Chemical Research in Toxicology

Background: In the United States, several states have restricted sales of flavoured tobacco products, including popular menthol- and mint-flavoured Oral Nicotine Pouches (ONP). In response, tobacco companies introduced "unflavoured" ONP containing odorless synthetic cooling agents. Since these, in turn, have become targets of legislative bans, the tobacco industry may seek out flavourants with other sensory effects to increase the appeal of "unflavoured" ONP. Methods: Online merchants were searched for "unflavored" ONP marketed to consumers in jurisdictions with flavour bans. Sensory effects of aqueous extracts from identified "heat", "spicy" and "unflavoured" ONP were analyzed by Ca2+ microfluorimetry in HEK293 cells expressing the human heat/chili pepper flavourant (capsaicinoid) receptor, hTRPV1. ONP were analyzed for capsaicinoids and sweeteners by Liquid Chromatography/Mass Spectrometry (LC/MS). Results: A new category of "heat" or "spicy" ONP was identified, including products marketed as "unflavoured". Extracts from all these ONP robustly activated TRPV1, with "unflavoured" Lucy Heat the most potent. Chemical analysis demonstrated that Lucy Heat contained the synthetic capsaicinoid nonivamide at high levels (~675 microgram/pouch), while others contained mixtures of capsaicinoids (5-25 microgram/pouch) combined with other characterizing flavours (tropical, fruit). All tested ONP contained sweeteners. Conclusions: The tobacco industry continues to probe regulatory loopholes by claiming that newly introduced capsaicinoid flavourants and sweeteners in ONP do not represent characterizing flavours. This is contradicted by industry and regulatory determinations assigning characterizing flavour properties to these additives. The toxicological health risks of repeated capsaicinoid exposures due to ONP use, in combination with nicotine and other constituents, need to be assessed.

2,4-Dinitrochlorobenzene (DNCB) is a well-characterized skin sensitizer that has been widely used in immunological and toxicological research and, historically, in clinical immunotherapy. Although it is a well-investigated chemical, this is the first study focusing on the dose response behavior at low-level concentrations. The aim was to reveal potential hormetic effects due to its known Nrf2 inducting activity. Therefore, THP-1 cells were treated with low doses of DNCB and two endpoints were evaluated for hormetic responses: metabolic activity using a resazurin-based assay and immune activation by measuring CD86 and CD54 expression using flow cytometry. The results showed a significant hormetic effect on the metabolic endpoint at the lower cell density for both analyzed time points, and a hormetic tendency at the higher cell density. Metabolic activity increased to approximately 125% of the control at 0.05 micromolar DNCB. For the immunological endpoint a slight decrease in CD86 and CD54 surface marker expression was observed, up to -16% and up to -12% compared to control at 0.5 micromolar DNCB. These findings highlight the importance of including low dose concentrations when characterizing chemical dose-response relationships and evaluating toxicological risk.

The presence of N-nitrosamine drug substance-related impurities (NDSRIs) in pharmaceuticals represents a significant regulatory and safety challenge due to their classification as "cohort of concern" compounds. This paper describes the toxicological evaluation of N-Nitrosotrimetazidine (NTMZ), performed to refine the initial default acceptable intake (AI) limits of 18 to 26.5 ng/day established by regulatory authorities. The evaluation followed a tiered approach: NTMZ was first confirmed as mutagenic in vitro via the standard Ames test. To further investigate its genotoxic potential, two in vivo studies were conducted in Wistar and transgenic rats. Detection of DNA strand breaks in the liver and duodenum (comet assay) together with positive results in the cII mutation assay confirmed an in vivo mutagenic mode of action. Benchmark Dose (BMD) analysis of the transgenic rat data yielded a BMDL50 of 7 mg/kg/day in the male liver. To characterize long-term carcinogenic risk, a GLP-compliant 2-year carcinogenicity study was conducted in Wistar rats. Chronic exposure induced dose-dependent increases in liver tumors (hemangiosarcomas, hepatocellular carcinomas and adenomas) and intestinal tumors (adenomas and adenocarcinomas), leading to a Tumor Dose 50 (TD50) of 23 mg/kg/day in male rats. Benchmark dose analysis of tumor incidence identified a lowest BMDL10 of 2.6 mg/kg/day in females, which served as the basis for deriving an AI of 13 microg/day. This assessment demonstrates a strong predictive correlation between the BMD derived from the in vivo transgenic model, the BMDL10 and the final TD50 values obtained in the 2-year carcinogenicity study. These findings provided the scientific basis for establishing a conservative AI of 13 microg/person/day based on the BMDL10 and further support the regulatory acceptance and use of BMD-derived approaches for the evaluation of nitrosamine impurities.

Integrating high-throughput in vitro data into next-generation risk assessment (NGRA) workflows requires screening strategies that yield quantitative potency estimates and mechanistically interpretable biological signals. Transcriptomic and morphological profiling are increasingly adopted for early-stage hazard identification by enabling triage of substances for resource-intensive follow-up and prioritizing candidates most likely to present meaningful risk. In this study, we aimed to characterize biological concordance and uncertainty by quantifying how well high-throughput transcriptomics (HTTr) and Cell Painting PLUS (CPP) bioactivity profiles recover target-relevant biological signals in immortalized human renal proximal tubule epithelial RPTEC/TERT1 cells using 313 reference chemicals with high-confidence target annotations. Through quality control procedures and biological activity filters we yielded 142 reference chemicals spanning 66 different targets, which were systematically evaluated for biological concentration-responses by HTTr and CPP. HTTr was evaluated using TXG-MAPr-based qualitative and quantitative gene network activity analysis. HTTr showed the most prominent activity for targets that were highest expressed in RPTEC/TERT1 cells. Active chemical-pairs showed strong gene network activity correlation albeit with different potencies. Similarly, the highest transcriptomic concordance was observed for reference chemicals acting in the same pathway, such as EGFR/MEK or PI3K/AKT/mTOR. CPP often showed high sensitivity primarily at the organelle level providing limited statistical power for chemical grouping. Collectively, the results support HTTr and CPP as complementary early-tier assays within an in vitro weight-of-evidence safety testing framework. Although CPP is suitable as a cost-effective screening modality, HTTr offers higher mechanistic resolution for mode-of-action inference in high-throughput bioactivity screening and therefore remains necessary for high-confidence mechanistic interpretation.

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.

People are widely exposed to the antimicrobial cetylpyridinium chloride (CPC) via consumer products, but CPC is a mitochondrial toxicant with potency comparable to that of canonical mitotoxicants. CPC is largely unregulated despite growing usage, bioavailability, and ability to cross the blood-brain barrier. Previously, we showed, in several cell types at non-cytotoxic and exposure-relevant doses, CPC inhibits ATP and OCR, endpoints of the electron transport chain (ETC). Mitochondrial toxicity is linked to multiple diseases (e.g., diabetes, Parkinsons, myalgic encephalomyelitis), but CPC has not been studied epidemiologically, and little mechanistic information is available. To determine why OCR and ATP are hampered by CPC, we hypothesized that CPC inhibits individual ETC components, cardiolipin, or TCA enzymes. Here, we show that, in primary human skin cells, an immune mast cell model, and isolated mitochondria, CPC apparently inhibits multiple ETC Complexes. Detailed investigation pinpointed the mechanism to the distal end of ETC: Complex III-cytochrome C-Complex IV. Using multiple approaches, we show that CPC does not directly inhibit any of the Complexes (not even Complex I as earlier reported), nor TCA enzymes, nor coenzyme Q. Yet, we found that CPC exhibits mitotoxicity as potent as cyanide. Anionic lipid cardiolipin attracts cytochrome C to the inner mitochondrial membrane so that it may shuttle electrons from Complex III to IV. Despite not altering levels of cardiolipin, CPC hinders cytochrome C by electrostatically interfering with cardiolipin. To aid epidemiology, risk analysis, and predictive toxicology, we have determined the precise biochemical mechanism of action of this ubiquitous compound.

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.

BackgroundCannabis derivatives have been reported to possess antinociceptive properties. However, oral delivery is limited by poor bioavailability, stability, and reliability of effects. Previously, we reported an analgesic effect of the aqueous complex {Delta}9-tetrahydrocannabinol/2-hydroxypropyl-{beta}-cyclodextrin (THC/HP{beta}CD) after intracerebroventricular administration in male rats. MethodsHere, we investigated the analgesic effects of the THC/HP{beta}CD complex after oral administration (0.3 and 3 mg/kg) by the tail flick test after both acute and chronic administration (15 days) in female rats. Locomotor activity and anxiety-like behavior were also evaluated at the same experimental conditions. Moreover, dopamine and glutamate content in the periaqueductal gray (PAG), a key area for the antinociceptive action of THC, were also measured by HPLC. ResultsAfter acute administration, the antinociceptive effect of the complex was seen at 3 but not 0.3 mg/kg THC, with a maximum effect observed at 30 min (MPE 60%). Similar results were obtained after 15 days of treatment, although partially reduced (max MPE 20%). Reductions in locomotor activity with the dose of 3 mg/kg and a slight biphasic effect of the two doses on anxiety-like behavior were also observed. Finally, neurochemical analyses revealed that the dose of 3 mg/kg significantly increased dopamine and glutamate content in the PAG, an effect no longer present after 15 days of treatment. ConclusionsOur results highlight the antinociceptive efficacy of the THC/HP{beta}CD complex also after oral administration, notably higher than that previously seen with other carriers, although with some degree of tolerance after chronic administration. From a translational point of view, these results are relevant for the development of THC-based oral formulations with analgesic properties for the treatment of pain in humans. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/742765v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@fff791org.highwire.dtl.DTLVardef@d672f4org.highwire.dtl.DTLVardef@1150b3forg.highwire.dtl.DTLVardef@956403_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

Cytochrome P450 (CYP) enzymes play a key role in the metabolism of both xenobiotics and endogenous compounds, and the activity of some CYP isoforms are susceptible to induction and/or inhibition by certain chemicals. As CYP induction and inhibition can significantly alter the in vivo fate of xenobiotics i.e., levels of parent chemicals and/or metabolites, and thus toxicity, CYP induction/inhibition data is needed for regulatory chemical toxicity hazard assessment. Utilizing available human in vivo pharmaceutical data, a successful validation was previously conducted on the in vitro HepaRG CYP induction test method for measurement of induction of three key human CYP enzymes CYP1A1/1A2, 2B6 and 3A4. However, further validation data was required to demonstrate applicability of the test method to also accurately detect CYP induction mediated by industrial and pesticidal chemicals. Here we report on the supplementary validation of the HepaRG CYP enzyme induction test method carried out in two laboratories under the auspices of the EU Horizon2020-funded project "GOLIATH", to expand the chemical applicability domain beyond pharmaceutical chemicals. Successful transfer was demonstrated and reproducibility assessed for the original 10 selected proficiency pharmaceuticals, plus three reference inducers together with six additional non-pharmaceutical augmentation chemicals. The method and chemical selection were found to be reliable and relevant for the routine assessment of human CYP induction. For the augmentation chemicals being proposed as additional proficiency chemicals, the test method achieved a reasonable but not optimum reproducibility. Recommendations are proposed to improve the test methods specificity, reflecting the inherent uncertainty around borderline CYP inducing chemicals. Plain language summaryCytochrome P450 (CYP) enzymes help break down drugs and other chemicals in the body. Their activity can be increased (induced) or decreased (inhibited), which can change how toxic a chemical is and when it is excreted. Because of this, CYP data is important for chemical safety assessments. A laboratory-based method using HepaRG cells was previously validated to measure induction of key CYP enzymes (CYP1A1/1A2, CYP2B6 and CYP3A4) using pharmaceutical chemicals. This study aimed to show that it also works well for industrial and pesticidal chemicals. In the EU funded GOLIATH project, two laboratories tested 10 pharmaceutical and 6 non-pharmaceutical chemicals. The method showed good reliability overall and strong reproducibility for pharmaceuticals. For non-pharmaceutical chemicals, results were acceptable but less consistent. The study concludes that the method is useful for routine testing, but improvements are needed to increase accuracy and better handle chemicals that show weak or borderline CYP induction effects.

Objectives The 2016 Draft National Institute for Occupational Safety and Health (NIOSH) universal protocol ''A Performance Test Protocol for Closed System Transfer Devices Used During Pharmacy Compounding and Administration of Hazardous Drugs.'' employing ATD GC MS with the 2016 Draft NIOSH proposed challenge agent 2 Phenoxyethanol (2 POE, 2.5% w/v in water) was used to assess the containment performance of two barrier and two air-cleaning CSTDs in a head-to-head test with replication (n=4) for both NIOSH tasks 1 (drug preparation) and 2 (drug administration). The study objective was to assess the containment performance of both physical barrier and air-cleaning CSTDs. Methods ChemoLock barrier (ICU Medical, USA), ChemoLock air-cleaning (ICU Medical, USA), PhaSeal barrier (Becton Dickinson, USA), and Tevadaptor air-cleaning (Simplivia, IL now marketed as Chemfort) CSTDs were evaluated using the 2016 Draft NIOSH universal test protocol for Task 1 (n=4) and Task2 (n=4). A 100 mL empty IV bag, accessed with a CH-10 ChemoClave bag spike was used with the IV administration set in Task 2 to avoid pressurisation of the occluded administration set during the simulated ''IV push''. Air samples were collected (GilAir pump) at 100mL/minute for 30 minutes on Tenax TA thermal desorption (TD) tubes (Markes, UK). 2 POE, a semi-volatile surrogate mimicking hazardous drugs was prepared at 2.5% w/v solution in MilliQ water. This was aliquoted (50mL) into drug vials and crimp sealed with a butyl rubber stopper (Adelphi, UK). 2 POE was analysed against a calibration curve (n=8) in the range 0.6ng to 200ng using analytical methods published previously. Chromeleon v7.3 CDS (Thermo Scientific, UK) was used for analysis based on detection of SIM ions (M/Z: 77, 94, 138). Blank TD tubes (n=85) were used to determine the experimental LOD and LLOQ. Negative control tests (employing MilliQ water as surrogate) were performed for each CSTD Task 1 (n=1) and Task 2 (n=1). An open ''needle and syringe'' positive control test was performed at 5 and 10 microlitre volumes in the NIOSH chamber to demonstrate system suitability. Results Chamber cleaning and analysis performed between tests, gave average blank values (n=85) of 0.05{+/-}0.01 ppbv (95% confidence interval). The experimental limit of detection (LOD) and quantification (LLOQ) for the tests was 0.16{+/-}0.01 ppbv and 0.41{+/-}0.01 ppbv respectively. The positive control tests produced signals of 3.13 ppbv (n=1) and 6.59 ppbv (n=1) for 5 microlitre and 10 microlitre releases of 2 POE respectively and demonstrated system suitability to quantify down to a 650 nanolitre liquid release volume. (1) ChemoLock barrier, (2) ChemoLock air-cleaning, (3) BD PhaSeal barrier and (4) Tevadaptor air-cleaning CSTDs all generated <LLOQ signals for both NIOSH tasks 1 and 2. No statistically significant difference was found between the containment performance of the two air-cleaning and the two physical barrier CSTDs evaluated in this study. Conclusions System suitability was demonstrated using deliberate releases of the 2 POE surrogate at 5 and 10 microlitre volumes producing 2-POE concentrations in the NIOSH chamber of 3.13 ppbv and 6.59 ppbv respectively. Evaluation of the containment performance gave <LLOQ (0.41{+/-}0.01ppbv) for all four of the CSTDs tested in the study: (1) ChemoLock barrier, (2) ChemoLock air-cleaning, (3) BD PhaSeal barrier and (4) Tevadaptor air-cleaning, based on testing of four replicates of each technology in both NIOSH Task 1 and NIOSH Task 2. There was no statistically significant difference in containment found between the two air-cleaning and the two barrier CSTDs evaluated in this study.

Evidence of negative health and environmental effects of glyphosate-based herbicides (GBHs) has led to marketing of glyphosate-free formulations. A frequent glyphosate replacement is pelargonic acid, which is rapidly degraded, leading to claims of greater safety and less environmentally damaging than GBHs. However, toxicity of commercial pelargonic acid formulations containing several co-formulants have not been determined. Using Roundup NL, a representative pelargonic acid-based herbicide, we undertook tissue culture cell assays measuring viability, plasma membrane integrity, DNA damage, and activation of stress-response pathways. In human hepatoma HepG2 cells, Roundup NL was more cytotoxic than pelargonic acid, and more toxic than the GBH Roundup ProBio and glyphosate as shown by reduced viability underpinned by plasma membrane damage. Pelargonic acid and Roundup NL did not induce oxidative stress. However, comet assays revealed that pelargonic acid but not Roundup NL caused a modest but significant increase in DNA damage at sub-cytotoxic concentrations. The murine embryonic stem cell-based ToxTracker system confirmed Roundup NL as not directly genotoxic but triggered oxidative stress and protein damage (ER stress, impaired proteostasis) indicating cell and assay dependency of oxidative stress pathway activation. Our results suggest that exposure to pelargonic acid-based herbicides constitutes a health hazard and that co-formulants present in Roundup NL contribute substantially to its overall toxicity.

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.

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.

The ubiquitously occurring food contaminants alternariol monomethyl ether (AME), tenuazonic acid (TeA) and altertoxin II (ATX-II) are recognized as emerging Alternaria mycotoxins, yet substantial data gaps remain regarding their toxicokinetic characteristics. The hepatic metabolism of these three substances was investigated in primary rat (PRH) and human (PHH) hepatocytes by monitoring parent compound depletion and, where applicable, metabolite formation. AME was initially evaluated at 5 {micro}M and subsequently investigated over a concentration range of 0.75-8 {micro}M (0.75, 1.5, 3, and 8 {micro}M), whereas TeA was assessed at 5 {micro}M, and ATX-II was assessed at 0.22 and 1.11 {micro}M, with additional short-term experiments conducted over 30 min for AME and ATX-II, and over 10 min for concentration-dependent AME depletion. For AME, time-dependent clearance was further evaluated in PRHs at two hepatocyte densities, 0.25 and 0.5 million cells/mL. In PRHs, AME metabolism followed Michaelis-Menten kinetics (Vmax = 150.9 pmol{middle dot}min-{superscript 1}{middle dot}10- cells, Km = 1.18 {micro}M), whereas no reliable kinetic model could be established for PHHs. In contrast, TeA exhibited high metabolic stability, with only 9-10% depletion after 4 h, indicating negligible hepatic clearance in both species. ATX-II was also rapidly depleted and became undetectable within 30 min, accompanied by transient formation of altertoxin I (ATX-I), which was more pronounced in PHHs than in PRHs. Substrate depletion revealed pronounced interspecies differences in hepatic clearance capacity and stability. Overall, these findings provide comparative insights in primary human and rat hepatocyte systems, offering a foundation for future studies on their toxicological relevance and impact on human health.

Chemotherapy treatment of colorectal cancers (CRC) using cisplatin (CDDP) encounters problems of drug resistance by the cancer cells and cytotoxicity to normal cells, highlighting the urgent need for joint therapeutical strategies. Selenium-enriched rapeseed extracts exhibit anti-cancer effects but the bioactive components and mechanisms remain unclear. Here, we applied different solvents to fractionate the extracts from Selenium-enriched rapeseed and found that the water extract (WE) fraction significantly enhanced the cytotoxic effect of CDDP on cancer cells but no damage on normal cells. HPLC-ICP-MS analysis revealed that methylselenocysteine (MSC) and selenocystine (SeCys2) were the main selenium speciation in WE. Through cell biology and integrative multi-omics analysis, we found a synergistic anti-CRC cell effect when combining CDDP with MSC, sulforaphane (SFN), celastrol (Cel), Indole-3-carbinol (I3C), -linolenic acid (ALA) or linoleic acid (LA). We propose that the CDDP-WE combination treatment holds the promise for improving curative efficacy for chemo-refractory CRC patients in the future.

BackgroundCaspase-3 and -7 are key effector caspases in the apoptotic pathway, a form of programmed cell death, and their activities serve as a well-established biomarker for evaluating environmental chemical toxicity and informing chemical risk assessment. Loss of mitochondrial membrane potential is a key event in the activation of Caspase-3/7 signaling and the subsequent induction of apoptosis. Therefore, simultaneous assessment of mitochondrial membrane potential and Caspase-3/7 activity enables elucidation of the mechanisms and pathways through which apoptosis is initiated.. Rapid and accurate assessment of the potential toxicity of environmental chemicals and drugs remains a major challenge. Quantitative Structure-Activity Relationship (QSAR) modeling have been widely used for toxicity prediction. Graph-based approaches encode compounds directly as molecular graphs, allowing structure-activity relationships to be learnt from molecular topology without the information loss in binary fingerprints. While advanced graph models such as graph transformers (GTs) have shown outstanding performance in many domains, they have not been fully leveraged in QSAR modeling on Caspase and mitochondrial toxicity. MethodsWe propose a QSAR modeling pipeline that encompasses assay data preprocessing, feature representations (fingerprints and molecular graphs), and benchmarking machine learning (ML) models, including classic ML models, graph neural networks (GNNs), GTs, and their consensus ensembles. Based on in vitro Caspase and mitochondrial assays in PubChem, we applied the pipeline to predict Caspase-3/7 activation and mitochondrial membrane potential (MMP). Beyond in vitro assays, we also built in vivo QSAR modeling for FDA Drug-Induced Liver Injury (DILI) gold standard on human hepatotoxicity. Moreover, mechanistic analysis on Caspase-3/7 activation was conducted by comparing with MMP disruption to identify chemical substructures that may be responsible for dual activations. We also investigated cell-line-specific responses by identifying structural motifs that selectively induce Caspase-3/7 activation in individual cell lines. ResultsExperimental evaluations show that GTs and GNNs outperformed classic ML models when the number of active compounds is large, such as MMP disruption, while classic ML models and GTs performed good for highly imbalance data with limited active compounds, such as Caspase-3/7 activation. For DILI prediction, the full consensus model achieved the highest AUC 0.69 and Graphormer had the highest F1 score 0.79, both surpassing the previous best model with AUC 0.63 and F1 0.65 with a large margin. Our mechanistic analysis shows that phenolic compounds bearing a para-hydroxyphenyl motif, as well as members of the lipophilic chain family with long alkyl chains can trigger the collapse of MMP, leading to the activation of caspases-3 and -7. Human embryonic kidney (HEK293) was the only cell line with a distinct structural motif: 1,1-dichloroethane and chlorobenzene. Human neuroblastoma (SK-N-SH) is uniquely impacted by an epoxide fragment and rat hepatoma (H-4-II-E) is uniquely impacted by a tetramethylcyclohexene motif and an acetaldehyde fragment. ConclusionsThe proposed pipeline for QSAR modeling, including data preprocessing, feature representations, and incorporation of advanced graph ML approaches, is highly effective in predicting not only on Caspase-3/7 activation and membrane potential collapse, but also on FDA DILI human hetatotoxicity. As future research directions, we will leverage extra information, e.g., biological activity and findings in existing toxicity literature, and recent advances in large language models and agentic AI to further improve the predictive performance and enable a sensitive and specific framework for assessing human hepatotoxicity of environmental compounds.

Excessive osteoclast activity contributes to pathological bone loss in osteoporosis, rheumatoid arthritis, and osteolytic malignancies. The effects of small catechol derivatives on receptor activator of nuclear factor-{kappa}B ligand (RANKL)-induced osteoclastogenesis remain poorly understood. This study investigated the effects of 4-methylcatechol (4-MC) on RANKL-induced NF-{kappa}B activation and osteoclast differentiation. 4-MC reduced RANKL-induced NF-{kappa}B luciferase activity in HEK-293T/RANK cells. 4-MC also suppressed RANKL-induced TRAP activity in RAW264.7 cells in a concentration-dependent manner and reduced the number of TRAP-positive multinucleated osteoclasts, without affecting cell viability. Molecular docking predicted non-covalent binding of 4-MC within the ATP-binding hinge region of IKK{beta} (PDB: 4KIK), forming a close polar contact with Glu97, predicted hydrogen bonds with Cys99, and a hydrophobic contact with Ile165, within the pocket occupied by the co-crystallized inhibitor K252a. Covalent docking predicted that the oxidized quinone form of 4-MC engages Cys179 in the IKK{beta} activation loop. Quantum chemical calculations confirmed a markedly higher electrophilicity index for the oxidized quinone than for the parent catechol, supporting this mechanism. In silico ADMET profiling indicated favorable drug-likeness and safety. These findings identify IKK{beta} as a plausible molecular target of 4-MC through both non-covalent and covalent mechanisms. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/741661v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@35a0d3org.highwire.dtl.DTLVardef@d19458org.highwire.dtl.DTLVardef@1623fadorg.highwire.dtl.DTLVardef@1429e8b_HPS_FORMAT_FIGEXP M_FIG C_FIG

Plastic products have greatly improved convenience in daily life. However, diverse pollutants released from these materials pose substantial risks to human health. Lactiplantibacillus plantarum PD01 has previously been demonstrated to reduce microplastic (MP) bioaccumulation and toxicity in murine model. In this study, we conducted a randomized, double-blind, placebo-controlled trial to further evaluate the efficacy of L. plantarum PD01 in reducing MPs and MP-associated chemicals in humans. A total of 106 participants were recruited in November 2025. Eligible participants were randomly assigned to either the placebo or intervention group, and orally received placebo or 1.0x1010 colony-forming units (CFU) L. plantarum PD01 after meals three times a day, respectively. Fecal, urinary, and blood samples were collected to determine the MPs contents, plasticizer levels, gut microbiota composition, blood metabolic profiles and biochemical indicators. Among these measurements, the analysis of urinary phthalate metabolites was completed first and revealed a significant reduction after the probiotic intervention. Compared with the placebo group, 6-week L. plantarum PD01 supplementation resulted in significant relative reductions in urinary levels of MCMHP by 60.2% (P = 0.0343), MMP by 51.2% (P < 0.001), MEHP by 49.6% (P = 0.0058), MiBP by 45.7% (P = 0.0085), MnBP by 35.8% (P = 0.0478), and {Sigma}DEHP by 46.2% (P = 0.0461). The interim results presented here provide the first clinical evidence that the probiotic strain PD01 can significantly reduce residual MP-associated chemicals in human body. To the best of our knowledge, this is the first randomized controlled trial (RCT) to evaluate probiotic intervention targeting MPs and MP-associated chemicals in humans.

IntroductionMusculoskeletal disorders remain a major cause of disability worldwide and require non invasive regenerative strategies that support tissue repair. Green-synthesized zinc oxide nanoparticles (ZnONPs) have attracted interest because of their biocompatibility and biological activity. This study investigated the synthesis of Mimosa pudica-derived ZnONPs (ZnOMP) and evaluated their effects on human bone marrow mesenchymal stromal cells (BM-MSCs). MethodologyZnOMP were synthesized using an aqueous extract of Mimosa pudica leaves and characterized by UV-Vis spectroscopy, FTIR spectroscopy, powder X-ray diffraction, SEM, EDS, and TEM. BM-MSCs isolated from human bone marrow were exposed to ZnOMP, plant extract, and synthesized ZnO nanoparticles. Cell metabolic activity was assessed by MTT assay after 1, 3, and 5 days. Cytoskeletal and nuclear morphology were analyzed by fluorescence microscopy and CellProfiler-based morphometry. Osteogenic differentiation was evaluated after 21 days using Alizarin Red S staining and quantification. ResultsSpectroscopic and microscopic analyses confirmed the successful formation of phytochemical-capped ZnOMP nanoparticles with nanoscale dimensions and specific elemental composition. ZnOMP maintained significantly higher metabolic activity than Mimosa pudica extract or ZnO at both 150 and 300 g/mL. Morphometric profiling revealed that Mimosa pudica extract induced the most pronounced changes in nuclear morphology, reflecting enhanced nuclear plasticity and substantial remodeling of nuclear architecture, whereas ZnOMP preserved cellular and nuclear features closer to untreated controls. During osteogenic induction, ZnOMP did not impair matrix mineralization and preserved the ability of BM-MSCs to form a mineralized extracellular matrix. ConclusionMimosa pudica-mediated ZnO nanoparticles combine favorable biocompatibility with preservation of mesenchymal stem cell morphology and osteogenic competence. These findings support their potential use as bioactive nanomaterials for musculoskeletal tissue engineering and regenerative medicine.