Frontiers in toxicology最新文献

Introduction: High-throughput screening (HTS) in vitro testing can be a powerful tool for evaluating chemicals across an abundance of mechanistic, targeted assay systems. This study reviewed HTS in vitro data for the systematic evaluation of endpoints relevant to carcinogenesis. To these means, we focused on assay endpoints from the ToxCast/Tox21 HTS program that have been mapped to Key Characteristics of Carcinogens (KCCs) to help focus our review on the ∼750 assay endpoints that have been previously identified as potentially informative for evaluating modes of action relevant to carcinogenesis.

Methods: Data for ToxCast/Tox21 HTS assay endpoints were retrieved from the publicly accessible invitrodb v4.2 and reviewed for five chlorotriazine herbicides (atrazine, cyanazine, propazine, simazine, and terbuthylazine) to evaluate any indication of cancer-relevant bioactivity. More specifically, we present a workflow comprising the use of a focused assay endpoint inventory based on KCC attribution, integration of assay flags to identify robust bioactivity, and putting in vitro mechanistic insights into context with literature-based context for toxicokinetic considerations and in vivo evidence.

Results and discussion: There were common targets consistently identified as bioactive across the chemical class including induction of estrone levels and potential CAR/PXR activation. These findings were put in context by utilizing in vivo data and knowledge about atrazine to weigh the evidence. Though the ToxCast/Tox21 HTS mechanistic assays did not yield novel insights into chlorotriazine carcinogenicity, our workflow exemplifies how starting from mechanistic screening data and integrating apical endpoints can be conducted. By providing context to in vitro ToxCast/Tox21 data with toxicokinetics information and available in vivo study outcomes demonstrates how the HTS data and KCC framework can be applied to review a chemical class for carcinogenicity potential.

Introduction: Fish cell lines represent a powerful tool for studying the biology and toxicology of aquatic species in compliance with the 3Rs principles. In addition, they hold potential for more advanced biotechnological applications. However, fish cell cultures are mainly cultivated with fetal bovine serum. Therefore, in this study, we investigated the impact of serum reduction and the effects of six growth factors and cytokines on a sturgeon larval cell line (AOXla7y), which has been previously proven to be a valuable model for climate change and toxicology studies.

Methods: The serum reduction (from 10% to 5% and 2%) and the addition of two concentrations (10 and 50 ng/mL) of six growth factors and cytokines (FGF-2, IGF-1, LIF, IFN-γ, IL-13, and IL-15) to the 2% serum growth medium were evaluated over 6 days of cultivation. The morphology and cell density were determined using phasecontrast images after the experiment ended, while real-time label-free cell impedance (xCELLigence) was recorded throughout the cultivation period. Moreover, the end-point oxygen consumption in basal and uncoupled respiration conditions was analyzed with the Seahorse XF Cell Mito Stress Test Kit.

Results: The results demonstrated a general adaptation of the sturgeon cell line to a serum-reduced environment and the modulatory effects of growth factor and cytokine supplementation on cell growth and metabolism.

Discussion: These findings suggest that the sturgeon cell line has the potential to transition to a serumfree medium without major observed morphological modifications and with a limited reduction in proliferation. Its metabolism was differentially modulated by the signaling of growth factors and cytokines and exhibited a variable metabolic phenotype under mitochondrial stress. This study provides a characterization of the Atlantic sturgeon cell metabolism and offers a preliminary assessment for developing an animal-free and chemically defined medium.

Background: Drug poisoning is a growing public health concern, affecting both adult and pediatric populations. The COVID-19 pandemic has further influenced the incidence and patterns of these episodes. This study describes the clinical and epidemiological characteristics of drug poisoning episodes in adult and pediatric patients treated at a tertiary hospital in Spain between 2018 and 2024.

Materials and methods: This retrospective, observational, single-center study used data from the Spanish Minimum Basic Data Set of Hospital Discharges (CMBD-AH). All hospitalizations coded with a diagnosis of drug poisoning (ICD-10-ES: T36-T50) were included. Variables analyzed included demographics, type of admission, drug class involved, intentionality, length of stay, ICU admission and duration, and clinical outcomes.

Results: A total of 2,989 episodes with at least one drug poisoning code were identified in 2,481 patients (85.7% adults; 14.3% pediatric). The median age was 55 years in adults and 14 years in pediatric patients. Females predominated in both groups. Self-poisoning was the most frequent intentionality (52.4% in adults; 54.7% in pediatric patients), while accidental poisonings were more common in pediatric patients under 12 and adults over 60. Benzodiazepines were the most frequently involved drug-class across all age groups; in pediatric self-poisoning, paracetamol was most commonly implicated. ICU admission was required in 9.6% of pediatric and 9.2% of adult episodes. Mortality was reported in 3.3% of adult and 0.5% of pediatric episodes. Additionally, 12.5% of patients experienced recurrent episodes. A significant post-COVID increase in poisoning episodes was observed (p < 0.001).

Conclusion: Although drug poisoning represented only 1.7% of all hospital discharges, it posed a substantial burden due to its frequency, recurrence, and ICU requirements. The CMBD-AH is a valuable tool for characterizing drug-related hospitalizations across age groups. Strengthened toxicovigilance, targeted prevention strategies, and early mental health interventions are essential to reduce its impact on healthcare systems.

The widespread use of fetal bovine serum (FBS) and other animal-derived reagents in cell culture raises ethical concerns and scientific limitations, including batch variability and undefined composition. To address these challenges and promote the adoption of xeno-free, human-relevant methods, we developed a graduate-level laboratory course based on animal-free workflows. The curriculum covers key molecular and cell biology techniques: HeLa cell culture and passaging, transfection, RNA interference (RNAi), quantitative RT-PCR (qRT-PCR), dual-luciferase assays, and Western blotting, using reagents selected to exclude animal-derived components. A chemically defined medium (CDM) was optimized for robust HeLa cell growth in the absence of FBS, and recombinant TrypLE was implemented as a substitute for porcine trypsin. Validated non-animal-derived antibodies are also introduced. The course has been successfully piloted and provides a scalable, ethical framework for modern bioscience education. A detailed, open-access protocol enables replication and dissemination. This initiative equips students with practical skills and educational foundation in animal-free methodologies, supporting a shift toward reproducible and ethically responsible biomedical research.

Background: Acetaldehyde and methylglyoxal are structurally related aldehydes produced by thermal degradation of the electronic cigarette (EC) solvents, propylene glycol and glycerin. Despite their presence in EC aerosols, the biological effects of these aldehydes when inhaled during vaping are largely unknown.

Methods: Three-dimensional (3D) human bronchial epithelial tissues (EpiAirway™) were exposed at the air liquid interface (ALI) to aerosols containing acetaldehyde or methylglyoxal at concentrations relevant to human vaping. PBS-exposed tissues served as controls. Comparative proteomic analyses were performed to assess global alterations in protein expression. Based on proteomics data, concentration-response experiments were conducted using BEAS-2B bronchial epithelial cells to evaluate reactive oxygen species, mitochondrial function, and cytoskeletal integrity.

Results: ALI exposure to acetaldehyde or methylglyoxal resulted in 79 and 76 differentially expressed proteins (DEPs), respectively, with 51 overlapping proteins exhibiting similar fold change directionality. Ingenuity Pathway Analysis (IPA) Toxicity Lists identified key affected pathways, including mitochondrial dysfunction, fatty acid metabolism, G2/M DNA damage checkpoint regulation, and mitochondrial biogenesis. Gene Ontology (GO) ontology analysis revealed substantial overlap in affected biological processes and cellular components. Findings were further supported and expanded in BEAS-2B cell concentration-response assays, which confirmed mitochondrial impairment, elevated ROS levels, and disrupted cytoskeletal organization. Notably, TRPM8 inhibition attenuated methylglyoxal-induced mitochondrial dysfunction (MTT assay), while both TRPM8 and TRPA1 inhibition partially rescued actin depolymerization.

Conclusion: Brief ALI exposure of EpiAirway™ tissues to vaping-relevant concentrations of acetaldehyde or methylglyoxal altered the bronchial epithelial proteome. Complementary concentration-response experiments with submerged BEAS-2B cells confirmed and extended the proteomics data. While both aldehydes exhibited similar proteomic and functional impacts, methylglyoxal was effective at substantially lower concentrations in all assays with some effects partially mediated via TRPA1 and TRPM8 channels.

With the increasing prevalence of alcohol-related diseases, expanding our understanding of the toxic effects of excessive alcohol consumption is critical for prevention and treatment of metabolic and inflammatory pathology. This review summarizes current knowledge on the metabolic dysfunction and inflammation caused by alcohol and their impact on the pathogenesis of alcohol-related liver disease (ALD), type 2 diabetes, cardiovascular disease, and obesity, and neurological damage. It highlights recent evidence that alcohol induces a cascade of reactive oxygen species (ROS)-mediated lipid peroxidation and nicotinamide adenine dinucleotide (NAD⁺) depletion, triggering mitochondrial dysfunction and metabolic imbalances in the liver, heart, pancreas, and brain. By integrating these mechanistic insights with emerging data on how disrupted lipid and glucose metabolism amplify immune dysregulation, the review underscores the interplay between metabolic and inflammatory pathways in exacerbating tissue injury across these organs. A deep understanding of these metabolic and inflammatory disruptions is therefore essential for developing novel therapeutic strategies, including metabolic and nutritional interventions, aimed at mitigating the health risks of excessive alcohol consumption.

Macrophages, essential components of the innate immune system, are considered to be involved in the regulation of Leydig cell steroidogenesis, though by mechanisms that remain uncertain. Mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of di-(2-ethylhexyl) phthalate (DEHP), has been shown to affect testosterone production directly via its effects on Leydig cells, but also has been implicated in immune system modulation. These observations raise the possibility that MEHP might affect male steroidogenesis both by its direct effects on Leydig cells and perhaps also indirectly through its effects on macrophages. As yet, however, MEHP effects on macrophages and the potential relationship between macrophage response and Leydig cell steroidogenic function are poorly understood. Using in vitro methodology, we investigated the effects of MEHP on macrophage function and of downstream effects of changes in macrophage function on Leydig cell steroidogenesis. Mouse macrophage RAW 264.7 cells were cultured with MEHP (0-300 µM) for 24 h. Significant dose-dependent changes were seen in these cells in response to MEHP exposure, including increased cell size and granularity, increased mitochondrial content and membrane potential, decreased ATP production and oxygen consumption, and elevated intracellular and mitochondrial-derived oxidative stress. These changes suggested a pro-inflammatory response of the RAW 264.7 cells to MEHP. MEHP exposure activated the p38 MAPK pathway linking oxidative stress to inflammatory signaling and induced a dose-dependent increase in TNF-α secretion. In vitro exposure of MA-10 Leydig cells to TNF-α was found to inhibit steroid (progesterone) production by these cells. The observations, taken together, that TNF-α was secreted by MEHP-activated macrophages and that exposure to TNF-α can inhibit LH-stimulated steroid (progesterone) production by MA-10 Leydig cells suggest the possibility of the involvement of an immune-mediated mechanism resulting from MEHP exposure on impaired Leydig cell steroid production.

Per- and polyfluoroalkyl substances (PFAS) are widespread anthropogenic contaminants known to the scientific community for their persistence and toxicity. Our research aims to evaluate the effect of chronic environmental exposure to PFAS on the antioxidant system of Squalius cephalus. In particular, to better understand how various components of the antioxidant system act together to counteract the adverse effects of PFAS, in the present study we evaluate the gene expression and enzymatic activity of two selenium-dependent glutathione peroxidases (namely GPx-1 and GPx-4) and the catalase, in the two major organs involved in the accumulation and detoxification from pollutants, including PFAS, i.e. liver and caudal kidney. Fish were sampled from four sites in the Veneto region with different concentrations of total dissolved PFAS. To better assess the acclimatisation to the environment, the morphological characteristics of the fish were also examined, as well as the development of organs, through the calculation of some somatic indices. Lipid accumulation was demonstrated histologically in both the liver and caudal kidney, which likely occurs to attenuate the high reactivity of PFAS toward protein content in these organs. The results demonstrate how Squalius cephalus can survive chronic PFAS exposure through cellular and systemic physiological responses.

The increasing misuse of benzodiazepines (BZDs) in drug-facilitated crimes (DFCs) has become a serious concern for forensic experts, healthcare professionals, and legal authorities. These drugs, which are commonly prescribed for anxiety and sleep disorders, are also used to commit crimes such as sexual assault and robbery. Their sedative and memory-blocking effects render them particularly dangerous. One of the biggest challenges is that BZDs are rapidly broken down in the body, limiting the time available for detection. This creates major problems in forensic investigations and reduces the chance of holding offenders accountable for their actions. In addition, memory loss caused by BZDs often affects a victim's ability to recall events, making legal cases more challenging. This review aims to comprehensively synthesize the current knowledge on the use of BZDs in DFCs, their neuropharmacological mechanisms, and the challenges associated with their detection. It also discusses legal issues and emerging forensic tools that may help overcome the current limitations. By addressing this issue from medical, forensic, and legal perspectives, this review aims to recommend better prevention strategies, more effective investigations, and stronger legal outcomes for cases involving benzodiazepine-facilitated crimes.

Introduction: Exposure to teratogenic compounds during pregnancy can lead to significant birth defects. Given the considerable variation in drug responses across species, along with the financial and ethical challenges associated with animal testing, the development of advanced human-based in vitro assays is imperative for effectively identifying potential human teratogens. Previously, we developed a human induced pluripotent stem cells (hiPSCs)-based biomarker assay, ReproTracker, that follows the differentiation of hiPSCs into hepatocytes and cardiomyocytes. The assay combines morphological profiling with the assessment of time-dependent expression patterns of cell-specific biomarkers to detect developmental toxicity responses.

Methods: To further increase the predictability of the assay in identifying potential teratogens, we added differentiation of hiPSCs towards neural rosette-like cells. We evaluated the performance of the extended assay with a set of 51 well-known in vivo teratogens and non-teratogens, including the compounds listed in the ICH S5 reference list.

Results: The optimized assay correctly identified (neuro)developmental toxicants that were not detected in the hepatocyte and cardiomyocyte differentiation assays. These compounds selectively downregulated gene and protein expression of the neuroectodermal marker PAX6 and/or neural rosette marker NESTIN in a concentration-dependent manner and disrupted the differentiation of hiPSCs towards neural rosette-like cells. Overall, based on the current dataset, the addition of neural commitment improved the assay accuracy (from 72.55% to 86.27%) and sensitivity (from 67.50% to 87.50%), when compared to the previously described assay.

Discussion: In summary, trilineage differentiation expanded the spectrum of teratogenic agents detectable by ReproTracker, making the assay an invaluable tool for early in vitro teratogenicity screening.