Archives Of Toxicology[JOURNAL]
Sun
200 papers
Triphenyl phosphate promotes lipid accumulation in human mesenchymal stem cells through metabolic stress pathways.
Gronske MC, Stutts W, Jima D … +3 more , Palumbo J, Stapleton HM, Kullman SW
Arch Toxicol · 2026 Jun · PMID 42228132 · Publisher ↗
Organophosphate esters (OPEs), used as both plasticizers and flame retardants, are increasingly recognized for their potential to disrupt cellular homeostasis and emerging evidence indicates their ability to induce metab...
Organophosphate esters (OPEs), used as both plasticizers and flame retardants, are increasingly recognized for their potential to disrupt cellular homeostasis and emerging evidence indicates their ability to induce metabolic stress in diverse biological systems. This study investigated the impact of Triphenyl Phosphate (TPhP) on human mesenchymal stem cell (hMSC) differentiation and cell stress. hMSCs were treated with DMSO (vehicle control), 10 µM rosiglitazone (a PPARγ agonist), or 25 µM TPhP for 14-21 days. Following treatment, cells were stained to assess nuclear integrity, lipid accumulation, and mineralization. Immunocytochemistry (ICC) for osteogenic and adipogenic markers was used to determine whether TPhP promotes adipogenesis under osteogenic conditions. TPhP exposure led to significant lipid accumulation without activation of adipogenic differentiation programs and markedly reduced mineralization. In particular, co-treatment with PPARγ inhibitors did not mitigate lipid accumulation or modulate the expression of adipogenic regulators. Gene expression analysis following TPhP exposures revealed upregulation of pathways involved in lipid biogenesis, triglyceride synthesis, phospholipid metabolism and endoplasmic reticulum (ER) stress, with minimal changes to classical adipogenic markers. These findings indicate that TPhP disrupts the metabolic balance between osteogenesis and lipid metabolism, with the potential to promote ectopic lipid deposition within the bone forming environment. This dysregulation is relevant for the development of metabolic disorders, including obesity, type 2 diabetes, and metabolic bone diseases, including osteoporosis.
E-cigarette aerosol exposure modulates crystalline silica-induced fibrotic responses in the lungs.
Twum Y, Goldsmith WT, Olanrewaju O … +2 more , Olfert IM, Gao W
Arch Toxicol · 2026 Jun · PMID 42228131 · Publisher ↗
Pulmonary toxicity of e-cigarettes (e-cigs), particularly their combined effects with occupational exposures like crystalline silica (S), remains largely unexplored. This study evaluated the combined effects, molecular m...
Pulmonary toxicity of e-cigarettes (e-cigs), particularly their combined effects with occupational exposures like crystalline silica (S), remains largely unexplored. This study evaluated the combined effects, molecular mechanisms, and differential responses in the lungs following co-exposure to e-cig aerosols and S. Subchronic silicosis was established by oropharyngeal aspiration of S (0.2 g/kg) into C57BL/6 mice. Mice were exposed to filtered air, S, or e-cig (E) + S with endpoints evaluated 28 days post-exposure. The count median diameter of e-cig aerosols generated for our mice exposure was 0.771 µm (GSD = 2.33). Enhanced pause (Penh) values increased significantly in male E + S group and female S and E + S groups, compared to their respective air controls, during the first and second weeks of exposure. Histological evaluations (hematoxylin and eosin and Masson's trichrome staining) revealed significantly increased fibrosis scores in both male and female S and E + S groups compared to respective air controls. Hematological analysis revealed a significant decrease in neutrophil counts/percentage in female E + S mice compared to S alone. Cytokine analysis revealed a significant increase in keratinocyte chemoattractant (KC) levels in male S and E + S groups compared to air controls, while in female mice, KC expression increased significantly in the E + S group compared to S alone. Protein analysis revealed distinct sex-specific changes in key protein markers associated with fibrosis and epithelial-mesenchymal transition. Taken together, these findings indicate that S-induced lung fibrosis with or without e-cig exposure occurred in both male and female mice, with different underlying molecular mechanisms.
Integrated human toxicokinetics of acetamiprid using urine, blood, and feces data in physiologically-based kinetic modelling for reverse dosimetry.
Wieland NC, Noorlander A, Zwartsen A … +7 more , Greupink R, Graumans MHF, Mol H, Dias J, Russel FGM, Ragas AMJ, Scheepers PTJ
Arch Toxicol · 2026 May · PMID 42185666 · Publisher ↗
Acetamiprid (ACE) is a widely used neonicotinoid insecticide with known neurotoxic potential, for which the European Food Safety Authority lowered the acceptable daily intake. Current physiologically-based kinetic (PBK)...
Acetamiprid (ACE) is a widely used neonicotinoid insecticide with known neurotoxic potential, for which the European Food Safety Authority lowered the acceptable daily intake. Current physiologically-based kinetic (PBK) models lack adequate kinetic data for ACE and its metabolite acetamiprid-N-desmethyl (ACE-DEM). The aim of this study was to generate detailed disposition data for ACE in a controlled volunteer setting to develop a robust PBK model suitable for reverse dosimetry. Four volunteers received an oral dose of ACE (90% of the acceptable daily intake, ADI) and a single dermal administration on a separate occasion. Concentration-time profiles of ACE and ACE-DEM were collected for urine, blood, and feces. A PBK model was developed using toxicokinetic parameters from the volunteer study. The model was validated using previously published human data. Sensitivity analyses identified key parameters of model performance. ACE was rapidly absorbed and extensively metabolized to ACE-DEM. ACE was undetectable in urine after 24 h, while ACE-DEM remained quantifiable for 96 h. Urinary excretion accounted for 8-24% of the dose, with < 2% in feces, indicating near to complete absorption. Dermal uptake was approximately 30%. The PBK model adequately described the relationship between external exposure, plasma concentrations, and urinary excretion. Three different reverse dosimetry methods were compared. The estimated oral doses fell within a threefold margin of the administered doses. In conclusion, a human-data-informed PBK model enables reverse dosimetry to estimate oral ACE exposure from urinary biomonitoring data.
Structure-based machine learning model for discovering pregnane X receptor (PXR) agonists and biological activity validation.
Huang FF, Luo Y, Chen H … +4 more , Meng YH, Li W, Liang H, Ai CZ
Arch Toxicol · 2026 May · PMID 42185665 · Publisher ↗
Pregnane X receptor (PXR), a nuclear receptor superfamily member, maintains bile acid homeostasis by regulating metabolic enzymes [e.g., cytochrome P450 3A4 (CYP3A4), uridine diphosphate glucuronosyltransferase 1A1 (UGT1...
Pregnane X receptor (PXR), a nuclear receptor superfamily member, maintains bile acid homeostasis by regulating metabolic enzymes [e.g., cytochrome P450 3A4 (CYP3A4), uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1)] and multidrug resistance protein 1 transporter, and alleviates liver/intestinal inflammation via inhibiting the nuclear factor kappa-B pathway, serving as a critical therapeutic target for cholestatic liver diseases and inflammatory bowel disease. In this study, we established a novel structure-based machine learning strategy to identify PXR agonists from a natural product database. With generated bioactive conformations binding with PXR, we integrated ligand-based and structure-based features to construct a comprehensive machine learning model using light gradient boosting machine. This model illustrated an R of 0.874 for the internal validation set and an R of 0.845 for the external test set, superior to the performance of other machine learning models, e.g. random forest regression, support vector regression, gradient boosting regression, K-nearest neighbors, and extreme gradient boosting. The model was used to predict the PXR agonistic activity of the candidate molecules screened out by the pharmacophore model. Promising candidates were selected out for further assay with HepG2 cell culture combined with a dual-luciferase reporter. Ultimately, natural products like schisantherin A, rhynchophylline, and irigenin were identified as potent PXR agonists, with half-maximal effective concentrations (EC) of 1.58 μM, 2.57 μM, and 20.67 μM, respectively. These PXR agonists act as potential candidates for targeted therapies against PXR-related diseases. We anticipate that this work will provide support for the design and discovery of PXR modulators.
Applicability of next-generation risk assessment generic PBK models and their performance within the chemical domain.
Spaenig M, Ashraf MW, Bouhifd M … +3 more , Drake C, Sobanski T, Escher SE
Arch Toxicol · 2026 May · PMID 42183848 · Publisher ↗
Physiologically based kinetic (PBK) models are a cornerstone of in vitro to in vivo extrapolation and are therefore central to next-generation risk assessment (NGRA). This study assesses the performance of a generic, hig...
Physiologically based kinetic (PBK) models are a cornerstone of in vitro to in vivo extrapolation and are therefore central to next-generation risk assessment (NGRA). This study assesses the performance of a generic, high-throughput PBK model for 26 organic chemicals spanning a wide range of physicochemical properties. Model predictions based on standard in silico and in vitro input parameters were benchmarked against published, chemical-specific PBK models and in vivo kinetic data. Predicted plasma maximum concentrations (Cmax) were within tenfold of in vivo values for 50% of the chemicals and within threefold for 31%. Published PBK models generally showed closer agreement with in vivo Cmax, likely because they incorporate in vivo-derived kinetic parameters or chemical-specific kinetic processes. An analysis of the chemical space revealed a lipophilicity-dependent bias, where Cmax for lipophilic compounds tended to be underpredicted, whereas hydrophilic compounds were overpredicted. No other consistent trends were observed with respect to physicochemical or ADME descriptors, possibly reflecting the limited dataset size. Overall, while the generic PBK model workflow appears suitable for NGRA applications, the observed lipophilicity-dependent bias indicates that chemical-specific corrections or refinements may be required for more accurate predictions.
Metabolic fate and detectability of three novel fluoroprolintane psychostimulants: in vitro and in vivo characterization by hyphenated mass spectrometry.
Wagmann L, Herter A, Dybek MB … +4 more , Adejare A, Wallach J, Brandt SD, Meyer MR
Arch Toxicol · 2026 May · PMID 42183847 · Publisher ↗
Amphetamine-type stimulants (ATS) represent a major segment of the global drugs of abuse market. Prolintane (1-(1-phenylpentan-2-yl)pyrrolidine), a substituted phenylethylamine ATS, was historically utilized as medicatio...
Amphetamine-type stimulants (ATS) represent a major segment of the global drugs of abuse market. Prolintane (1-(1-phenylpentan-2-yl)pyrrolidine), a substituted phenylethylamine ATS, was historically utilized as medication for several therapeutic indications. This study aimed to investigate the metabolic fate and urinary detectability of the three novel derivatives 2-, 3-, and 4-fluoroprolintane, which were recently shown to act as monoamine reuptake inhibitors. Liquid chromatography (LC)-high-resolution (HR) tandem mass spectrometry (MS/MS) was used for tentative identification of metabolites in rat urines (collected over a 24 h period following oral administration of 2 mg/kg fluoroprolintane isomer, each) or incubations with pooled human liver S9 fraction (1 h and 6 h, 25 µM fluoroprolintane isomer, each). Isozyme mapping was performed using individual incubations with 11 human phase I monooxygenases. The same rat urines were used for detectability studies using standard urine screening approaches (SUSA) by gas chromatography (GC)-mass spectrometry (MS), LC-ion trap MS, and LC-HRMS/MS. The fluoroprolintanes were extensively metabolized, with a total of 79 metabolites identified. Hydroxylations and subsequent oxidations were primarily mediated by CYP1A2, CYP2B6, CYP2C19, and CYP2D6, while glucuronidation and O-methylation were observed as main follow-up phase II reactions. All three SUSA allowed detection, primarily via metabolites. However, the high degree of metabolic overlap will make isomer differentiation challenging. These findings contribute to a comprehensive risk assessment and provide critical reference data for clinical and forensic laboratories to identify these substances in biological specimens.
Assessment of 2-aminothiazoline-4-carboxylic acid (ATCA) formation and identification of robust serum metabolic biomarkers for acute cyanide exposure in mice fed diets with varying methionine and cystine content.
Hisatsune K, Asano T, Taniguchi M … +1 more , Zaitsu K
Arch Toxicol · 2026 May · PMID 42178406 · Publisher ↗
Accurate assessment of cyanide (CN) exposure is challenging because direct CN measurement is unstable, and the formation of the CN metabolite 2-aminothiazoline-4-carboxylic acid (ATCA) may be influenced by dietary sulfur...
Accurate assessment of cyanide (CN) exposure is challenging because direct CN measurement is unstable, and the formation of the CN metabolite 2-aminothiazoline-4-carboxylic acid (ATCA) may be influenced by dietary sulfur amino acids, potentially confounding its utility as an exposure indicator. Here, we evaluated the effect of dietary methionine/cystine on ATCA formation and investigated diet-robust endogenous metabolic biomarkers of CN exposure using MS-based metabolomics and bioinformatics. Male ICR mice were fed a control diet, a high-methionine/cystine diet, or a low-methionine/cystine diet for 7 days and then administered CN (5 mg/kg, intraperitoneally) or vehicle. Metabolomics showed that the low-methionine/cystine diet induced a distinct baseline metabolome profile; however, serum ATCA concentrations did not differ significantly among dietary groups after CN exposure, indicating a negligible dietary effect on ATCA biosynthesis under the conditions tested. Multivariate analyses demonstrated clear separation between CN-administered and vehicle cohorts regardless of diet, suggesting the presence of metabolic signatures of CN exposure that are relatively less susceptible to dietary variation. Twenty-five metabolites were identified as candidate biomarkers by multivariate and univariate analyses, and their discriminatory performance was evaluated using a random forest classification model, which supported their specificity for CN exposure. Overall, these results suggest that ATCA formation and selected serum endogenous metabolic biomarkers are minimally influenced by variation in methionine/cystine intake, thereby potentially improving biomarker-based CN exposure assessment.
Isatin-pyridine oxime hybrids as potential reactivators of A-242-inhibited acetylcholinesterase.
Pinto AMV, Bhattacharyya D, da Silva MCJ … +8 more , Kitagawa DAS, Dos Santos MC, Cavalcante SFA, Islam ST, LaPlante SR, Forgione P, Diz JSF, França TCC
Arch Toxicol · 2026 May · PMID 42171685 · Publisher ↗
Three new isatin-pyridine-2-oxime hybrids (AM01-03) were synthesized and experimentally evaluated as reactivators of acetylcholinesterase (AChE) inhibited by a surrogate of the nerve agent A-242, in comparison with their...
Three new isatin-pyridine-2-oxime hybrids (AM01-03) were synthesized and experimentally evaluated as reactivators of acetylcholinesterase (AChE) inhibited by a surrogate of the nerve agent A-242, in comparison with their 4-oxime isomers previously reported by our research group. Furthermore, the interactions of these molecules within the AChE/A-242 complex were assessed through docking and molecular dynamics (MD) simulations. Our results show that the 2-oxime isomers, unlike their 4-oxime analogues, can reactivate AChE inhibited by the A-242 surrogate at 100 µM and are even capable of outperforming the commercial reactivator HI-6. These findings suggest that isatin-oxime hybrids may exhibit a specific biological activity-similar to that observed for obidoxime and trimedoxime-in the reactivation of AChE inhibited by A-242.
Points of Departure for BPDE-induced cellular senescence and cell death are characterized by altered DNA damage signaling and regulated by unrepaired DNA double-strand breaks.
Schmidt A, Schöneis A, Sallbach J … +6 more , Pöschmann A, Lippold R, Rasenberger B, Hofmann TG, Tomicic MT, Christmann M
Arch Toxicol · 2026 May · PMID 42168583 · Publisher ↗
The existence of thresholds for carcinogenic compounds is an important topic in toxicology and regulatory science. Traditionally, genotoxic carcinogens are thought to exhibit no thresholds. However, cellular defense mech...
The existence of thresholds for carcinogenic compounds is an important topic in toxicology and regulatory science. Traditionally, genotoxic carcinogens are thought to exhibit no thresholds. However, cellular defense mechanisms like DNA repair and apoptosis can neutralize low levels of genotoxic stress implying different Points of Departure (PoDs) for different cellular endpoints. Moreover, since cellular PoDs are regulated by the DNA damage response (DDR) and the associated DNA damage signaling cascades, the question arises whether the DDR and its cellular outcome change, depending on the level of DNA damage. Here we analyzed whether PoDs for distinct cellular processes induced by benzo[a]pyrene-9,10-diol-7,8-epoxide (BPDE) are observed at the same or different level of DNA damage and whether these PoDs correlate with activation of different DNA damage signaling routes. BPDE represents the active metabolite of the polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P) which is a product of incomplete combustion and therefore ubiquitously present in the natural environment. Our data indicate a PoD with a LOAEL (lowest observed adverse effect level) between 0.1 and 0.25 µM for DNA strand break formation, DDR activation, induction of cell death and cellular senescence. A high amount of cell death was observed at a dose of 1 µM and was accompanied by accumulation of DNA strand breaks and mediated by a switch from the p53 signaling axis to the p53 axis of the DDR. Importantly, BPDE-induced mutagenicity was observed predominantly at low BPDE concentration that failed to trigger the DDR and cellular senescence. These results suggest that low BPDE concentrations, which are unable to activate the DDR, are especially harmful in relation to mutation formation and carcinogenesis, eventually even more than DDR-activating concentrations.
Lentiviral-mediated expression of cytochrome P450 2D6 in HepaRG cells: new means for in vitro xenobiotic biotransformation and cellular response to chimeric viral mRNA.
Coppens-Exandier H, Mackanga F, Darde T … +12 more , Bonhomme C, Angenard G, Leroyer P, Ribault C, Gicquel T, Pelletier R, Roshchina F, Schaefer E, Chesné C, Jamin A, Corlu A, Loyer P
Arch Toxicol · 2026 May · PMID 42166010 · Publisher ↗
The human HepaRG cell line is the closest surrogate to primary culture of hepatocytes (PHH) for toxicology studies. However, differentiated HepaRG cells express low levels of the cytochrome P450 2D6 (CYP2D6) involved in...
The human HepaRG cell line is the closest surrogate to primary culture of hepatocytes (PHH) for toxicology studies. However, differentiated HepaRG cells express low levels of the cytochrome P450 2D6 (CYP2D6) involved in the biotransformation of many drugs. Herein, progenitor HepaRG cells were transduced using lentiviral particles encoding both human CYP2D6 and GFP proteins. The resulting transgenic HepaRG cells stably expressed catalytically active CYP2D6 at levels close to those observed in PHH from rapid metabolizers and HepaSH™ hepatocytes. In CYP2D6 transgenic HepaRG cells, tramadol was metabolized into both N- and O-desmethyl tramadol as seen in humans while parental HepaRG cells produced only N-desmethyl tramadol. Following treatment with perhexiline, the CYP2D6 expressing HepaRG cells exhibited higher IC values and reduced mitochondrial damages compared to those found in parental cells. Transcriptomic analysis revealed that the expression of CYP2D6 did not significantly affect the cells' ability to proliferate and differentiate or compromise key hepatocyte-specific functions. However, we identified a small number of genes, including NXF3 and TRIM63, which were up-regulated in transgenic cells. Using CRISPR/Cas9-mediated knockdown of GFP and/or CYP2D6 sequences, we demonstrated that NXF3 mRNA and protein inductions were triggered by the lentiviral mRNA encoding GFP and CYP2D6 rather than by genomic transgene integration. Together, these findings establish CYP2D6-transgenic HepaRG™ cells as an optimized and reliable hepatocyte-like model for studying the metabolism and toxicity of CYP2D6 substrates. Our results also support the hypothesis that the NXF3 gene may be a marker of cellular response to the expression of a lentiviral chimeric mRNA.
AIComet: a reliable automated scoring program for DNA damage assessment in the comet assay.
Germot A, Elie N, Ibazizene L … +6 more , Guillemot S, Evenden P, Møller P, Delépée R, Meryet-Figuiere M, Lebailly P
Arch Toxicol · 2026 May · PMID 42166009 · Publisher ↗
The alkaline comet assay is widely used to assess genotoxicity in biomonitoring studies but lacks standardized scoring system and shows high inter-laboratory variability. Visual scoring is time-consuming, operator-depend...
The alkaline comet assay is widely used to assess genotoxicity in biomonitoring studies but lacks standardized scoring system and shows high inter-laboratory variability. Visual scoring is time-consuming, operator-dependent, and limited in sensitivity to low DNA damage levels. We developed a fully automated, open-access scoring model for comet assay analysis to address these limitations. The AIComet model performances were assessed and the model was compared to manual scoring performed in-house on the lymphocytes of 327 healthy volunteers, and compared to visual scoring by 11 trained investigators on open-access images from (Møller et al. 2023). The influence of a larger number of nuclei scored on the sensitivity of the results was evaluated. AIComet showed excellent results for comet classification and detection. Correlation with manual scoring by our laboratory investigator was excellent with a y = 1.02x - 0.155 linear regression equation and 0.92 R. AIComet scores (51.0, 40.2, and 55.0 a.u) on the three datasets consistently fell close to the median values from the 13 investigators (50.4, 41.0, and 61.7 a.u). Regarding its sensitivity, N = 310 nuclei scored are needed to obtain a median relative difference of 10% between the score for N nuclei and the score based on all nuclei available, with a [0.4%-42.1%] 95% confidence interval.AIComet is a standardized, reproducible, time-efficient technique for comet assay scoring, reducing operator-dependent variability while improving sensitivity for detecting subtle differences in DNA damage. These findings could be important, primarily in biomonitoring studies with low-exposure settings, but also for reproducing comet assay results overall.
Embryotoxicity of pyrethroid insecticides and their mixtures in a human induced pluripotent stem cell-based model in vitro.
Ma Y, Gutierrez CT, Scholze M … +2 more , Valente MJ, Vinggaard AM
Arch Toxicol · 2026 May · PMID 42159750 · Publisher ↗
Pyrethroid insecticides are used worldwide, yet their potential effects on early human development remain poorly understood. We applied the PluriLum assay, a human induced pluripotent stem cell-based 3D in vitro model, t...
Pyrethroid insecticides are used worldwide, yet their potential effects on early human development remain poorly understood. We applied the PluriLum assay, a human induced pluripotent stem cell-based 3D in vitro model, to evaluate the embryotoxicity of three commonly used pyrethroids (α-cypermethrin, deltamethrin, and etofenprox), and their shared metabolite 3-phenoxybenzoic acid (3-PBA). Embryoid bodies (EBs) were repeatedly exposed to these compounds throughout their differentiation into cardiomyocytes, and effects on cardiomyocyte beating, expression of the early cardiac marker NKX2.5, as well as their impact at a transcriptional level was assessed. The three pyrethroids were tested in combination at two different mixture compositions to investigate their potential for additivity. All three pyrethroids impaired cardiomyocyte differentiation at micromolar levels, with the following potency ranking: α-cypermethrin > etofenprox > deltamethrin. At higher concentrations, α-cypermethrin also reduced cardiomyocyte contractility. In contrast, 3-PBA showed no significant effects on neither differentiation nor contractile function. The pyrethroid mixtures followed the principle of concentration addition. An exploratory transcriptomic analysis revealed that α-cypermethrin and deltamethrin significantly altered the EBs gene expression profiles, affecting pathways related to ion channel activity, receptor signalling, and extracellular matrix organization. These findings suggest that exposure to pyrethroids may interfere with early human cardiac development through effects on multiple molecular targets. This study also highlights the value of the PluriLum assay as a human-relevant platform for assessing the embryotoxic potential of environmental chemicals.
Differential induction of hepatic CYP enzymes by tobacco products and e-cigarettes: recommendation to rethink smoking status in clinical pharmacology.
Lenich AK, Litz A, Reindl LM … +1 more , Ruez S
Arch Toxicol · 2026 May · PMID 42159749 · Publisher ↗
Smoking status is often recorded as "Yes/No", but this binary approach overlooks the complexity of tobacco use and limits the precision of clinical data interpretation. Cigarette smoke is a known inducer of cytochrome P4...
Smoking status is often recorded as "Yes/No", but this binary approach overlooks the complexity of tobacco use and limits the precision of clinical data interpretation. Cigarette smoke is a known inducer of cytochrome P450 (CYP) enzymes, yet effects of other tobacco products on drug interactions remain poorly understood. This study addresses the gap by evaluating CYP1A1, CYP1A2, CYP2B6, CYP2C8 and CYP3A4 induction in primary human hepatocytes by 6 cigarette brands, 1 heated tobacco product (HTP), 6 cigar brands, 2 smokeless tobacco brands, and 3 e-cigarette brands (20 flavors). Cigarettes and cigars induced CYP1A1 strongest (4-29-fold mRNA; 29-95-fold enzyme activity), but also mRNA of CYP1A2 (4-10-fold), CYP2B6 (2-18-fold) and CYP3A4 (3-18-fold). HTPs demonstrated weaker CYP1A1 induction than cigarettes (3-4-fold mRNA, 6-16-fold enzyme activity), at 10-times higher concentrations, what clearly distinguishes them from cigarettes. Smokeless tobacco led to stronger mRNA induction of CYP1A2 (12-26-fold) than CYP1A1 (4-14-fold). E-cigarettes induced mRNA of CYP3A4 (2-108-fold), CYP2B6 (3-39-fold), and CYP2C8 (2-14-fold) more strongly than CYP1A1/CYP1A2 (2-7-fold), with brand-dependent differences for CYP3A4 and CYP2C8 (p < 0.01). Relevance of e-cigarette interactions was supported by CYP2B6 and CYP3A4 induction on enzyme activity and protein expression levels. Nicotine content did not influence induction outcome. These product-specific differences underscore that tobacco products should be distinguished in clinical pharmacology. It is highly recommended to collect detailed tobacco product use data in clinical studies, as provided in this work in an example. This would enable targeted in vitro testing of prevalent products, population specific trial planning and improve clinical data interpretation.
Lipid‑disrupting effects of an organochlorine mixture in Sertoli TM4 cells reveal testicular obesogen mechanisms.
Virmani I, Sychrová E, Řehůřková E … +3 more , Gadara D, Spáčil Z, Sovadinová I
Arch Toxicol · 2026 May · PMID 42159748 · Publisher ↗
Male reproductive health is declining globally, reflected by reduced sperm counts, impaired semen quality, increasing infertility, and a rising incidence of testicular cancer. Endocrine‑disrupting chemicals include metab...
Male reproductive health is declining globally, reflected by reduced sperm counts, impaired semen quality, increasing infertility, and a rising incidence of testicular cancer. Endocrine‑disrupting chemicals include metabolic disruptors, within which obesogens represent a specific subgroup that dysregulate lipid metabolism and cellular energy homeostasis and are increasingly implicated in these adverse trends. Although obesogens act across multiple tissues, their lipid‑metabolism‑related effects on testicular cells remain comparatively understudied. We investigated male reprotoxicity of an environmentally and human‑relevant organochlorine mixture (OC‑MIX), consisting of 20 persistent contaminants, including polychlorinated biphenyls (PCBs), chlordane, and dichlorodiphenyltrichloroethane/dichlorodiphenyldichloroethane (DDT/DDE), originally identified in ringed seal blubber and representative of Arctic food‑chain exposures. Although OC‑MIX has shown male reproductive toxicity in animal models, its cellular mechanisms in Sertoli cells remain insufficiently defined. Using immature murine Sertoli TM4 cells, we demonstrated that OC‑MIX (5-50 µg/mL) disrupted lipid homeostasis and induced oxidative stress, culminating in lipotoxicity, processes known to compromise Sertoli cell support of spermatogenesis. These responses differed from those elicited by a fatty acid mixture or the lipotoxicant amiodarone and did not appear to involve androgen receptor, aryl hydrocarbon receptor, or peroxisome proliferator‑activated α signaling, suggesting distinct lipid‑centered mechanisms. Comparative profiling in immature murine Leydig TM3 cells revealed cell‑type‑specific responses, likely reflecting intrinsic differences in lipid composition. Notably, OC‑MIX selectively altered phospholipid and lysophospholipid species in Sertoli TM4 cells, indicating interference with membrane remodeling and lipid‑dependent signaling. Overall, these findings support the hypothesis that persistent organochlorines act as testicular obesogens, contributing to male reproductive dysfunction through selective, lipid‑disrupting mechanisms.
Development of novel 2-[(8-hydroxyquinolin-7-yl)(phenyl)methylamino]benzoic acid derivatives for inhibiting the catalytic activity of botulinum neurotoxins type B and F: in silico, in vitro & in vivo evaluation.
Agnihotri S, Kushwah VC, Dhaked RK
Arch Toxicol · 2026 May · PMID 42159747 · Publisher ↗
Botulinum neurotoxins (BoNTs) are the most pernicious toxin and category 'A' bioterrorism agent, responsible for 'botulism', a rare but fatal disease. Toxins are divided into seven toxinotypes from A-G, where A, B, E, an...
Botulinum neurotoxins (BoNTs) are the most pernicious toxin and category 'A' bioterrorism agent, responsible for 'botulism', a rare but fatal disease. Toxins are divided into seven toxinotypes from A-G, where A, B, E, and F are accountable for human botulism. The available treatment strategies are inadequate and ineffective for post-neuronal intoxication. We report novel 2-[(8-hydroxyquinolin-7-yl)(phenyl)methylamino]benzoic acid derivatives targetingthe catalytic site of VAMP-acting serotypes (BoNT/B and F). Inhibitory potential of the synthesized compounds was studied using a framework of in silico and experimental approaches. Inhibitory activity and binding affinity were evaluated using substrate-based cleavage and SPR assays. Compound efficacy was tested in mice through pre-mixed, prophylactic, and therapeutic strategies. Molecular dynamics simulations analyzed binding interactions, structural fluctuations, and complex stability. Endopeptidase assay revealed that the selected compounds displayed ≥ 80% inhibition of BoNT/B and F catalytic activity, with IC values ranging from 17.58 to 34.05 µM. SPR analysis displayed binding affinity of these molecules ranging from 8.13E-06 to 7.69E-04 for both the proteins. In the mouse bioassay, the selected molecules displayed complete protection and extension in survival of up to 20-fold. MD simulation study supported the experimental finding, revealing key interactions with HExxH and other active site residues forming stable conformation throughout the simulation time. Among them, compounds A15 and A36 were predicted to be more effective inhibitors of BoNT/B and F serotypes, respectively. These findings could lay a promising way for the development of novel therapeutics by reducing disease severity, enhancing survivability, and recovery where no post-exposure therapy presently available.
Acute cell‑death and lysosomal stress responses to nicotine and cigarette smoke extract in human mesenchymal stromal cells.
Junttila A, Heikkinen J, Kylmäoja E … +4 more , Hawash A, Åström P, Lehenkari P, Palosaari S
Arch Toxicol · 2026 May · PMID 42159746 · Publisher ↗
Mesenchymal stromal cells (MSCs) are essential for connective tissue repair, and impaired healing is well documented in tobacco users. MSCs are one plausible target for these adverse effects, but the underlying cellular...
Mesenchymal stromal cells (MSCs) are essential for connective tissue repair, and impaired healing is well documented in tobacco users. MSCs are one plausible target for these adverse effects, but the underlying cellular mechanisms of high localized nicotine exposure remain poorly understood. This study investigated how short-term nicotine and cigarette smoke extract (CSE) exposure affect human MSC function, viability, and inflammatory signaling in vitro. MSCs isolated from bone marrow were exposed to CSE containing 4-40 µM nicotine or to 100 µM-10 mM pure nicotine. CSE produced markedly stronger cytotoxicity than nicotine, reducing proliferation and rapidly inducing necrotic cell death at 20-40 µM nicotine equivalents. Pure nicotine elicited a biphasic response: concentrations below 5 mM slightly increased proliferation, while 5 mM caused apoptotic cell death with prominent lysosomal vacuolization, and 10 mM shifted cell death toward necrosis. Sublethal exposures that generated pre-apoptotic cells were associated with significant IL8 induction and MMP2 activation, whereas IL6 remained largely unchanged. Nicotine induced lysosomal disruption suggests broader impacts on MSC homeostasis beyond viability, potentially influencing lineage commitment. These findings elucidate short-term effects of nicotine and CSE; high-dose nicotine and CSE are toxic to MSCs, while the lower doses perturb the inflammatory signaling and lysosomal function. Such alterations may compromise tissue regeneration, wound healing, and periodontal stability in users of potent localized nicotine delivery products.
CpG-ODN/Coa-ASC16 nanostructured adjuvant platform as a safe and effective alternative in Bothrops diporus antivenom production.
Lopez GL, Hernández D, Costa CRC … +7 more , Toyama MH, Maslovski F, Palma S, Morón G, Leiva LC, Maletto BA, Fusco LS
Arch Toxicol · 2026 May · PMID 42159745 · Publisher ↗
In the field of vaccines, adjuvants have been optimized to enhance immune responses while reducing adverse effects. Transferring these advances to antivenom production could improve antibody quality and animal welfare. H...
In the field of vaccines, adjuvants have been optimized to enhance immune responses while reducing adverse effects. Transferring these advances to antivenom production could improve antibody quality and animal welfare. Here, we evaluated a novel adjuvant platform, CpG-ODN/Coa-ASC16, which combines immunostimulatory CpG-ODN oligodeoxynucleotides with the biodegradable nanostructure Coa-ASC16, co-formulated with a hemorrhagic antigenic model based on Bothrops diporus venom (B.dV). Immunization with B.dV/CpG-ODN/Coa-ASC16 elicited IgG titers equivalent to those induced by Freund's adjuvant, with significantly higher IgG1 levels. Antibodies generated with both formulations exhibited progressively higher avidity, reflecting effective affinity maturation. Sera from B.dV/CpG-ODN/Coa-ASC16-immunized mice recognized major venom proteins and neutralized key toxic activities-proteolytic, coagulant, and indirect hemolytic-at levels comparable to Freund's-induced sera. Functionally, sera from B.dV/CpG-ODN/Coa-ASC16-immunized mice conferred protection against a lethal venom challenge, resulting in survival of a fraction of animals and prolonged time to death in non-survivors. Importantly, histopathological analyses revealed minimal tissue alterations in the mice treated with CpG-ODN/Coa-ASC16, in sharp contrast to the severe abscesses and granulomas caused by Freund's adjuvant. Overall, this study provides new evidence that CpG-ODN/Coa-ASC16 can be effectively combined with hemorrhagic Bothrops venom to generate robust, high-affinity antibodies that efficiently neutralize the major venom toxins while markedly reducing local tissue damage. These findings position CpG-ODN/Coa-ASC16 as a safer and ethical alternative platform for antivenom production.
6PPD metabolic activation by CYP3A initiates cytotoxicity in mouse primary hepatocytes.
Sun Y, Zheng J, Zhao Y … +4 more , Li W, Hu Z, Peng Y, Zheng J
Arch Toxicol · 2026 May · PMID 42159744 · Publisher ↗
N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) is a widely used rubber antioxidant and an emerging environmental pollutant, yet its hepatotoxic potential and the underlying role of metabolic activation remain...
N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) is a widely used rubber antioxidant and an emerging environmental pollutant, yet its hepatotoxic potential and the underlying role of metabolic activation remain poorly understood. Here, we integrated metabolite profiling, liver microsomal incubations, chemical oxidation synthesis, and mouse primary hepatocytes and animal exposure to examine the metabolic and toxicological outcomes of 6PPD. A total of 54 metabolites were identified in mice, including phase I oxidation products and phase II conjugates. Importantly, the reactive metabolite p-benzoquinone diimide (6PPD-QDI) was detected and shown to form conjugates with biological thiols. In microsomal incubations containing glutathione (GSH), N-acetylcysteine (NAC), or cysteine conjugates, 6PPD produced the corresponding conjugates, supporting the generation of 6PPD-QDI. The conjugate formation was NADPH-dependent and substantially suppressed by the CYP3A inhibitor ketoconazole, indicating that CYP3A mediates the bioactivation of 6PPD. In vivo, identical GSH and NAC conjugates were detected in bile and urine of exposed mice, and hepatic proteins contained dose-dependent protein adduction at cysteine residues. In primary hepatocytes, 6PPD induced concentration- and time-dependent cytotoxicity associated with GSH depletion and protein adduction, which was mitigated by CYP3A inhibition or thiol supplementation. Notably, CYP3A4-overexpressing HepG2 cells exhibited enhanced susceptibility to cytotoxicity and protein adduction compared to wild-type cells.
Human serum-supplemented cell culture conditions improve xenobiotic metabolism in hepatoma cell lines HepG2 and Huh7.
Kroesen MD, Wagmann L, Gehl AL … +2 more , Kiemer AK, Meyer MR
Arch Toxicol · 2026 May · PMID 42159743 · Publisher ↗
Hepatoma cell lines are established in vitro models for xenobiotic metabolism, yet their enzymatic capacity is typically lower than that of primary human hepatocytes. Although widely used as standard culture supplement,...
Hepatoma cell lines are established in vitro models for xenobiotic metabolism, yet their enzymatic capacity is typically lower than that of primary human hepatocytes. Although widely used as standard culture supplement, fetal bovine serum (FBS) poses ethical concerns and may not provide the optimal nutritional environment for human-specific metabolic functions. This study compared the metabolic activity of the hepatoma cell lines HepG2 and Huh7 cultured in medium supplemented with either human serum (HS) or FBS. Metabolic performance was tested using a dual cytochrome P450 (CYP) substrate cocktail in HepG2, with results compared to literature data for Huh7. For comprehensive evaluation of metabolic capacity, both cell lines were exposed to six synthetic cathinones: 4MeO-αP-VP (4'-methoxy-α-pyrrolidinovalerophenone), 4MeO-αP-BP (4'-methoxy-α-pyrrolidinobutyrophenone), 4MeO-NE-BP (4'-methoxy-N-ethylbutyrophenone), 4MeS-αMor-PrP (4'-methylthio-2-morpholinopropiophenone), 4MeS-αP-BP (4'-methylthio-α-pyrrolidinobutyrophenone), and 4MeS-NE-BP (4'-methylthio-N-ethylbutyrophenone) under both supplement conditions. HepG2 showed unchanged CYP2D6 activity and increased CYP3A4 activity following HS incubation. HS further led to a general increase in metabolite abundance across both cell lines. Particularly the N-oxide formation consistently increased for every compound tested and abundance of most hydroxy and oxo metabolites was higher, most likely due to elevated CYP3A4 activity. Compared to FBS, metabolite formation in the HS group increased significantly, up to fourfold in 12 of 13 reactions for HepG2, and up to fivefold in 8 of 11 reactions for Huh7. Although HepG2 is a more common hepatoma cell line in metabolic research, Huh7 demonstrated higher normalized metabolite formation for most reactions, suggesting it as preferred or highly complementary model. Supplementing culture media with HS significantly enhanced the metabolic competence of the tested hepatoma cell lines. HS can be recommended as a cost-efficient, ethically sound, and physiologically relevant alternative for improving in vitro metabolism assays.
Cholera toxin as a model AB toxin: functional significance of B-subunit pentamerization in GM1 recognition, pathogenicity, and delivery efficiency.
Park YJ, Lee KS, Lim D … +1 more , Kim CU
Arch Toxicol · 2026 May · PMID 42159742 · Publisher ↗
AB toxins constitute a major family of bacterial exotoxins. Their pathogenicity depends on the coordinated actions of two components: an enzymatically active A subunit and a pentameric B subunit. The B subunit mediates h...
AB toxins constitute a major family of bacterial exotoxins. Their pathogenicity depends on the coordinated actions of two components: an enzymatically active A subunit and a pentameric B subunit. The B subunit mediates host cell recognition and intracellular trafficking. Among them, cholera toxin represents a representative model for investigating structure-function relationships governing toxin entry and delivery. While the catalytic activity of the A subunit has been extensively characterized, growing evidence indicates that pathogenic outcomes are critically shaped by trafficking strategies encoded within the B subunit pentamer. This review focuses on the cholera toxin B subunit (CTB) as a model system to elucidate how pentameric organization enables multivalent GM1 recognition, receptor clustering, and efficient intracellular transport. We summarize current knowledge on the molecular basis of CTB pentamerization, its thermodynamic and structural advantages, and the role of multivalency in amplifying delivery efficiency and pathogenicity. In addition, we examine how CTB-mediated intracellular trafficking influences uptake pathway selection and intracellular routing, thereby contributing to consistent and robust toxic outcomes. Beyond pathogenic mechanisms, we briefly discuss the potential implications of CTB-mediated trafficking for translational applications. By integrating structural, cellular, and functional perspectives, this review demonstrates how CTB-mediated mobility governs intracellular routing and uptake pathway selection. This framework provides a basis for understanding AB toxin pathogenicity and its broader biological significance.
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