro cytotoxicity of ten commonly used pesticides and their mixtures (lambda-cyhalothrin, cypermethrin, deltamethrin, tebuconazole, glyphosate, acetamiprid, cyprodinil, piperonyl butoxide, fluopyram, and imazalil) on human intestinal Caco-2 cells. METHODS: Cytotoxicity was assessed using the MTT assay, as a measure of metabolic activity, and the trypan blue exclusion test, as an indicator of cell membrane integrity. FINDINGS: Results showed that high concentrations (100 mg/L) of all pesticides significantly reduced cell viability and vitality. Notably, glyphosate and tebuconazole exhibited significant toxicity even at lower concentrations, respectively 0.1 mg/L and 10 mg/L. Combination treatments (Top 3 and Top 8 pesticide mixtures) retained the cytotoxic effects observed for individual compounds, showing additive (non-synergistic) effects. CONCLUSIONS: Overall, these findings indicate that certain pesticides-based herbicides can exert cytotoxic effects on intestinal cells even at relatively low concentrations and highlight the importance of using the component-based approach in mixture risk assessment for humans. This study was performed as part of the EU SPRINT (Sustainable Plant Protection Transition: A Global Health Approach) project.

Environmental factors including chemical exposures are important contributors to Parkinsons disease (PD). Nearly all well-validated chemicals involved in PD affect mitochondria, and the great majority of those identified inhibit mitochondrial complex I, causing ATP depletion and oxidative stress. We hypothesized that inhibition of mitochondrial complex III would also cause dopaminergic neurotoxicity. Using Caenorhabditis elegans to evaluate the in vivo effects of complex III-inhibiting pesticides antimycin A and pyraclostrobin, we found that both caused selective dopaminergic neurotoxicity. We evaluated exacerbation of dopaminergic neurotoxicity by the presence of -synuclein, and pdr-1/PRKN and pink-1/PINK1 mutant backgrounds and found increased neurotoxicity for pdr-1. Complex III inhibition caused a more-oxidized cellular environment in those neurons and pharmacological and genetic antioxidant interventions rescued neurotoxicity, but energetic rescue attempts did not. Finally, optogenetic production of superoxide anion specifically at complex III caused dopaminergic neuronal damage. Thus, redox stress at complex III following chemical exposure causes dopaminergic neurotoxicity in vivo in C. elegans.

Inorganic arsenic is a widespread environmental contaminant and known human carcinogen, yet the mechanisms by which nutritional status modulates arsenic toxicity remain poorly understood. Here, we investigated the main and interactive effects of environmentally relevant concentrations of arsenic, low food quantity, and low dietary phosphorus supply on genome-wide gene expression in aquatic grazer Daphnia pulex. Differential gene expression analysis identified a total of 1,213 differently expressed genes with interactions of arsenic x nutrient stressors accounting for approximately 70% of the transcriptomic response. Low phosphorus emerged as a dominant main effect stressor and it also had a profound impact on transcription as a co-stressor. The low phosphorus x arsenic interaction exhibited the greatest transcriptional impact (435 DE genes), revealing that phosphorus limitation rather than food quantity influences arsenic toxicity at the gene expression level. Gene ontology and Pathway Activation Analysis revealed that main effects elicited simple yet distinct functional responses, whereas arsenic x nutrient interactions induced complex pathway-level disruptions including cell signaling, detoxification metabolism, DNA repair mechanisms, and energy homeostasis. Further assessment of gene expression revealed that all arsenic x nutrient interactions are antagonistic supporting previous literature that found arsenic behaves antagonistically as a co-stressor. Our results provide mechanistic insight into how nutritional status modulates arsenic toxicity and highlights the importance of considering arsenic x nutrient co-stressor interactions.

Current insecticide toxicology still lacks an integrated understanding of how lethal and sublethal exposure shape organismal performance across biologically and environmentally relevant contexts. In particular, insecticide susceptibility may vary with physiological state, environmental conditions, and recent exposure history, factors that are only partially captured by standardized toxicity assessments. Here, using Drosophila melanogaster, we systematically compared responses to three insecticides with distinct modes of action - acetamiprid, chlorantraniliprole, and deltamethrin - across developmental stages, sexes, adult ages, and ambient temperatures. Insecticide sensitivity varied substantially across these conditions, with temperature modulating toxicity in a compound-dependent manner. Notably, deltamethrin caused substantial lethality at concentrations more than 10,000-fold below approved field application rates. Beyond these context-dependent differences in acute toxicity, sublethal pre-exposure to each insecticide rapidly reduced lethality during a subsequent challenge with the same compound, with increased tolerance already detectable after 24 h. Importantly, these exposure conditions that enhanced insecticide tolerance were associated with reduced resistance to environmental challenges, including heat and nutrient starvation, revealing a potential trade-off in organismal stress resilience. Acetamiprid exposure further altered reproductive performance in a concentration-dependent manner, with higher exposure reducing egg production while increasing developmental success among the resulting offspring, such that the number of offspring reaching adulthood remained largely unchanged. Together, these findings demonstrate that insecticide susceptibility is highly context dependent and can be rapidly modified by recent exposure history. We identify short-term, compound-specific, and potentially costly tolerance as an underappreciated consequence of insecticide exposure and highlight the importance of incorporating biological context, environmental conditions, and sublethal exposure history into pesticide risk assessment.

Per-and polyfluoroalkyl substances (PFAS) constitute a diverse class of persistent synthetic chemicals utilized across industrial, medical, and consumer sectors that are pervasive global pollutants. Exposure to PFAS is linked to adverse impacts on both innate and adaptive immune systems. Human lactoferrin (hLF) is a key antimicrobial component of the developing innate immune system present in colostrum and breast milk. We hypothesized that hLF is a potential PFAS binding protein related to PFAS immunotoxicity. The results of thermal stability experiments indicated that all 11 tested PFAS bind and destabilize the structure of hLF. Notably PFBA, PFOS, HFPO-DA, and 6:2 FTSA decreased apo-hLF melting temperatures from 64oC to [&le;] 37oC, suggesting that PFAS exposures destabilize the native hLF protein under physiological conditions. Relative binding affinities (Kd) ranged from 0.2-11 mM across tested PFAS. Molecular docking was used to confirm experimental binding affinities and identify molecular interactions involved with PFAS binding. Calculated Gibbs Free Energies of binding ranged from -4.4 to -8.8 kcal/mol. Together, these results demonstrate that PFAS bind hLF at affinities comparable to human serum albumin and other PFAS binding proteins, and that some PFAS can destabilize hLF protein structure at physiologically relevant temperatures and conditions.

Bio-based plastics, such as polylactic acid, offer an alternative to petroleum-based plastics and a prospect to address the plastic pollution problem. While mounting evidence suggests that microplastics pose a human health threat, the risk posed by bio-based plastics remains unknown. Here, we use Caenorhabditis elegans to investigate the effects of secondary microplastics from 3D-printed polylactic acid on fertility and lifespan. We have created and characterized microplastics from a 3D-printed item using cryogenic milling. Using these microplastics, we exposed C. elegans and assessed lifespan, reproduction, and various stress responses. Our studies demonstrate that exposure to 1 {micro}g/L polylactic acid microplastics reduces fertility and alters the gonad structure. When examining germline integrity, we find chromosomal disorganization in the gonad after polylactic acid microplastic exposure and increased apoptotic cell death, which correlates with a DAF-16/FOXO and gst-4 oxidative stress responses. While lifespan has been observed to decrease with exposure to microplastics of different polymer types, we did not observe a change in lifespan with exposure to polylactic acid microplastics. The germline is likely more sensitive than somatic tissues under our exposure conditions. The reduction in fertility is driven by alterations to the germline, characterized by chromosome aberrations, oxidative stress, and cell death.

Several studies have been conducted on human exposure to ultrafine particulate matter (UFPM), Black carbon (BC), and polystyrene nanoplastics (PS-NPs). However, it remains unclear whether different chemical types of environmental nanoparticles induce a similar mitochondrial stress response or a unique particle-specific response. In the present study, we examined the molecular mechanisms underlying nanoparticle-induced mitochondrial stress response and immunotoxicity using human peripheral blood mononuclear cells exposed to UFPM, BC, and PS-NPs under similar experimental conditions. Oxidative stress, mitochondrial adaptation, respiratory chain integrity, mitochondrial integrated stress response, inflammatory signaling, and systems-level interactions between molecules were analyzed through the evaluation of the expression of NRF2, HIF-1, PGC-1, TFAM, OMA1, DELE1, mitochondrial ND1, Complex I-V, NF-{kappa}B, TNF-, and NLRP3 and the use of principal component analysis, hierarchical clustering, and correlation networks. All three nanoparticles caused oxidative stress and mitochondrial dysfunction with different kinetics and mechanisms. UFPM mostly induced an acute antioxidant response and mitochondrial adaptation; BC led to chronic mitochondrial dysfunction, chronic activation of the OMA1-DELE1-mediated mitochondrial ISR pathway, and inflammation; while PS-NPs induced low but chronic mitochondrial adaptation along with mitochondrial biogenesis and stress responses. Our systems-level analysis showed that oxidative stress, mitochondrial adaptation, mitochondrial ISR, and inflammation represent a highly connected molecular network regardless of the physicochemical nature of the nanoparticles, with the OMA1- DELE1 axis being a key regulatory node connecting mitochondrial stress response and inflammation. Overall, we have found that mitochondrial stress response is a common mechanism underlying the toxicity of chemically different nanoparticles and have also revealed particle-specific stress-response dynamics responsible for the degree and persistence of cellular damage. The current work presents novel insights into the molecular mechanisms of nanoparticle-induced immunotoxicity and suggests OMA1, DELE1, NRF2, PGC-1, TFAM, ND1, and Complex I-V as potential biomarkers.

Hexafluoropropylene oxide dimer acid (GenX), a replacement for legacy per- and polyfluoroalkyl substances (PFAS), is increasingly detected in the environment, yet its chronic toxicity remains poorly characterized. Current safety assessments rely largely on short-term, high-dose studies that may not capture the biological consequences of long-term, low-dose exposure. To address this gap, we employed 3D human liver (HepG2/C3A) spheroids cultured in a continuously rotating bioreactor system (ClinoStar) to systematically evaluate dose- and time-dependent mRNA changes in response to GenX under environmentally relevant conditions. Spheroids were exposed to GenX (0.08-50 M, spanning environmentally relevant to mechanistically informative concentrations) for acute (4 days) and chronic (4 weeks) durations, followed by genome-wide TempO-Seq transcriptomic profiling and benchmark dose (BMD) modeling. GenX elicited pronounced non-monotonic mRNA changes in acute exposure conditions, with the greatest number of differentially expressed genes (DEGs) observed at an intermediate concentration (0.4 M). In contrast, chronic exposure exhibited a generally concentration-dependent increase in DEGs, except for the 10 M condition, indicating a more consistent dose-response relationship than acute exposure. Notably, acute and chronic exposures elicited qualitatively distinct mRNA changes with low concordance across matched concentrations, demonstrating that exposure duration was a major determinant of mRNA changes. Acute low-dose GenX exposure preferentially modulated mRNA encoding components of cell cycle-related pathways, whereas acute higher dose exposures suppress mRNA levels of the constituents of lipid metabolic pathways and increase expression of mRNA encoding proteins involved in stress- and toxicity-associated signaling. Chronic exposure revealed a different pattern of changes in mRNA expression not observed under acute exposure conditions, including suppression of cellular components involved in lipid-related pathways at the lowest concentration tested. At higher concentrations, mRNA levels of components of multiple metabolic pathways were altered. Benchmark dose modeling identified a significantly lower transcriptomic point of departure (tPOD) for chronic exposure as compared to acute exposure, suggesting increased cellular sensitivity to prolonged GenX exposure and supporting the relevance of chronic models for human exposure assessment. Collectively, these findings demonstrate that GenX elicits time-dependent and non-monotonic changes in mRNA levels of human liver (HepG2/C3A) spheroids, with distinct responses depending on the exposure duration and dose. This study, therefore, highlights the importance of incorporating chronic, human-relevant in vitro models and transcriptomic endpoints into PFAS risk assessment and suggests that conventional short-term assays may underestimate the biological impact of sustained low-dose exposure. Key message (Impact of the study)This study provides systematic comparisons of short term (4 day) versus longer term (4 weeks), environmentally relevant GenX exposure in human liver spheroids, revealing non-monotonic, time-dependent changes in mRNA levels encoding cellular components of lipid metabolism-related pathways with potential implications for appropriate dose and time exposure parameters for use in New Approach Methods to be applied in risk assessment.

Background: Population-based biomonitoring of contemporary-use pesticides remains limited in the United States, particularly in rural agricultural regions, and few studies have repeated measurements within the same individuals over time. Methods: We analyzed 28 urinary pesticide-related biomarkers among 600 adults from the population-based Survey of the Health of Wisconsin with archived urine collected during 2008-2016; 296 participants provided repeat urine and updated exposure information in 2025. Detection frequencies, co-detection, and within-person detection patterns were characterized. Generalized estimating equations were used for stacked, repeated-measures analyses of factors associated with detection of aminomethylphosphonic acid (AMPA), glyphosate, 2,4-dichlorophenoxyacetic acid (2,4-D), and any of these three. Prospective-only analyses evaluated more detailed agricultural and recent exposure measures. Results: Glyphosate, AMPA, and 2,4-D were detected in 7.7%, 6.2%, and 4.3% of retrospective specimens and 5.4%, 3.1%, and 4.1% of prospective specimens, respectively. Co-detection and persistent detection across the 9 to 17-year interval was rare. In repeated-measures models, greater fruit and vegetable intake, older age, and male sex were associated with higher 2,4-D detection. Lower household income was associated with lower AMPA detection, while afternoon/evening collection was associated with higher AMPA detection. In prospective analyses, working on field-crop agricultural land showed the strongest agricultural associations, particularly for 2,4-D and detection of any of the three pesticides. Associations were not seen with self-reported conventional versus organic produce consumption. Conclusions: Urinary pesticide detections were generally infrequent in this Wisconsin population. Diet and direct agricultural activities may be more informative exposure pathways than residing near cropland or private well drinking-water characteristics.