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.

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.

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.

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.

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.

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.

Nanoplastics (NPs) are being used increasingly in cosmetics, personal care products, foods, automotive products, and cleaning products, as well as being by-products of some industrial processes. The aim of this study was to examine the effects of a sub-chronic exposure of NPs on various biological systems, using mice as a model. Adult male mice were administered 500 nm polystyrene (PS) NPs at 0.15 mg/day and 1.5 mg/day, to mimic subchronic exposure to NPs. Control mice were gavaged in the same manner but with sterile water instead of NPs. The mice were weighed weekly and treated daily for 60 days. The mice were then euthanized, and multiple tissues were retrieved and fixed or frozen for subsequent analyses. The intestines were rinsed and divided into pieces of the three regions: duodenum, jejunum, and ileum. Hematoxylin and eosin (H&E) staining was performed to evaluate histopathology, and RNA-Seq was conducted on tissues from all three regions. H&E staining revealed few effects on the duodenum, but increasingly pronounced disruption and epithelial alterations in the jejunum and ileum, respectively. RNA-Seq analysis of controls compared to the high-dose (HD; 1.5 mg/day) group supported these observations. The number of differentially expressed genes (DEGs) was low (19) in the duodenum, higher in the jejunum (114 DEGs), and significantly greater in the ileum (4982 DEGs). Genes associated with immune response, ion transport, and junctional proteins were among the groups of genes most differentially expressed. These results demonstrate the potentially harmful effects of PS NPs on the intestinal system of mice.

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

Here we report genotoxicity and 90-day oral toxicity evaluations of a high-purity, fermentation-derived mogroside preparation rich in mogroside V, the principal sweetener of monk fruit (Siraitia grosvenorii). The test article ([≥]95% total mogrosides, 70.3% mogroside V) is produced by a modified Escherichia coli from glucose, offering a higher-purity alternative to traditional monk fruit extracts. The test article was non-mutagenic in a bacterial reverse mutation test (OECD TG 471) and non-clastogenic in an in vitro human lymphocyte micronucleus test (OECD TG 487), up to the maximum recommended concentration. In the 90-day study (OECD TG 408), the test article was given by daily oral gavage at 0, 500, 1000, and 2000 mg/kg body weight/day to Sprague-Dawley rats. There were no deaths and no test article-related effects on clinical signs, ophthalmology, functional observational battery, body weight, food consumption, clinical pathology, thyroid hormones, oestrous cyclicity, or sperm parameters. Minor liver-weight increases lacking any clinical chemistry or histopathological correlates were determined to be non-adverse. No effects were seen on testis weight, sperm endpoints, or spermatogenesis. All other statistically significant differences were minor and considered incidental. The no-observed-adverse-effect level (NOAEL) was 2000 mg/kg body weight/day, the highest dose tested, supporting its safety as a food ingredient.

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.

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.

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.

Objective To systematically evaluate the predictability of drug chemical structures against the complete set of MedDRA Preferred Terms (PTs), construct a comprehensive structure adverse reaction landscape, validate the utility of molecular fingerprint representations in pan-screening tasks, and establish quantitative tools for drug safety pre-screening. Methods Drug adverse event association data were compiled from SIDER 4.1 and its derivative compilation Nsider, encompassing 2,005 approved drugs, of which 1,120 small-molecule drugs with successfully retrieved chemical structures were retained for analysis. For each Preferred Term (PT) meeting the minimum sample-size threshold ([&ge;]25 positive and [&ge;]30 negative drugs; n = 768), XGBoost classifiers were trained using MACCS 166-bit fingerprints, with model performance evaluated via 5-fold stratified cross-validation. A PT was designated as high-confidence if it achieved a validation AUC [&ge;] 0.75, a cross-validation AUC [&ge;] 0.70, and passed Benjamini Hochberg false discovery rate (FDR) correction (q < 0.05). The entire analytical pipeline was replicated using Morgan ECFP4 2048-bit fingerprints to assess the robustness of fingerprint selection. The robustness of significance screening was independently validated by permutation testing (500 random label permutations), and model generalizability was evaluated through Bemis-Murcko scaffold-based stratified split validation. SHAP (SHapley Additive exPlanations) analysis was employed to identify critical structural features, and mechanism-based case studies further demonstrated concordance between model-identified features and established pharmacophores. Results Among the 768 modelable PTs, 71 (9.2%) met the high-confidence significance criteria (FDR q < 0.05), predominantly comprising nervous system disorders (10 PTs) and psychiatric disorders (9 PTs). The toxicities exhibiting the strongest structural predictability included hyperprolactinaemia (AUC = 0.968), dermatitis acneiform (AUC = 0.966), tardive dyskinesia (AUC = 0.952), and breast tenderness (AUC = 0.979). Under a unified XGBoost hyperparameter configuration, the low-dimensional MACCS fingerprint (166-bit) demonstrated near-equivalent performance to the high-dimensional Morgan fingerprint (2048-bit) in pan-screening tasks ({Delta}AUC = -0.012, Cohen's d = 0.16), and achieved comparable performance at a substantially lower dimensionality across 18 of 22 organ systems. SHAP analysis identified key structural fragments corresponding to established pharmacophores (six-membered rings, tertiary amine cores, heteroatomic bonds, and bridged heteroatomic chains), and mechanism-based case studies further demonstrated that the model captured structural patterns concordant with known pharmacophores of D2 antagonists, EGFR inhibitors, and related agents. Bemis-Murcko scaffold-based stratified split validation revealed a median AUC decrease of merely 0.042, with 66% of PTs maintaining an AUC [&ge;] 0.70 under scaffold-split conditions. Immune-mediated and multifactorial endpoints remained structurally opaque under the current data conditions. Conclusion This study constructed a comprehensive structure-adverse reaction predictability landscape encompassing 768 MedDRA PTs; seventy-one PTs achieved high-confidence criteria. The MACCS 166-bit fingerprint achieved near-equivalent performance to the Morgan 2048-bit fingerprint at a markedly lower dimensionality, and its chemically explicit features enable direct translation of model outputs into structural alerts, providing a computational tool for prospective screening of potential adverse reaction profiles for any novel compound. A tiered assessment strategy is proposed: machine learning ensemble models for high-confidence screening, substructure alert rules for high-specificity exclusion, and pharmacovigilance signal detection for complementary coverage of structurally unpredictable endpoints. Bemis-Murcko scaffold-based stratified split validation further demonstrated that the median generalization performance decrease was merely 0.042 AUC, with 66% of PTs maintaining an AUC [&ge;] 0.70 under scaffold-split conditions, corroborating the existence of a genuine chemical basis underlying structure-adverse reaction mappings.

Breast cancer is the most common cancer in women worldwide and chemicals disrupting estrogen or progesterone signaling are recognized as potential risk factors. However, chemicals that alter the mammary gland (MG) development and function remain understudied, highlighting the need for additional research in this area. To address this gap, we investigate the relevance of using high-content imaging assays and more specifically, Cell Painting technology to measure cell morphology perturbation caused by chemical exposure and identify morphological features that could characterize mammary carcinogens (MC) risk factors. Using a dataset of MC and non-mammary carcinogens (Non-MC) with Cell Painting profiles from the JUMP-CP dataset, we retrieved 51 compounds: 28 MC, 23 non-genotoxic Non-MC. We characterized the morphological data by non-linear dimensionality reduction (UMAP) and hierarchical clustering. We, then, developed a Guilt-By-Association (GBA) framework comparing multiple configurations of similarity metrics, risk-score aggregation approaches and features representations. Morphological profiles clustered by mechanism of action rather than carcinogenicity label: genotoxic MC produced strong perturbations in endoplasmic reticulum, mitochondria, nucleus, and RNA compartments, whereas hormonally active compounds were indistinguishable from controls, reflecting the lack of functional steroid hormone receptors in the cell line used. Our best GBA configuration achieved an AUC-ROC of 0.630 and an AUC-PR of 0.696. Applied prospectively to endocrine disruptors chemicals, it prioritized clofentezine, 3-methylpyrazole, resorcinol, 2-tert-butyl-4-methoxyphenol, and thiabendazole as candidates for confirmatory testing. This study provides a transparent, interpretable tool for prioritizing chemicals in mammary carcinogenicity assessment, highlights limitations and clarifies where Cell Painting datasets must be complemented by hormone-sensitive models.

The delivery of small-molecule therapeutics across cellular membranes remains a fundamental challenge in drug development, particularly for compounds whose physicochemical properties limit intracellular bioavailability. Inspired by the low toxicity, biodegradability, and synthetic accessibility of triethanolamine-derived lipids, we investigated a series of C18 aminotriester lipids as excipients for the codelivery of anticancer therapeutics. Four structurally related triester lipids bearing saturated, cis-monounsaturated, trans-monounsaturated, and di-unsaturated C18 tails were synthesized through straightforward esterification reactions with triethanolamine. These lipids were evaluated for their ability to enhance the antiproliferative activity of several anticancer agents, including paclitaxel, doxorubicin, amonafide, homoharringtonine, camptothecin, and SN-38. Across multiple drug classes, the aminotriester lipids exhibited minimal intrinsic cytotoxicity and generally produced limited enhancement of drug potency. However, the fully saturated tris-stearate aminotriester lipid selectively enhanced the antiproliferative activity of amonafide in HCT-116 colorectal cancer cells, particularly at lower concentrations. In contrast, unsaturated lipid analogues displayed negligible enhancement effects, and the activity of the tris-stearate lipid was substantially attenuated in A549 lung adenocarcinoma cells, weakly observed in HT-29 colorectal cancer cells, and absent in CT26 murine colorectal cancer cells. Lactate dehydrogenase release assays indicated that these effects were not associated with membrane lysis or nonspecific cytotoxicity. Further, fluorescence microscopy time course experiments suggest improved membrane trafficking of amonafide when codelivered stoichiometrically with fully saturated C18 aminotriester lipids. Collectively, these findings demonstrate that saturated triethanolamine-based aminotriester lipids may function as selective and biocompatible codelivery excipients for certain small-molecule therapeutics. The observed dependence on both lipid saturation state and cellular context suggests that membrane organization and lipid packing may play important roles in mediating these effects. These results establish a foundation for further mechanistic investigations into aminotriester lipid-assisted small-molecule delivery systems.

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.

Breast cancer is a heterogeneous disease comprising molecular subtypes with distinct therapeutic vulnerabilities. Among emerging therapeutic strategies, copper-based nanoparticles have shown anticancer activity. However, whether these responses differ across breast cancer subtypes and which molecular programs underlie copper sensitivity remain poorly understood. We investigated subtype-specific responses to Polyvinylpyrrolidone-assisted copper nanoparticles (CuNP-PVP) in breast cancer cell lines and the molecular programs underlying copper adaptation. CuNP-PVP were synthesized and evaluated in luminal (MCF-7), HER2-positive (SKBR3), and triple-negative (MDA-MB-231) breast cancer cell lines, with transcriptomic analyses integrated with CCLE and TCGA-BRCA datasets. UV-Vis spectroscopy revealed a plasmon resonance band at 591 nm, while TEM showed spherical nanoparticles with an average diameter of 76.1 +/- 21.0 nm. DLS indicated a larger hydrodynamic diameter, consistent with PVP coating; {zeta}-potential measurements showed a surface charge of -13 mV, and XPS confirmed mixed reduced and Cu(I)/Cu(II) oxidation states. Exposure to 100 g/mL CuNP-PVP induced subtype-dependent effects on cell viability, with MCF-7 showing greater sensitivity, whereas MDA-MB-231 and SKBR3 were less affected. Baseline transcriptomic analyses revealed enrichment of oxidative stress, metabolic adaptation, and lysosomal programs in less sensitive cells, whereas MCF-7 showed higher expression of lipoylation-related genes. These features supported the development of a Potential Biological Copper-Response Index (PBCRI), which captured subtype-associated transcriptional patterns across independent cell lines and patient tumors, with luminal models showing lower scores and basal/TNBC models showing higher scores. Together, these findings identify candidate transcriptional programs associated with subtype-specific responses to CuNP and provide a framework for investigating potential molecular determinants of copper sensitivity.