Previously, the MetAbolomics ring-Trial for CHemical groupING (MATCHING) demonstrated high inter-laboratory reproducibility in the blinded application of rat plasma metabolomics to group eight substances (aniline, 2-chloroaniline, 4-chloro-3-nitroaniline, dichlorprop-p, fenofibrate, 17α-methyl-testosterone, trenbolone, WY-14643). Each ring-trial partner applied their preferred metabolomics workflows to acquire, process, quality assess and statistically analyse their data. All five partners whose datasets passed quality control correctly clustered the substances into three groups according to previously established toxicological effects, both for female and male rats. However, to ensure high confidence in each grouping hypothesis, metabolomics data should support a plausible toxicological interpretation by linking changes in annotated metabolite profiles to known toxicological effects associated with the substances. In this follow-up study, our first data-driven objective was to determine the consistency of group-specific discriminatory metabolites (including lipids) across partners. For all six cases (three groups in female and male rats), statistically significant consistency was observed, confirming that all partners discovered similar metabolic perturbations underpinning each group. Strikingly, this was achieved even though a range of statistical and computational approaches were used for both discovering and annotating discriminatory metabolites. Secondly, we evaluated whether pre-existing toxicological knowledge, organised using an Adverse Outcome Pathway-informed framework of metabolomic associative events (mAEs), could provide plausible toxicological interpretations of the partner's discriminatory metabolite profiles, for each toxicological effect (androgen receptor activity (AR), peroxisome proliferation (PP), anaemia). Binomial distribution analysis revealed statistically significant detection of characteristic mAEs for all six cases, albeit with insufficient specificity across the three effects. Consequently, a third objective was developed, to refine the mAEs by combining the pre-existing toxicological knowledge with partner-to-partner detection reproducibility of metabolites and their directional change, thereby significantly enhancing mAE detection specificity. Overall, the discovery by the five partners of significantly consistent metabolic perturbations underpinning each group further demonstrates the reliability of metabolomics for chemical grouping. Moreover, an integrated knowledge and data-driven approach is presented as a basis for deriving metabolomic signatures that were used to support plausible toxicological interpretations of the metabolomics data. Considering the broader implications of this study, we propose the development of a collaborative framework to facilitate community-driven contributions to a shared knowledgebase of metabolomic signatures.

Oxidative stress is a major contributor to xenobiotic-induced tissue injury, mitochondrial dysfunction, inflammation, ferroptosis, and carcinogenesis. Ursolic acid (UA), a naturally occurring pentacyclic triterpenoid, has emerged as a context-dependent regulator of redox homeostasis. This review summarizes current evidence regarding the dual effects of UA on oxidative stress and its toxicological implications. Under non-malignant conditions, UA generally exerts antioxidant and cytoprotective effects in the majority of experimental models examined. Reduced reactive oxygen species (ROS) accumulation, modulation of nuclear factor erythroid 2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) signalin, preservation of mitochondrial homeostasis, and attenuation of inflammatory and apoptotic responses appear to underlie these protective activities. Protective effects have been reported in experimental models of hepatic, renal, cardiovascular, neurological, and radiation-associated injury. By contrast, in tumor cells characterized by elevated basal oxidative stress, UA has frequently been shown to exert pro-oxidative actions, though this response is influenced by concentration, exposure duration, and cellular context. In malignant models, UA has been shown to enhance ROS accumulation, disturb redox balance, suppress antioxidant defenses, and promote cell death associated with ferroptosis and apoptosis, thereby increasing sensitivity to chemotherapy and radiotherapy. Despite promising preclinical findings, several limitations continue to restrict clinical translation, including poor oral bioavailability, variable pharmacokinetics, potential herb-drug interactions, and insufficient long-term toxicological evaluation. Overall, current evidence supports UA as a context-dependent redox modulator with dual antioxidant and pro-oxidant activities, although further mechanistic, pharmacokinetic, and translational studies remain necessary.

Chronic exposure to inorganic arsenic (iAs) has been linked to diabetes in both humans and laboratory mice. Prior studies highlight that the mechanism underlying the diabetogenic effects of iAs and the manifestation of iAs-associated diabetes depend, in part, on the exposure window. We have shown that exposure to iAs prior to conception or in utero consistently produced diabetic phenotypes in wild-type (WT) C57BL/6J mice and that this phenotype was characterized primarily by insulin resistance. The effects of postnatal iAs exposures have been less consistent. However, significance of these findings for human exposure remains unclear because WT mice metabolize and detoxify iAs more efficiently than humans. The present study describes the diabetogenic effects of iAs in humanized C57BL/6 N mice that express the human arsenic methyltransferase and exhibit human-like patterns of iAs metabolism. We examined diabetes indicators in male and female offspring of parents exposed to 0 (controls) or 200 ppb iAs (arsenite) prior to conception (PC), and offsprings exposed to iAs in utero (IU), or during the whole life, including the PC and IU windows. We found that only PC exposure produced diabetic phenotype in male offspring. This phenotype was characterized by elevated fasting blood glucose (FBG) and decreased HOMA-B, the indicator of beta cell function. However, fasting plasma insulin and HOMA-IR, the indicator of insulin resistance, were not affected. FBG was also higher in male mice after IU exposure as compared to control male mice, but this difference was not statistically significant. LL exposure had no significant effects in male offspring. No differences in diabetes markers were found between iAs-exposed and control female mice regardless of exposure window. These results suggest that PC exposure produced fasting hyperglycemia in the humanized male mice by impairing the function of insulin secreting beta cells. However, postnatal iAs exposure appeared to attenuate these effects, suggesting that activation of compensatory mechanisms may take place after birth. These findings may inform designs of population studies that examine iAs-associated diabetes, as well as the interpretation of data collected in these studies.

The credibility of scientific journals is often attributed to rigorous peer review and editorial independence. In reality, however, scientific publishing, like every human institution, is shaped by competing interests, institutional incentives, and errors of judgment. Its credibility therefore depends less on the presumed virtues of authors, reviewers, editors, and publishers than on transparent procedures, clearly defined responsibilities, and institutional arrangements that make departures from established standards visible and open to scientific scrutiny. This Editorial examines a revealing recent case in which publication of a Guest Editorial that had successfully completed the established editorial procedures of Archives of Toxicology was ultimately prevented by the Publisher. Rather than discussing the scientific merits of the underlying manuscript, the present analysis focuses on how the publication decision changed after the manuscript had already been formally accepted. The documented events show how the publication process was progressively compromised through a succession of additional procedural requirements, each introduced only after the preceding requirement had been fulfilled. As a consequence, publication no longer depended on completion of the journal's published editorial process but on successive interventions outside that process. The case illustrates that transparent editorial procedures alone are insufficient to safeguard editorial integrity. Effective institutional self-correction requires a second, equally important safeguard: the guaranteed possibility that alleged departures from editorial procedures can themselves be subjected to unobstructed public scientific scrutiny through mechanisms such as Commentaries or Opinion Articles. In the present case, both safeguards were compromised simultaneously. The established editorial procedures were progressively displaced, while the very mechanism intended to permit public examination of alleged procedural failures was itself prevented from operating. The present analysis argues that departures from editorial procedures are not corrected automatically. They become self-correcting only when independent publication pathways remain available in which transparent editorial governance is preserved. The long-term credibility of scientific publishing therefore rests not primarily on confidence in individuals but on institutional structures that make procedural departures visible, increase the expected cost of abusing institutional power, and guarantee that alleged failures of governance can themselves be subjected to public scientific scrutiny. Transparency without the guaranteed possibility of such scrutiny remains incomplete.

Heterocyclic aromatic amines (HAAs) are procarcinogens formed during the cooking of meat and fish and could potentially contribute to hepatocellular carcinoma. Although several HAAs are classified as possible or probable human carcinogens, most mechanistic studies have relied on rodent models, despite well-documented interspecies differences in HAA bioactivation by cytochrome P450 (CYP) isoforms. In this study, we investigated the regulation of CYP1A by the HAA 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) using complementary primary human hepatocytes and Hepoid® with HepaRG cells, a long-term 3D liver spheroid model. IQ at concentrations ranging from 0.1 to 20 µM strongly induced CYP1A1 and CYP1A2 expression and enzymatic activity following acute and chronic exposure, whereas other major hepatic CYP isoforms remained unaffected. This response was specific to IQ among other HAAs tested and to human hepatocytes and persisted for up to 28 days in 3D spheroids. IQ-induced CYP1A expression required transcriptional activation and involved the aryl hydrocarbon receptor (AhR). Using complementary pharmacological and genetic approaches, luciferase reporter assays and ligand-binding assays, we demonstrate that IQ is a direct AhR ligand that activates the canonical AhR-xenobiotic response element (XRE) signaling pathway in human hepatocytes. These findings indicate that IQ is a potent and selective inducer of CYP1A through the AhR and induces a sustained human-specific response upon repeated treatment. Persistent CYP1A induction and AhR activation may enhance the bioactivation of IQ and other co-occurring CYP1A substrates, highlighting the value of long-term human-relevant models to improve risk assessment of chemical carcinogens.

It is increasingly recognized that redox injury, one of the most extensively studied processes in disease pathophysiology, pharmacology, and toxicology, is rarely explained by an indiscriminate excess of free radicals alone, but rather by a combination of macromolecular injury, maladaptive redox signaling, ferroptosis, and dysregulated inflammation and metabolism. This paradigm shift has revealed several limitations of traditional antioxidants, including a focus on scavenging effects, poor target engagement, unfavorable or uncertain pharmacokinetics, failure to reach specific subcellular compartments, and lack of robust biomarkers of response. Concomitantly, greater attention is currently being paid to natural compounds combining antioxidant, anti-inflammatory, and metabolic effects. Fucoidans, structurally diverse, fucose-rich, sulfated polysaccharides derived mainly from brown seaweeds, are emerging as promising candidates because they target multiple pathways relevant to oxidative injury, including activation of antioxidant systems, preservation of mitochondrial function, attenuation of ferroptosis, and modulation of gut-organ axes that amplify oxidative tissue damage. This review critically appraises studies conducted over the last ten years, with particular emphasis on newly identified mechanisms, quality of fucoidan characterization, disease-model relevance, and translational potential. Current evidence supports an antioxidant effect of fucoidans, particularly in diseases affecting the liver, kidneys, intestines, central nervous system, and skin/wound healing. However, translation is constrained by heterogeneity across source species, extraction, purification, molecular weight, and analytical reporting. Standardized chemistry, robust pharmacokinetic characterization, and comprehensive assessments of biomarkers of response and organ biology are required to validate the role of fucoidans in mitigating oxidative stress.

Glutathione (GSH), a tripeptide composed of glutamate, cysteine, and glycine, is the most abundant intracellular low-molecular-weight thiol and serves as a central regulator of cellular redox homeostasis. In addition to its well-established roles in antioxidant defense and detoxification, GSH is essential for maintaining mitochondrial function and cellular viability. Since its discovery more than a century ago, extensive research has progressively revealed the diverse biological functions of GSH. More recently, growing evidence from experimental and clinical studies has demonstrated that disruption of GSH homeostasis is a common feature of multiple neurodegenerative diseases. Because the central nervous system consumes large amounts of oxygen, contains abundant oxidizable lipids, and possesses limited regenerative capacity, maintenance of neuronal GSH homeostasis is indispensable for protecting the brain against oxidative injury. Recent advances have further demonstrated that neuronal GSH synthesis is tightly regulated through specialized mechanisms, including astrocyte-neuron metabolic coupling and excitatory amino acid carrier 1-dependent cysteine transport. This review summarizes the historical development of GSH research, current understanding of the molecular mechanisms regulating brain GSH homeostasis, and how these mechanisms are disrupted in major neurodegenerative diseases. Finally, we discuss promising therapeutic strategies aimed at restoring endogenous neuronal GSH homeostasis and highlight the potential of brain GSH as both a biomarker and therapeutic target for oxidative stress-related neurodegenerative diseases.

Colistin and polymyxin B are last-resort antibiotics for the treatment of infections caused by multidrug-resistant Gram-negative bacteria; however, their clinical use is limited by considerable nephrotoxicity. Mitochondrial dysfunction has been proposed as one mechanism that contributes to polymyxin-induced kidney injury, but the precise mitochondrial target remains unclear. In the present study, we investigated the effects of colistin and polymyxin B on mitochondrial respiratory complexes. NMRI mice were treated subcutaneously with colistin or polymyxin B (20 mg/kg) every 8 h for a total of six times, which resulted in the accumulation of colistin and polymyxin B 29 and 40 μg/g kidney tissue, respectively. Both polymyxins increased the expression of markers of tubular damage up to 29-fold and induced mitochondrial dysfunction in the kidney. In in vitro experiments using mitochondria from intact kidneys, low concentrations of colistin inhibited Complex I-dependent respiration in the OXPHOS state, with an IC50 of approximately 5 μg/mL. This effect was Complex I-specific because at 5 μg/mL, colistin and polymyxin B did not impair respiration supported by other respiratory chain complexes or compromise mitochondrial membrane integrity. Similar Complex I inhibition by polymyxins was detected in vitro in rat kidney mitochondria. In conclusion, our findings identify mitochondrial Complex I as a sensitive target of colistin and polymyxin B and suggest that inhibition of Complex I-dependent respiration is a key driver of polymyxin-induced nephrotoxicity. This mechanism provides a toxicological framework for understanding interactions between polycationic peptide antibiotics and mitochondrial respiration and may support the selection and optimisation of safer antimicrobial peptides.

This work explores the applicability of new approach methodologies (NAMs) using a read-across approach to predict systemic toxicity with liver as the critical target organ, and derive a point of departurefor the target compound (TC) difenoconazole. Eight conazoles were used as source compounds (SCs), alongside metabolites hydroxyitraconazole and 1,2,4-triazole. The case study was structured according to the Alternative Safety Profiling Algorithm (ASPA) workflow developed within the RISK-HUNT3R project. Hierarchical clustering using chemical fingerprints identified similarity of different SCs to the TC, supporting their inclusion in the read-across analysis. A broad screening panel of NAMs, including nuclear receptor reporter assays, ToxProfiler, Cell Painting PLUS, transcriptomics in primary human hepatocytes and H295R steroidogenesis assays, was used to characterize biological activity of the TC and SCs and to substantiate the structure-based read-across. In a first prediction round, the nearest neighbour quantitative read-across approach (based on allometrically-scaled human lowest-observed-adverse-effect levels (LOAELs) of the SC) was used to predict 6.57 mg/kg/day as the LOAEL of the TC. This closely aligned with the in vivo-based value of 4.88 mg/kg/day. In a second approach, data from NAMs were used to predict human equivalent dose (HED), and the respective LOAELs of SC and TC, based on physiologically based kinetic modelling with quantitative in vitro to in vivo extrapolation. The HED derived from NAMs was approximately ten-fold lower than the LOAELs from traditional approaches. This case study illustrates suitable (ASPA-guided) approaches as well as the toxicological applicability of a NAM-based read-across assessment to obtain safe/conservative estimates of toxicity.

New approach methodologies (NAMs) are transforming chemical safety assessment by shifting focus from apical toxicity endpoints toward mechanistic, human-relevant prediction of adverse outcomes. Here, we present a human lung air-liquid interface (ALI) alveolar epithelial model coupled to an omics-to-adverse outcome pathway (AOP) analysis, linking acute sublethal exposure to potential long-term toxicity outcomes. A non-tumorigenic, monoclonal lung alveolar epithelial cell line (Arlo), capable of forming a tight monolayer barrier, was basolaterally exposed to sublethal concentrations (0, 20, 60, 100 µg/mL) of the profibrotic compound bleomycin. Dose-dependent modelling of transcriptomic signatures recapitulated known mechanisms of bleomycin-induced epithelial injury, consistent with early key events in fibrotic AOPs, enabling prediction of downstream adverse outcomes. The transcriptomic signature was overlayed on a network-based representation of human pulmonary fibrosis patient biopsies. When compared to a bleomycin exposed mouse lung model, the in vitro Arlo NAM performed comparably, especially for observing effects on extracellular matrix (ECM) restructuring in an acute exposure setting. Epigenomic profiling identified methylation changes associated with epithelial-mesenchymal transition (EMT), suggesting potential long-term gene expression regulation through epigenetic control. By linking functional endpoints with dose-response transcriptomic and epigenetic signatures, this work provides a reproducible schema for omics-to-AOP next-generation safety assessment and demonstrates that acute exposures can be modelled with a resource and time efficient NAM centred on toxicogenomics, informing on longer-term consequences.

Cholestasis is a pathological impairment of bile acid homeostasis resulting in accumulation of bile acids in the liver and systemic circulation. Cholestasis is of major concern, in particular in pharmaceutical industry, substantiating the need for human-relevant mechanistic screening tools to identify cholestatic liability early in the drug developmental pipeline. New approach methodologies (NAMs) centred around adverse outcome pathway (AOP) networks are promising tools in this respect. Leveraging human-relevant in vitro models integrated in a battery of activity-based in vitro assays allows to target specific key events along the cholestatic pathway. This approach not only enhances human relevance, but also provides deep mechanistic insights essential for human-relevant hazard assessment. The present paper focuses on the practical development of an AOP network-driven in vitro testing platform tailored for cholestatic hazard assessment. In a first part, the mechanistic foundation of cholestasis is discussed based on state-of-the-art toxicological knowledge. In a second part, a standardized workflow for NAM establishment is proposed, detailing the selection of human-relevant in vitro models, and a curated library of activity-based in vitro assays as well as reference chemicals, alongside optimized exposure parameters.

Developmental neurotoxicity (DNT) potential of (agro)chemicals is assessed in rodents according to OECD Test Guidelines (TG) 426 (DNT) or 443 (Extended One-Generation Reproductive Toxicity; EOGRTS) incorporating a DNT cohort. While the EOGRTS evaluates reproductive and neurotoxicity endpoints across multiple life stages, key differences in study design, dose selection, and exposure duration between OECD 426 and OECD 443 can complicate data interpretation and impact hazard identification and risk assessment. This manuscript evaluates current DNT testing strategies and examines the feasibility of a combined DNT-EOGRTS approach aimed at generating data for regulatory decision making. This combined approach requires substantial modifications to the EOGRTS design, and its implementation should be approached with caution. Given the increased complexity and logistical challenges, additional cognitive assessments such as learning and memory (L&M) should only be included when scientifically justified on a substance specific basis. Retrospective analyses indicate that L&M is among the least sensitive DNT endpoints, and its inclusion in an EOGRTS does not yield a functional equivalent of a standalone DNT. The review also examines regulatory triggers and recent regulatory decisions relevant to inclusion of cognitive assessments. Finally, the status and limitations of alternative approaches, including the in vitro DNT battery and zebrafish embryo models, are discussed. Overall, OECD 426 remains the scientifically preferred approach for DNT assessment. While a combined design may be feasible, it is inherently suboptimal for DNT evaluation. Collectively, this comprehensive review provides recommendations for generating robust DNT data while supporting animal welfare-conscious, future-oriented regulatory testing strategies.

Chronic, low-dose inorganic arsenic (iAs) exposure is a major environmental risk factor for lung cancer, yet the early cellular events that link low-dose exposure to carcinogenesis remain poorly understood. Because most people are exposed to iAs at low, chronic doses rather than acutely toxic levels, the metabolic events during this early window are especially relevant to human cancer risk. In this study, we investigated metabolic responses to non-cytotoxic, low-dose iAs exposure (0.5-2.0 µM) in human lung epithelial cells using short (48 h) and long (24-week) exposure models. Integrated transcriptomic and metabolomic analyses revealed that metabolic reprogramming begins within 48 h of exposure, characterized by increased glutathione synthesis, increased glycolysis, and triglyceride accumulation. The early adaptations mirrored the Warburg effect and dynamic lipid remodeling we observed in our chronic model of iAs exposure (0.5 µM). These findings indicate that iAs induces early metabolic rewiring that precedes cell transformation in human lung cells and suggest that low-dose iAs exposure may metabolically prime cells to support later oncogenic events and phenotypes. Metabolic networks underpinning the cellular response to iAs may therefore provide therapeutic targets for preventing or treating iAs-induced lung cancers.