Archives of Toxicology

Slaby, Sylvain; Duflot, Aurélie; Maillet, Géraldine; Couteau, Jérôme; Minier, Christophe; Allonier-Fernandes, Anne-Sophie; Pinto, Patrícia I. S.; Knigge, Thomas; Monsinjon, Tiphaine

doi: 10.1007/s00204-026-04416-wpmid: 42086802

Statistical analysis of in vitro assay data is a critical step towards a good interpretation of biological responses. However, it is still frequently undermined due to inappropriate statistical procedures, misinterpretation, insufficient statistical power—often resulting from small sample sizes—or poorly defined methodologies, in case of standardized tests. In line with practices commonly adopted in clinical studies and more recently in biomonitoring research, the use of thresholds for interpreting results may improve the robustness of conclusions. This work presents the application of a methodology for defining thresholds using a normal distribution–based approach. As a case study, these thresholds were applied to analyze data obtained from the DLES test (Dicentrarchus labrax estrogen screen test), an in vitro screening tool designed to detect interactions between chemicals and nuclear estrogen receptors in D. labrax. The results were subsequently compared with methods derived from the OECD TG 455, as well as with non-parametric statistical analyses. By applying normal distribution–based thresholds, data analysis was simplified and the reliability of the DLES test results was increased, especially when compared with hypothesis tests. Also, this was especially true when studying non-model species, for which standard reference substances are rarely available. However, special attention should be paid to the size of the initial dataset used to define the thresholds. The methodology implemented here could provide insight for other in vitro assays. Overall, this article encourages the reflection on approaches to in vitro data analysis.

Ma, Tingkun; Zhai, Pan; Ning, Shiwei; Shi, Yulong; Chen, Yusheng; Xu, Dongxue; Li, Yiming

doi: 10.1007/s00204-026-04423-xpmid: N/A

Sepsis-associated acute kidney injury (S-AKI) is a frequent and severe complication of sepsis in which metabolic dysfunction and inflammatory activation develop in parallel and reinforce each other. Although mitochondrial dysfunction is widely recognized in S-AKI, the structural mechanisms linking mitochondrial bioenergetic failure to innate immune signaling remain incompletely understood. Emerging evidence suggests that mitochondrial lipid remodeling—particularly alterations in cardiolipin (CL)—may represent a structural interface connecting these processes. As a signature phospholipid of the inner mitochondrial membrane, cardiolipin supports cristae architecture, stabilizes respiratory chain supercomplexes, and maintains efficient oxidative phosphorylation. In renal tubular epithelial cells, which depend heavily on mitochondrial oxidative metabolism, disruption of cardiolipin homeostasis may have particularly profound consequences. Under septic stress, oxidative injury and dysregulated lipid remodeling alter cardiolipin composition and distribution, contributing to respiratory chain instability, impaired ATP production, and increased reactive oxygen species generation. In parallel, cardiolipin oxidation or externalization can transform mitochondrial membranes into signaling platforms that promote inflammasome activation and mitochondrial danger-associated molecular patterns (mtDAMPs) release. In this review, we propose a cardiolipin-centered framework that integrates mitochondrial bioenergetic failure with inflammatory amplification in S-AKI. By positioning mitochondrial lipid remodeling at the intersection of metabolism and innate immunity, this framework highlights mitochondrial lipid homeostasis as a potential mechanistic node and therapeutic entry point in septic kidney injury.

Jin, Congying; Ma, Yumei; Chen, Li; Kong, Xiangbing; Zhang, Changqing; Zhang, Meihua; Ma, Wanli; Zhu, Xiaoxiao; Guo, Honghui; Jiang, Luyao; Li, Xia; Tian, Yuejie; Yu, Dianke; Jin, Yuan

Tatarczuch, Aleksandra; Kotarska, Katarzyna; Polański, Zbigniew; Ptak, Anna

doi: 10.1007/s00204-026-04439-3pmid: 42128920

Per- and polyfluoroalkyl substances (PFAS) are persistent environmental pollutants that can accumulate in the human body, including follicular fluid (FF), potentially affecting the function of reproductive cells. We used the mixture of these chemicals at concentrations previously reported in human FF to assess their effects on in vitro maturation, DNA integrity, and mitochondrial function of mouse oocytes. Exposure to PFAS led to meiotic abnormalities, manifested by an increase in the percentage of oocytes arrested at the germinal vesicle breakdown (GVBD) stage and a decrease in the number of oocytes reaching the metaphase II (MII) stage. We demonstrated that arrest at GVBD is associated with Spindle Assembly Checkpoint (SAC) activation as experimental inhibition of SAC restores normal maturation to MII. In addition, we observed increased DNA damage and changes in oxidative balance, including an increase in reduced glutathione (GSH) and a decrease in reactive oxygen species (ROS) levels. However, we found an increase in mitochondrial activity, basal oxygen consumption (OCR), and mitochondrial respiration intensity in immature GV stage oocytes, indicating disturbances in bioenergetic homeostasis. These findings show for the first time that PFAS mixtures, at concentrations relevant to human exposure, interfere with oocyte maturation, genome integrity, and mitochondrial function. The data obtained highlight the need for further research on the toxicity of PFAS mixtures, especially at doses mirroring their environmental presence, and their potential impact on female fertility.

Norman, Caitlyn; Acreman, Dean; Bissram, Meera; Curtis, Blake; Hamer, Sebastian; Westphal, Folker; Putz, Michael; Stefan, Cristiana; Delaney, Sarah R.; Lines, Rick; McLeod, Malcolm D.; McDonald, Karen; Green, Henrik

doi: 10.1007/s00204-026-04433-9pmid: N/A

Designer benzodiazepines (DBZDs) are a class of new psychoactive substances (NPS) designed as legal alternatives to prescription BZDs. Bromazolam has been the most prevalent DBZD detected on the recreational market around the world; however, a new DBZD, ethylbromazolam (8-bromo-1-ethyl-6-phenyl-4 H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepine; also known as bromoethylazolam) has recently emerged. In this study, the emergence of ethylbromazolam in Canada, the UK, and Australia is reported based on analysis of samples from drug checking services and in Germany based on analysis of samples seized by customs and mail services. Since November 2024, ethylbromazolam has been increasingly detected with a concurrent decrease in bromazolam detections, suggesting that its emergence is likely in response to the international control of bromazolam on 3rd December 2024. Additionally, increased detections of other DBZDs, including desalkylgidazepam (bromonordiazepam) and clobromazolam (phenazolam) have been recently observed. The in vitro α1β2γ2 GABAA receptor activity of ethylbromazolam was determined using an automated patch clamp assay. Ethylbromazolam was found to have similar in vitro GABAA receptor activity as bromazolam (EC50 of 10.1 nM and 15.2 nM, respectively), indicating comparable pharmacological activity and potential for harm. The market should continue to be monitored closely as it continues to evolve in response to the control of bromazolam.

Junttila, Anni; Heikkinen, Janne; Kylmäoja, Elina; Hawash, Ayman; Åström, Pirjo; Lehenkari, Petri; Palosaari, Sanna

doi: 10.1007/s00204-026-04440-wpmid: N/A

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.

Schmidt, Ariane; Schöneis, Anna; Sallbach, Jason; Pöschmann, Alexandra; Lippold, Rebekka; Rasenberger, Birgit; Hofmann, Thomas G.; Tomicic, Maja T.; Christmann, Markus

doi: 10.1007/s00204-026-04426-8pmid: 42168583

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 p53Ser15 signaling axis to the p53Ser46 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.

Wagmann, Lea; Herter, Anica; Dybek, Michael B.; Adejare, Adeboye; Wallach, Jason; Brandt, Simon D.; Meyer, Markus R.

doi: 10.1007/s00204-026-04462-4pmid: N/A

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.

Hammour, Mohammad Majd; Herzberger, Lisa; Xin, Yuxuan; Chen, Guanqiao; Tombaz, Melike; Ehnert, Sabrina; Springer, Fabian; Damm, Georg; Vosough, Massoud; Hengstler, Jan G.; Müller-Vieira, Ursula; Nüssler, Andreas K.; Aspera-Werz, Romina H.

doi: 10.1007/s00204-026-04431-xpmid: N/A

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a systemic condition associated with compromised bone integrity. Emerging evidence suggests that disturbances in hepatic vitamin D metabolism may contribute to these skeletal impairments. However, the hepatic mechanisms driving bone deterioration remain poorly defined. This study aimed to establish a human 3D in vitro model of MASLD and demonstrate that hepatic vitamin D dysregulation adversely affects bone homeostasis. Liver spheroids composed of HepaRG cells, LX-2 stellate cells, and HUVECs were stimulated with 600 µM free fatty acids (2:1 oleic: palmitic acid) to induce MASLD-like features, validated by BODIPY staining and gene expression. MASLD model induction led to downregulation of hepatic genes regulating lipid and vitamin D metabolism. ELISA confirmed significantly reduced 25-hydroxyvitamin D levels, aligning with downregulation of CYP2R1 and CYP27A1. Transcriptomic profiling of human MASLD liver biopsies validated these molecular changes. To evaluate MASLD’s systemic impact on bone, THP-1-derived macrophages and SCP-1 mesenchymal stem cells were seeded onto bone scaffolds and co-cultured with spheroids. Bone scaffolds co-cultured with MASLD spheroids showed impaired mineralization and elevated expression of bone resorption markers. These findings mirror skeletal dysfunction observed in MASLD patients and suggest a mechanistic link between hepatic vitamin D dysregulation and bone pathology. This study introduces a pioneering 3D human liver-bone co-culture model that reveals MASLD-driven disruption of hepatic vitamin D metabolism as a direct contributor to bone deterioration. This 3D model develops a powerful translational platform for decoding systemic disease mechanisms and targeting the liver-bone axis therapeutically.

Hisatsune, Kazuaki; Asano, Tomomi; Taniguchi, Masaru; Zaitsu, Kei

doi: 10.1007/s00204-026-04435-7pmid: 42178406

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.