Toxicological Sciences

Lead (Pb) exposure is strongly associated with cardiovascular diseases, but the effects of developmental Pb exposure on the electrophysiological properties of human cardiomyocytes have not yet been investigated. Using human induced pluripotent stem cell (iPSC)-derived cardiomyocytes, we investigated the effects of human-relevant Pb exposure during ventricular cardiomyocyte differentiation on action potential properties and calcium dynamics in differentiated myocytes. We exposed 2 iPSC lines, from 1 male and 1 female donor, to Pb (0.5 or 5 micromolar) or control during only the first 8 days of differentiation, prior to functional maturation. Differentiation was carried out using an established protocol involving temporal modulation of Wnt signaling, followed by purification and maturation of contractile myocytes. We measured action potential properties and calcium dynamics using fluorescent dyes and optical mapping. We hypothesized that Pb exposure would adversely affect cardiomyocyte function, with effects persisting after cessation of exposure. In keeping with this hypothesis, we found significant Pb-induced changes in calcium (Ca) transients and action potential dynamics that were concentration and cell line specific and persisted well after exposure had ended. In the female cell line, beat frequency significantly increased with Pb exposure for both action potentials and Ca transients, while in the male cell line, beat frequency significantly decreased with Pb exposure for action potentials and increased for Ca transients. For voltage measurements, we observed significantly increased arrythmias with Pb exposure in both cell lines. Significant changes in repolarization and recovery time (for voltage and Ca, respectively) were present with Pb exposure in both cell lines. Gene expression of Ca transporters/channels and K channels were not affected, suggesting that persistent effects of Pb exposure on cardiomyocyte electrophysiology may occur through post-translational mechanisms. Overall, this study paves the way for future work to examine how exposure to Pb impacts long-term cardiac function through developmental programming.

Genetic variation impacts biological response to chemical and environmental exposures, contributing to differences in resistance or susceptibility. Forward genetic screens of genetically diverse cell populations can be used to identify the precise genetic variants that drive response variation. Cell lines from laboratory mouse genetic reference populations like the Diversity Outbred (DO) population are well powered for genetic mapping. Diverse stem cell panels are especially beneficial for studying early developmental effects of chemical exposures like triphenyl phosphate (TPHP), an organophosphate flame retardant linked to adverse developmental effects, including altered cell cycle in stem cells. Self-renewal and pluripotency are defining properties of stem cells during development; therefore, it is important to understand how chemicals like TPHP may alter these critical characteristics. To better understand the influence of genetic variation on TPHP exposure response, we utilized a genetically diverse panel of DO induced pluripotent stem cells (DO iPSCs). Our analyses characterize interline variation in gene expression through differential gene expression analysis and expression quantitative trait locus (eQTL) mapping. We identified genomic loci that contribute to variation in gene expression following TPHP exposure, including a regulatory hotspot on chromosome 15. Gene set enrichment highlighted several pathways affected by TPHP including lipid metabolism, steroid hormone signaling, and cell cycle. Our study shows that genetic variation modulates the effects of TPHP on gene expression and cell cycle in stem cells. Additionally, our work demonstrates that genetically diverse cell panels like the DO iPSC resource offer a tractable and scalable approach for identifying the genetic determinants of chemical toxicity.

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.

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.

Current health risk assessments from thirdhand exposure to methamphetamine (meth) to occupants of contaminated residences when no cooking or use is still occurring assume that inhalation exposure is insignificant. After Wright et al. (2021) reported substantial meth concentrations in a contaminated residence, the present study was conducted to further test the hypothesis that concentrations were high enough to contribute to total occupant dose. Air sampling was conducted in contaminated residences that had been previously sampled for surface contamination. Samples were collected on personal sampling pumps in fixed locations on treated glass fiber filters is cassettes and analyzed by gas or liquid chromatography with mass spectroscopy. Sixty-nine samples were collected from eighteen residences for the study. Meth concentrations ranged from 0.02 to 15 ug/m3. Concentrations in supply air vents were significantly higher than in return vents or in ambient air. Residences with no HVAC systems (baseboard heat) had airborne concentrations similar to residences with HVAC systems turned off, and lower than when HVAC was on. Whether a residence was occupied or not did not show differences. All data from residences with HVAC had a significant correlation with surface concentrations (R2 = 0.61) but some factors that affected results were not determined in the study. In the context of the risk assessment and regulatory standard established by California and adopted in several other states, the inhalation exposure would approximately double total occupant dose for infants, and this population would exceed the California reference dose by approximately 100%. Further studies should be conducted to refine these estimates and determine the factors that influence airborne concentrations.

Objective: Environmental pollutants are increasingly recognized as potential risk factors for metabolic dysfunction-associated steatotic liver disease (MASLD), yet the effects of mixed exposures and underlying mechanisms remain unclear. We evaluated associations between environmental chemical mixtures and MASLD and the mediating role of inflammation-related biomarkers. Methods: We analyzed 60 environmental chemicals across six categories among 39,061 U.S. adults from the National Health and Nutrition Examination Survey (NHANES) 1999-2020. Principal Component Pursuit with Principal Component Analysis (PCP-PCA) identified latent exposure patterns. MASLD was identified using multisociety Delphi consensus criteria and the Hepatic Steatosis Index (HSI [&ge;] 36). Logistic regression, restricted cubic spline models, and mediation analysis were applied. Results: PCP-PCA generated 5-17 dimensions per chemical category, with first-dimension variance contributions ranging from 30.70% to 98.88%. Six dimensions from four categories were retained, with phthalate esters (PAEs) and organophosphate flame retardants (OPFRs) showing the most consistent associations with MASLD. PAEs Dim.1, explaining 90.71% of variance, showed a significant non-linear dose-response association (P-nonlinear = 0.0001). Significant associations were identified for mono-isobutyl phthalate (MiBP; OR 1.12, 95% CI 1.07-1.19), mono(carboxyoctyl) phthalate (MCOP; OR 1.08, 95% CI 1.02-1.14), mono(carboxynonyl) phthalate (MCNP; OR 1.12, 95% CI 1.03-1.21), and bis(1,3-dichloro-2-propyl) phosphate (BDCP; OR 1.15, 95% CI 1.08-1.23). C-reactive protein (CRP) and red cell distribution width (RDW) were the primary mediators, accounting for 32.42% and 19.62% of the MiBP-MASLD association, respectively (both p < 0.0001). Conclusion: Environmental co-exposure, particularly to PAEs and OPFRs, was associated with higher MASLD risk, with systemic inflammation potentially contributing to these associations.

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.

Experimental assessment of developmental neurotoxicity (DNT) is costly, time-consuming, and difficult to scale across large chemical inventories. New approach methodologies (NAMs), including computational approaches based on machine learning, deep learning, and large language models (LLMs), offer new ways to screen and prioritize chemicals. However, these emerging computational approaches have largely focused on predicting toxicity endpoints, with less attention to the mechanisms underlying those predictions. For DNT assessment, understanding this mechanistic basis is equally important. In this study, we integrated LLM inference with the adverse outcome pathway (AOP) framework to connect DNT predictions with plausible mechanisms. We constructed an AOP-informed benchmark linking ChEMBL target-activity measurements for 5,861 compounds to 14 AOPs representing five neurodevelopmental outcomes and evaluated ten LLMs for the prediction of molecular initiating events (MIEs), key events (KEs), key event relationships (KERs), and adverse outcomes (AOs), as well as the generation of mechanistic explanations. Explanation quality was evaluated using both a naive LLM judge and a rubric-based LLM judge. Across the zero-shot models, the highest observed macro-F1 scores were 0.818 for MIEs and 0.463 for AOs, but only 0.056 for KEs and 0.034 for KERs, identifying downstream pathway reconstruction as the principal challenge. Few-shot prompting substantially improved performance, with Gemini 3.8 Flash under four-shot prompting achieving macro-F1 scores of 0.956 for AOs, 0.813 for MIEs, 0.492 for KEs, and 0.457 for KERs. Both LLM-judge configurations indicated improved mechanistic explanation quality with few-shot examples, although strong outcome prediction did not consistently ensure recovery of the recorded pathway. Our results demonstrate the potential of AOP-informed LLMs to support DNT assessment by linking predicted neurodevelopmental outcomes to plausible biological mechanisms and enabling predictions to be evaluated at both the outcome and pathway levels.

BACKGROUND Botanicals are widely consumed as herbal remedies and plant-derived products, but their complex and variable chemical composition presents major challenges for safety assessment. Although hepatotoxicity has been reported for several botanicals, the constituent compounds responsible and the underlying mechanisms often remain poorly characterised. OBJECTIVES We aimed to determine whether Cell Painting could identify candidate constituent drivers of botanical bioactivity and generate hypotheses about potential mechanisms of action in primary human hepatocytes. METHODS We profiled 13 botanical extracts and 85 known constituents across multiple concentrations using Cell Painting alongside assays of metabolic activity and membrane integrity. To interpret morphological phenotypes, we trained machine-learning models using a Cell Painting reference atlas of >1,000 chemicals linked to ToxCast/Tox21 molecular and cellular bioactivity annotations. RESULTS Cell Painting detected bioactivity in 11/13 botanical extracts and 46/85 constituents, compared with 9/13 extracts and 40/85 constituents in the cell-health assays, and generally detected activity at lower concentrations. For 9/13 extracts, at least one constituent was active at a lower concentration than the corresponding botanical extract, supporting the contribution of a subset of potent constituents to mixture-level responses. Aristolochia and goldenseal were the most potent extracts across Cell Painting and cell-health assays and contained highly active constituents. Reference-atlas models generated specific mechanistic hypotheses, including cytotoxicity-, endocrine-, and genotoxicity-associated responses for several botanicals and constituents. CONCLUSION These results support Cell Painting combined with machine learning as a sensitive approach for characterising cellular responses to botanicals, prioritising candidate constituent drivers of hepatotoxicity-associated phenotypes, and generating testable hypotheses about potential mechanisms of action.

Background: Milrinone, a phosphodiesterase-3 (PDE-3) inhibitor, is widely used to improve cardiac output in pediatric and adult patients. Yet, developmental differences in myocardial responsiveness to milrinone remain incompletely understood. In this study, we examined the impact of postnatal maturation on the acute cardiac effects of milrinone using an intact guinea pig heart model. Methods: Neonatal (0-2 days), juvenile (4-10 days), and adult (> 6 months) guinea pig hearts were excised, Langendorff-perfused, and cardiac metrics were evaluated under basal conditions and in response to acute milrinone treatment (15 minutes, 10 and 100 M sequential concentrations). Pseudo-electrocardiograms were recorded continuously and left ventricular pressure measurements were performed under sinus rhythm and in response to external pacing. Post-rest potentiation was used to assess contractile reserve, and optical action potentials and calcium transients were recorded. Results: Baseline left ventricular developed pressure (LVDP), action potential duration (APD), and calcium transient duration (CaD) increased with postnatal maturation, consistent with developmental cardiomyocyte remodeling and refinement of excitation-contraction coupling. Milrinone increased heart rate in all age groups, with the greatest chronotropic response at 100 M. Milrinone also increased ventricular contractility and relaxation across developmental stages, but the magnitude of the response varied with age and was attenuated during high frequency pacing. Adult hearts had the greatest increase in LVDP during sinus rhythm and robust post-rest potentiation, which were less pronounced in neonatal hearts. APD and CaD were shortened in neonatal and adult hearts, but were minimally affected in juveniles, indicating a non-linear developmental pattern. Conclusions: Milrinone exerts positive chronotropic, inotropic, and lusitropic effects throughout development, but the magnitude and frequency dependence of these responses vary with age. These findings suggest that postnatal maturation influences the cardiac response to PDE3 inhibition. These developmental differences highlight the importance of considering age as a biological factor in pediatric drug selection and dosing.

Human metabolism converts inorganic arsenic to the pentavalent methylated species MMA(V) and DMA(V), the forms most people excrete, and the forms long read as the end of a detoxification pathway. Whether a transporter sets how much of these metabolites reaches the genome has not been tested in a mammalian cell. We expressed human AQP3, AQP7, AQP9 or AQP10 in HEK293T and MRC5-SV40 cells and measured gamma-H2AX by flow cytometry across dose series of As(V), MMA(V) and DMA(V), pairing every aquaporin with a GFP-Tubulin control and an untransfected mock acquired in the same replicate. As(V) was inactive in HEK293T cells and only weakly active in MRC5-SV40 cells to 20 micromolar, and both methylated species damaged DNA only in the millimolar range, DMA(V) being the more potent of the two in both cell lines. Against that weak baseline, AQP9 and AQP10 raised DMA(V)-induced gamma-H2AX in HEK293T cells by roughly 17 percentage points over the matched control, more than doubling the damage the same exposure produced in control cells, whereas AQP3 and AQP7 changed it not at all. AQP9 alone remained active with MMA(V). The ranking held in MRC5-SV40 fibroblasts at one-sixth the size, and within single wells the damage rose with the amount of AQP9 a cell carried while the control was flat. Aquaglyceroporins therefore discriminate among arsenic species, and AQP9 and AQP10 turn a weakly genotoxic metabolite into a substantially more genotoxic one.

Peroxisome proliferator-activated receptor (PPAR) is a nuclear receptor that plays a central role in metabolic adaptation by regulating gene expression programs involved in lipid utilization and energy homeostasis. Although the hepatic functions of PPAR and its target genes are well characterized, less is known about its transcriptional activity in extrahepatic tissues. Pemafibrate (K-877), a selective PPAR modulator (SPPARM), is a high-affinity PPAR ligand with improved potency and selectivity compared with conventional fibrates. Here, we investigated pemafibrate-induced tissue-specific transcriptional responses comparing wild type and whole-body PPAR-deficient littermate mice. Twelve-week-old male PPAR+/+ and PPAR-/- mice were treated daily with pemafibrate (0.1 mg/kg) or vehicle by oral gavage for 14 days. We performed genome-wide transcriptomic analyses in the liver, jejunum, kidney, soleus muscle, and BAT. Pemafibrate-induced transcriptional changes were entirely dependent on PPAR, as no differentially expressed genes were detected in PPAR-/- mice. The magnitude and nature of the response were tissue-specific, with the strongest transcriptional effects observed in the liver and jejunum, whereas the kidney, soleus muscle, and BAT showed limited responses. In the liver, pemafibrate primarily induced the expression of genes involved in fatty acid metabolism, mitochondrial function, and energy production. In the jejunum, pemafibrate activated lipid metabolic programs together with tissue-specific pathways related to cellular homeostasis. Collectively, our findings show that pemafibrate is a highly selective PPAR activator with a preferential transcriptional activity in the liver and intestine after oral administration. This study provides a resource for defining tissue-specific PPAR-responsive gene networks and expands our understanding of PPAR functions beyond the liver.

Per- and polyfluoroalkyl substances (PFAS) and phthalate metabolites are understudied in follicular fluid of women undergoing assisted reproduction. We characterized these in previously collected follicular fluid from patients at a Nebraska fertility clinic (August 2022 to June 2025) and retrieved demographics and clinical characteristics from fertility records. Spearman correlations and nonparametric tests evaluated PFAS and phthalate correlations and exposure differences by age, race, BMI, and urbanicity. Seven PFAS and four phthalate metabolites were detected in at least half of participants. PFOS and PFHxS concentrations were higher in younger women, and mono-isobutyl phthalate (MiBP) was higher in younger and non-White women. PFAS and phthalate correlations were weak, and exposures did not differ by urbanicity, indicating multiple chemical exposures in follicular fluid with sociodemographic disparities.

Hospital readmission is used in cirrhosis research as if a return to hospital meant the disease had worsened: it is a trial outcome, a quality indicator and a prediction target. We asked what those readmissions were. Sixty-one adults with alcohol-related cirrhosis, selected because they had been hospitalized more than once, contributed all 326 later admissions to one department. A clinician read each full record and classified it for acute decompensation and for admission mode, blinded to diagnostic codes and structured flags. Fewer than two in five admissions (125, 38.3%) were decompensations. One in three (114) had been booked in advance; none of these was a decompensation, yet they used 29.1% of all bed-days. Among 124 returns within 30 days, planned admissions outnumbered decompensations, 48 to 43. Counting all admissions tracked a patients decompensations only moderately (correlation 0.50) whereas counting urgent admissions tracked them closely (0.93). In crude analysis the sicker patients had fewer admissions, not more, because planned care goes to those well enough to receive it. A liver severity score, computable for 165 admissions, did not separate planned from urgent admissions or decompensations from other stays, even within one patient. Readmission was not the same thing as decompensation.

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.

Objectives: Social media trends such as the "Benadryl Challenge" have increased adolescent misuse of diphenhydramine (DPH), an over-the-counter antihistamine with anticholinergic activity. Whether co-ingested anticholinergic medications compound cardiac risk is untested. Using the Anticholinergic Cognitive Burden (ACB) scale, this study examined whether co-medication ACB burden is associated with outcome severity and cardiac toxicity in adolescent DPH cases reported to the FDA Adverse Event Monitoring System (AEMS). Methods: This study extracted all adolescent reports (age 13-19) mentioning DPH in any drug role from AEMS, yielding 3,168 cases from 217,507 deduplicated records. Given non-normality (Shapiro-Wilk, all p < 0.001), non-parametric tests were used. A multivariable logistic regression predicted cardiac toxicity; logistic and random-forest models were compared by cross-validated AUC. Results: Co-medication ACB burden correlated positively with case severity ({rho} = 0.142, p < 0.001) and differed by age group (H = 19.52, p < 0.001). However, burden and cardiac risk diverged: cardiac cases carried a lower burden (mean 0.55 vs 1.27) and were younger, and burden did not independently predict cardiac toxicity (OR = 0.927, 95% CI [0.841, 1.022], p = 0.128). Younger age was the strongest predictor (OR = 0.865 per year, p < 0.001); cardiac events occurred in 10.2% of 13-15-year-olds versus 4.9% of older adolescents. Conclusion: Anticholinergic co-medication burden and cardiac risk dissociate: burden tracks overall severity, but cardiac events concentrate in the youngest, least co-medicated adolescents, a profile consistent with deliberate single-agent overdose rather than polypharmacy. A low co-medication burden should not reassure clinicians triaging young adolescents.

Carbaryl, a widely used carbamate pesticide, has been epidemiologically associated with genotoxic and carcinogenic outcomes in exposed human populations, including elevated cancer risk at several tissue sites and, in some agricultural communities, chronic tubulointerstitial kidney disease of unknown etiology (CKDu). However, the cellular mechanisms underlying these associations, and whether carbaryl acts uniformly across tissues, remain poorly defined. Screening a panel of cell lines of distinct species and lineage, we found that carbaryl elicits pronounced, dose-dependent cytotoxicity and cell cycle arrest selectively in a renal tubular epithelial line (IMCD3) and, to a lesser extent, a cervical epithelial line (HeLa), while a fibroblast line (NIH3T3) was unaffected. In IMCD3 cells, carbaryl induces S- and G2/M-phase arrest through direct binding to DNA and consequent DNA damage, triggers Bcl-2-mediated late apoptosis independently of reactive oxygen species, and drives irreversible mitotic arrest. Molecular dynamics simulations, cellular thermal shift assays, and kinase measurements converge on CDK1 as a direct target of carbaryl: carbaryl destabilises the CDK1 active site and inhibits its catalytic activity, whereas CDK2 is engaged through an opposing, rigidifying mode with no functional consequence. This asymmetry provides a coherent mechanistic basis for the combined S-phase and G2/M arrest observed. Together, these findings establish carbaryl as a genotoxic, cell-cycle-disrupting agent with tissue-selective potency acting directly on CDK1, and provide a cell-based mechanistic framework for interpreting its reported associations with renal and other tissue pathologies in exposed human populations.

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.

Inhalation of airborne per- and polyfluoroalkyl substances (PFAS) represents an important yet understudied exposure route. While volatile organic compounds (VOCs) have been widely explored as biomarkers of toxicant exposure, their potential for early detection of PFAS-induced pulmonary toxicity remains largely unexplored. This study investigated VOC profiles in human alveolar (A549) and bronchial (BEAS-2B) epithelial cells following exposure to environmentally relevant PFAS concentrations (1, 5, and 10 ppb) for 12, 24, and 48 h. VOCs were analyzed using headspace solid-phase microextraction coupled with gas chromatography-Orbitrap mass spectrometry. We observed significant concentration- and time-dependent alterations in VOC profiles in both cell models. The principal VOCs associated with PFAS exposure include butanal, acetone, 2-butanone, 2-nonanone, (E)-2-nonenal, (E)-2-octenal, (E)-2-heptenal, 2,4-heptadienal, 2,4-nonadienal, 2,4-decadienal, 1-pentanol, and 1-hexanol. Cell type-specific regulations were observed, reflecting differential metabolic responses between transformed and non-transformed lung epithelial cells. Metabolite set enrichment analysis indicated perturbations in ketone body metabolism, consistent with altered cellular energy metabolism. Complementary qPCR analysis revealed altered expressions of CYP1A1, CYP2E1, ALDH1A1, and ALDH2, supporting a mechanistic link between PFAS exposure and oxidative stress, lipid peroxidation, and impaired aldehyde detoxification. These findings demonstrate that VOC profiling, combined with transcriptional analysis, can reveal PFAS-induced metabolic perturbations in pulmonary epithelial cells.

Nanoplastics (NPs) are widespread environmental contaminants, yet most toxicity studies rely on short-term, single-dose exposures that poorly reflect daily human exposure. The kidney, particularly the proximal tubule, is a likely site of repeated NP exposure given its role in filtration and reabsorption, but chronic exposure effects are largely unexplored. Here, a long-term in vitro model was developed in which human proximal tubule (HK-2) cells were continuously exposed to 20 nm or 100 nm carboxylated polystyrene NPs (100 {micro}g/mL) over 12 days. Continuous 20 nm NP exposure caused progressive loss of viability whereas 100 nm NPs left viable cell numbers largely unaffected. RNA-sequencing showed 20 nm NPs drove extensive, time-dependent transcriptional dysregulation (1,548 differentially expressed genes at day 6, rising to 4,431 at day 9), far exceeding the minimal alterations in gene expression (35 genes) caused by 100 nm NPs. Cell-cycle analysis and pathway enrichment revealed KRAS pathway activation and S-phase arrest in 20 nm-treated cells, with ANGPTL4 the most upregulated gene. Validation with qRT-PCR confirmed increased ANGPTL4, HMOX1 and FN1 expression, genes implicated in renal fibrosis and chronic kidney disease. These findings show that continuous, size-dependent NP exposure elicits progressive cytotoxic and transcriptional responses in kidney proximal tubule cells and indicate ANGPTL4 as a candidate early biomarker of NP-induced renal stress.