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.
This study aimed to identify early carcinogenic markers to predict the hazard potential of mineral fibres using a human 3D bronchial model. Traditional 2D cell models inadequately mimic lung complexity, prompting the use of more physiologically relevant 3D systems. We investigated the effects of crocidolite (CRO) and two size fractions of chrysotile (≤ and > 5 μm length: CHR S and CHR L) to elucidate early toxicological mechanisms and relate them to the IARC key characteristics (KCs) of carcinogens. Initial analyses (MTT, TEER, and histology) at 24-48 h showed a transient acute toxicity and tissue resilience, indicating limited predictive value at this time point. However, after 12 d of exposure, fibre-treated tissues exhibited persistent alterations corresponding to established IARC KCs, suggesting the early onset of cell transformation pathways. Key findings included: sustained inflammatory signalling, characterized by prolonged overexpression of IL-1β, IL-6, and IL-8; ii) persistent genotoxicity, marked by the prolonged nuclear positivity of γH2AX, indicating DNA double-strand breaks without significant cell death; and iii) induction of fibrotic and epithelial-to-mesenchymal transition (EMT) signals, including the increased expression of key markers such as TGF-β, VEGF, ZEB-2, N-Cadherin, Vimentin, and Mesothelin. These alterations correspond to IARC KCs 2, 6, and 8. They were observed within a short time frame of tissue treatment and could serve as reliable, early predictive endpoints for in vitro toxicological tests. We propose that integrating these specific KC-based biosignatures in 3D lung models with physicochemical fibre characterization may provide a robust framework for predictive assessment of inhalable fibre carcinogenicity.
Heritable mutations in male germ cells pose a critical risk to human health and future generations, however standardized methods for assessing germ cell genotoxicity remain limited. We refined the in vivo alkaline comet assay (proof-of-concept (Dirven et al. 2023); protocol (Olsen et al. 2024)) to detect DNA damage in testicular germ cells, with selective addressment of haploid spermatids and primary spermatocytes. Measurements of DNA damage (% Tail DNA) and DNA content (total fluorescence intensity) in individual comets were combined with visual comet identification to distinguish testicular comet populations based on differences in DNA content and appearance. To verify the method's functionality and reliability, DNA damage was assessed in rats exposed to the direct-acting, well-characterized genotoxicants X-rays and ethyl methanesulfonate across distinct testicular cell populations, alongside liver and blood. To minimize experimental variation, the protocol included stringent standardization of animal handling, tissue processing, and comet assay procedures. Both X-rays and EMS induced significant DNA damage in testicular germ cells, with comparable responses across testicular cell types and similar (X-rays) or higher levels observed in somatic tissues. The low inter-animal variability observed supports the robustness of the method. Importantly, inclusion of testicular germ cells in OECD test guideline 489 would provide a valuable tool for hazard identification and mutagenicity classification of chemicals under the Globally Harmonized System of Classification and Labelling of Chemicals. This versatile, sensitive, and resource-efficient assay enhances the assessment of male-mediated genetic risks and supports regulatory efforts to protect reproductive health and safeguard the genetic integrity of future generations through the use of safer chemicals.
2,5-Dimethylfuran (DMF) is a heat-induced contaminant found in various thermally processed foods. Due to its structural similarity to furan, a well-known hepatotoxin and potential human carcinogen, the presence of DMF in food poses a potential risk to consumers. DMF undergoes cytochrome P450 (CYP)-mediated biotransformation leading to the formation of two primary phase-I metabolites: the reactive cis-3-hexene-2,5-dione (HDO), which can react with cellular nucleophiles, and the primary alcohol 5-methylfurfuryl alcohol (MFA), formed after hydroxylation of the alkyl moiety. To deduce the in vitro formation kinetics of these two phase-I metabolites, we utilised a newly developed HPLC-ESI-MS/MS method to quantify HDO after scavenging with glutathione and monitored the formation of MFA by GC-MS in parallel. Metabolic activation and the formation of HDO was the predominant biotransformation pathway in human liver microsomes, whereas the parallel formation of MFA was significantly less efficient. In line with data on the metabolic activation of furan, CYP2E1 was the most active human CYP-isoform in the potential metabolic activation of DMF. However, CYP3A4 and CYP2D6 also contributed to the HDO formation in vitro. Hydroxylation of DMF and formation of MFA were exclusively catalysed by CYP2E1. Despite the formation of MFA as an alternative metabolic pathway, our kinetic data indicate that DMF is primarily metabolised by CYP2E1 to the reactive cis-enedial intermediate HDO, particularly at physiologically relevant concentrations. Therefore, exposure to DMF may contribute to the overall risk associated with dietary exposure to furans.
The traditional linear no-threshold model of toxicity and stress fails to capture the complex, adaptive nature of biological systems. Hormesis-a biphasic dose-response phenomenon where low-dose stress induces beneficial, adaptive responses while high doses cause harm-offers a more comprehensive paradigm. This article asserts that hormesis is a major, unifying explanatory principle of life that efficiently integrates evolutionary theory, genetics, and epigenetics. From an evolutionary perspective, hormesis is an energy-efficient strategy of adaptive homeostasis, permitting organisms to build biological capital during mild hardships/stresses to survive subsequent acute or chronic environmentally and/or age-related life-threatening challenges. Mechanistically, this survival strategy is hardwired into the genome via highly conserved stress-response networks (e.g., Nrf2, FOXO, Sirtuins) that upregulate cytoprotective pathways upon the detection of mild stressors. Furthermore, epigenetics provides the temporal bridge, recording these stress events via chromatin remodeling to create a lasting 'memory' of resilience, which may even be transmitted transgenerationally to prime/adapt offspring for future adversity. Ultimately, synthesizing these three biological domains through the lens of hormesis redefines biomedically based understandings of health, aging, and disease, demonstrating that biological resilience is actively maintained through manageable environmental and age-related challenges rather than the absence of stress.
Amphotericin B (AmB) is a polyene antifungal that, despite its severe dose-limiting toxicity, is the most effective drug to treating invasive fungal infections. The drug's toxicity arises from its poor selectivity for ergosterol over cholesterol and its tendency to aggregate. Understanding the relationship between toxicity and aggregation is challenging due to the drug's complex mechanism of action, yet imperative to improve the safety profiles of existing and new polyene-based antifungal drugs. In this review, we critically examine and summarise studies that investigate the aggregation of AmB and its relationship to toxicity. As the aggregation behaviour of AmB is highly sensitive to environmental conditions, we provide an in-depth analysis of sample preparation, characterisation of aggregates and control experiments. While data from haemolysis, animal, and cell-based studies consistently show that oligomeric AmB is more toxic than larger aggregates, findings on the toxicity of monomeric AmB inconsistent. Our collated data underscores the need for careful consideration of sample preparation and proper use of vehicle controls when studying the complex relationship between the supramolecular organisation of AmB and its biological effects.
N-nitrosamine (NA) impurities in pharmaceuticals represent "cohort of concern" compounds under ICH M7(R2), due to their mutagenic/carcinogenic potential, involving cytochrome P450 (CYP)-mediated metabolic activation. Increasing interest in mammalian cell-based genotoxicity/mutagenicity assays prompted our assessment of the in vitro alkaline comet assay regarding its predictive power for NAs. Here, precision-cut liver slices (PCLiS), primary human hepatocytes (PHH), primary rat hepatocytes (PRH), and HepG2 cells with rat or hamster S9-mix were investigated as in vitro model systems. Metabolic competence was characterized beforehand. For performance evaluation, a panel of known-mutagenic [N-nitroso-dimethylamine (NDMA), N-nitroso-diethanolamine, N-nitroso-methylaniline, S-N-nitroso-nornicotine, N-methyl-N-nitroso-2-propanamine] and reported non-mutagenic (methyl-t-butylnitrosamine, N-nitrosoproline) was tested, together with Nitrosamine Drug Substance-Related Impurities [N-nitrosodesloratadine, N-nitrosofolic acid, N-nitrosofluoxetine (NFluo)] at a concentration range of 0.005-10 mM. After 2 h (PCLiS, PHH and PRH) or 4 h (HepG2), NDMA concentration-dependently induced DNA strand breaks in all in vitro models. Sensitivity/specificity of the various liver cell models for prediction of carcinogenic NAs were 100%/50% (HepG2 with hamster S9-mix), 50%/100% (PHH, PRH), and 50%/50% (HepG2 with rat S9-mix), respectively. Benchmark dose modeling indicated a higher relative in vitro comet assay response for NFluo compared to NDMA in all cell systems. In conclusion, the in vitro comet assay represents a sensitive and/or specific tool for complementing regulatory in vitro tests in prediction of mutagenic NAs. However, further optimization work is needed, using expanded training sets of compounds and thorough validation of liver cell models, before the in vitro comet assay could be incorporated in the standard battery for genotoxicity testing.
Cocaine remains one of the most widely consumed illicit drugs globally, representing a significant public health challenge. While its acute reinforcing effects are mediated by the facilitation of dopaminergic and serotonergic neurotransmission, chronic exposure leads to pervasive neurobiological adaptations and systemic toxicity. Beyond its psychoactive properties, cocaine exerts multifaceted cytotoxic effects across several organ systems, including the brain, heart, and liver, primarily through the induction of oxidative stress, mitochondrial dysfunction, and the activation of apoptotic pathways. This review provides a comprehensive analysis of these cellular and molecular mechanisms and introduces novel evidence regarding the toxicological impact of seized cocaine. Original in vitro data demonstrate that the association of cocaine with common adulterants, levamisole, phenacetin, and caffeine, markedly exacerbates cytotoxicity through synergistic interactions. Furthermore, this review examines the pivotal role of the sigma-1 receptor (σ1R) in cocaine-induced toxicity, supported by molecular docking analyses that characterize specific interactions between the cocaine, adulterants, and conserved receptor residues. This receptor-mediated framework suggests a central mechanism contributing to the drug's combined toxic and reinforcing properties. Collectively, these findings integrate experimental, computational, and literature-based evidence to offer a broader mechanistic understanding of cocaine-induced toxicity and its modulation by adulterants, providing compelling evidence for the role of σ1R in these cytotoxic processes.
Developmental immunotoxicology (DIT) is emerging as a critical area in regulatory toxicology, driven by the recognition that the developing immune system is particularly vulnerable to xenobiotic exposure. Disruptions occurring during fetal or early postnatal life may result in long-lasting alterations in immune competence, tolerance, and disease susceptibility. This review provides a comprehensive overview of immune system development, highlighting key developmental stages from embryogenesis to postnatal maturation and identifying windows of heightened immune system sensitivity to toxicants. By integrating mechanistic insights and methodological advances, this review aims to support the improvement and extension of DIT testing frameworks and the development of predictive tools for regulatory and research applications. Recent advances in New Approach Methodologies offer promising alternatives for modeling human immune ontogeny, while highlighting the challenge of ensuring adequate coverage of critical developmental mechanisms and windows of susceptibility relevant to immunotoxicity. The integration of physiological maps and multi-omics technologies enhances mechanistic understanding, while epidemiological associations between exposures and functional endpoints underscore the real-world relevance of DIT and can identify biomarkers to guide the further development of relevant and sensitive models. Despite these advances, challenges remain, including the scarcity of human reference data, the lack of standardized protocols, and the need for validated test batteries covering diverse mechanisms once the tests have been refined. Addressing these gaps is essential to support the regulatory uptake of DIT data and to advance predictive, mechanistically anchored, and ethically sound strategies for DIT testing.
Carbamazepine is a commonly applied anti-neurosis drug, which meanwhile severely pollutes the global environment. It induced DNA/chromosome damage and gene mutations in vitro following metabolic activation, however, evidence for its genotoxicity in intact mammalians remains absent. In this study, 6-week old male C57BL/6J mice were exposed to carbamazepine by gastric gavage at doses of 8, 20, and 50 mg/kg/d for 7 consecutive days, followed by a micronucleus test in bone marrow polychromatic erythrocytes, a comet assay in hepatocytes, and Western blot assay of hepatic proteins, including a phosphorylated histone 2AX (γ-H2AX, indicator of double-strand DNA breaks), aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR), constitutive androstane receptor (CAR), and several Cyp enzymes. In some experiments, ticlopidine (4 mg/kg/d, specific inhibitor of Cyp2b) was used as a modulator. The results demonstrated no obvious toxicity of carbamazepine to the liver or bone marrow, while it induced micronucleus formation in the bone marrow and hepatic DNA damage (indicated by positive comet assay results and elevated γ-H2AX protein) at 20 and/or 50 mg/kg/d doses, all of which were nevertheless abolished or alleviated by ticlopidine. Meanwhile, carbamazepine induced hepatic AhR and CAR at 50 mg/kg/d, and PXR, Cyp2b10 and Cyp3a11 at doses ≥ 20 mg/kg/d, with no effect on Cyp1a2; furthermore, coexposure of ticlopidine blocked the induction of PXR, Cyp3a11 and Cyp2b10 by carbamazepine, while enhanced that of AhR. This study provides evidence for the genotoxicity of carbamazepine in mammalians in vivo, its dependence on Cyp2b activity, and the activation of relevant nuclear receptor/Cyp-regulating pathway.
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 IC50 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.
Primary human bronchial epithelial cells (pHBECs) of the airways of smokers are chronically exposed to cigarette smoke, which may induce chronic obstructive pulmonary disease (COPD) ranked fourth among the most common global causes of death. Using an established protocol for differentiation of pHBECs to a pseudostratified epithelium at an air liquid interface (ALI), we analyzed functional expression of transient receptor potential vanilloid 4 (TRPV4) proteins after application of cigarette smoke extract (CSE), which upregulated seven smoke exposure regulated genes (SERGs). TRPV4 protein expression in the plasma membrane and localization next to the cilia of ciliated cells was reduced, while cell barrier function was not altered after chronic exposure to CSE for 28 days compared to untreated control cells. Accordingly, TRPV4-mediated Ca2+ influx was blocked in pHBECs after CSE exposure. Moreover, Os-9 protein, which after binding mediates protection from degradation of TRPV4 protein by polyubiquitination, was significantly less expressed in pHBECs upon CSE exposure. Most interestingly, overexpression of OS-9 in pHBECs rescued reduced TRPV4 protein levels induced by CSE. Our study identifies a novel molecular mechanism of toxicity by CSE interfering with TRPV4 and OS-9 expression in pHBECs, which may blaze the trail for new therapeutic options in COPD.
Tattooing is an invasive procedure that has gained popularity, particularly among individuals. Growing attention has been directed toward the safety of tattoo ink ingredients, which are complex mixtures of synthesis precursors and byproducts. These inks may contain classified carcinogens, heavy metals, polycyclic aromatic hydrocarbons (PAHs), and phthalates, often at concentrations exceeding permissible limits. Tattooing involves the insertion of needles 1-3 mm into the dermis. While part of the pigment is eliminated with epidermal turnover, approximately 25% is transported to lymph nodes and subsequently into the bloodstream. The remaining pigment is engulfed by phagocytic cells involved in innate immunity. Repeated needle punctures trigger local inflammation, recruitment of immune cells, and release of inflammatory mediators, followed by tissue regeneration and remodelling driven by keratinocytes, fibroblasts, and endothelial cells. Cases of malignant melanoma, squamous cell carcinoma, keratoacanthoma, and lymphoma have been reported in individuals with tattoos. However, the relationship between tattooing and cancer is not fully understood. It is suspected that the cause may be the transfer of toxic substances to lymph nodes and then to organs. The possible carcinogenic effects of tattoo ink ingredients will be discussed based on available scientific reports.
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.
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.
N-ethylpentedrone (NEP) is a New Psychoactive Substance (NPS) of the cathinone class that has raised public health concerns, scheduled by both the United Nations Office on Drugs and Crime and the European Union Drugs Agency, since 2024. We report here a fatal case involving a 29-years-old man who used NEP in a chemsex context. Three plastic bags containing off-white to beige powders and crystals were found near the body. Proton and carbon nuclear magnetic resonance spectrometry (1H and 13C NMR) and ultra-high performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) analyses confirmed all three bags contained NEP with purity above 98%. UHPLC-HRMS data analyses using molecular networking and in silico prediction allowed to propose NEP metabolic profile in peripheral and cardiac blood, bile, gastric fluid, urine and hair. We described here six Phase I metabolites, and we proposed NEP (C13H19NO) as the principal biomarker in NEP intake, metabolites M4 (C11H17NO) and M1 (C11H15NO) serving as biomarkers of consumption, as all of which were detected in all postmortem body fluid samples as well as in hair. Predominant metabolic pathways involved keto-reduction, N-dealkylation, hydroxylation, and oxidation, while no Phase II metabolites were detected under the applied analytical conditions. NEP was quantified at 18 mg/L and 20 mg/L in peripheral and cardiac blood, respectively, and 654-755 pg/mg in hair. These exceptionally high concentrations in biological fluids indicate an acute NEP intoxication, while hair analyses confirm repeated exposure over several months, consistent with chronic use. In summary, the identification of these metabolites in various postmortem body fluid matrices and in hair will improve our understanding of potential drug consumption markers and contribute to better monitoring and detection of N-ethylpentedrone use and abuse.
Preclinical toxicology study reports contain the expert interpretations required to distinguish test article-related effects from incidental findings, yet these conclusions often remain embedded in unstructured text form that limit systematic reuse and integration with computational safety approaches. To address this gap, we developed a Large Language Model (LLM) - supported pipeline that converts toxicology reports into structured, machine-readable datasets harmonized with SEND terminology. The pipeline combines automated document preprocessing, section identification, schema-constrained information extraction, and semantic harmonization, complemented by targeted human curation. We evaluated the system performance using 200 Roche toxicology study reports, encompassing clinical pathology, histopathology, organ weights, exposure data, and study-level conclusions. Across domains, extraction performance was strong, characterized by consistently high sensitivity and precision for most parameters. Histopathology, organ weight, and NOAEL-related endpoints demonstrated the greatest robustness, with sensitivity typically above 95% and precision frequently exceeding 97%. Lower performance for parameters such as route of administration and substance identifiers reflected heterogeneous reporting practices rather than LLM-based method limitations. The structured datasets generated by this pipeline enable cross-study querying, identification of compounds with defined toxicological liabilities, integration with raw SEND data, and development of high-quality labels for predictive toxicology models. Practical utility has been demonstrated through representative use cases. These results demonstrate that LLM-assisted extraction can reliably capture expert toxicological interpretations at scale and provide a foundation for data-centric safety assessment, strategic decisions, reverse and forward translational toxicology research.
Diazinon (DZN) is a widely used organophosphorus insecticide whose toxicity has traditionally been attributed to acetylcholinesterase inhibition after metabolic activation to diazoxon. However, a growing body of rodent evidence indicates that this mechanism alone does not adequately explain the breadth and persistence of DZN-induced organ injury, particularly under subacute and subchronic exposure conditions. This narrative review synthesizes current evidence on the toxicokinetic, mechanistic and organ-specific basis of DZN-induced oxidative stress in rodent models, with emphasis on biomarker behaviour and the protective effects of antioxidant and pharmacological interventions. Relevant studies were identified from PubMed/MEDLINE, Scopus and Web of Science up to December 2025, with inclusion limited to in vivo rodent and rodent-derived ex vivo studies that assessed oxidative or nitrosative stress endpoints alongside organ injury or functional outcomes. Across the reviewed literature, DZN consistently increased reactive oxygen and nitrogen species, lipid peroxidation and protein oxidation, while depleting glutathione and disrupting antioxidant defenses such as superoxide dismutase, catalase and glutathione peroxidase. These redox disturbances were closely associated with inflammatory activation, mitochondrial dysfunction, and oxidative DNA damage and tissue injury in the liver, kidney, cardiovascular system, brain, reproductive organs, pancreas, adipose tissue and blood. Time-course and correlation data further indicate that oxidative stress develops rapidly, may precede or coincide with peak cholinesterase inhibition, and often persists beyond the overt cholinergic phase, supporting a partially dissociated but mechanistically intertwined oxidative axis. Intervention studies show that antioxidants, phytochemicals and selected pharmacological agents can attenuate DZN-induced redox imbalance and ameliorate biochemical and histopathological damage, although protection is typically incomplete. Overall, the evidence positions oxidative stress as a central cross-organ mechanism of DZN toxicity rather than a secondary by-product of cholinergic injury. A more standardized and translationally integrated research framework is now needed to refine biomarker selection, improve hazard characterisation and guide the development of adjunctive strategies for organophosphate-related toxicity.
The hepatic capacity for xenobiotic detoxification exhibits pronounced circadian rhythmicity. The molecular basis of this rhythm lies in the autonomous transcriptional-translational feedback loops constituted by clock-controlled genes (CCGs). These loops temporally regulate the expression of a vast array of drug-metabolizing enzymes (DMEs) and transporters, thereby determining the liver's detoxification efficacy at different times of day. Dysfunction of CCGs, such as reverse erythroblastosis virus α (Rev-erbα), brain and muscle ARNT-like 1 (Bmal1), circadian locomotor output cycles kaput (Clock), and period (Per), directly disrupts this metabolic timing and alters the liver's susceptibility to specific toxicants. Therefore, this review elaborates on the circadian characteristics of hepatic detoxification function, analyzes the complex network through which CCGs regulate hepatotoxicity, and further explores how external zeitgebers (e.g., light, food) and the timing of drug administration influence the outcomes of liver injury by either synchronizing or disrupting endogenous rhythms. These findings provide a theoretical basis for applying chronopharmacological principles to prevent and mitigate drug-induced liver injury through time-based intervention strategies, such as optimizing drug administration timing and managing lifestyle factors.
Polyhexamethylene guanidine phosphate (PHMG-p), a cationic disinfectant previously used in humidifiers, has been linked to severe pulmonary diseases in Korea. This study aimed to elucidate the molecular mechanisms underlying PHMG-p-induced lung toxicity using an integrated multi-omics approach. BALB/c mice were intratracheally instilled with PHMG-p (0, 0.03, 0.1 mg/kg, twice weekly for 4 weeks). Histopathology revealed dose-dependent pulmonary lesions, including inflammatory infiltration, alveolar wall hyperplasia, and fibrosis. Transcriptomic profiling identified 213 and 1,506 differentially expressed genes (DEGs) in the low- and high-dose groups, respectively, with enriched pathways related to immune activation, cytokine signaling, and cellular stress responses. Proteomic analysis detected 148 and 1,168 differentially expressed proteins (DEPs), many of which overlapped with DEGs and were associated with chemokine signaling, protein refolding, and ion transport dysregulation. Metabolomic profiling of serum samples identified dose-responsive alterations in amino acid and energy metabolism, with notable increases in glutamate, leucine, serine, and related metabolites. Integrated omics analysis revealed consistent up-regulation of CDKN1A, HSP90AA1, HSPA1A, HSPA8, and HSPH1, and down-regulation of FPR1, suggesting their roles as potential biomarkers of PHMG-p-induced pulmonary injury. Pathway convergence indicated activation of inflammatory and fibrotic remodeling processes, as well as metabolic reprogramming involving glutamate and branched-chain amino acid pathways. These findings provide mechanistic insight into PHMG-p-induced lung toxicity and highlight multi-omics signatures that may serve as biomarkers for monitoring or predicting pulmonary damage caused by cationic polymer biocides.