Toxicological Research最新文献

Oxidative stress plays an essential role in homeostasis, cell signaling, and host defense mechanisms. However, excessive levels are harmful and cause DNA damage, lipid peroxidation, and mitochondrial dysfunction, ultimately causing cell death. Oxiapoptophagy, a cell death mechanism driven by excessive reactive oxygen species (ROS), involves both apoptosis and autophagy. This study investigated the mechanisms underlying bisphenol AF (BPAF)-induced cell death in mouse GC-1 spermatogonia (spg), using 7-ketocholesterol (7KC) as a reference oxiapoptophagy inducer. Both 7KC and BPAF inhibited GC-1 spg proliferation with comparable half-maximal inhibitory concentration (IC₅₀): 16.9 µM for 7KC and 16.5 µM for BPAF. However, BPAF induced significantly higher ROS levels than 7KC. At 20 µM, BPAF predominantly triggered apoptosis, whereas 7KC mainly promoted autophagy. BPAF evidently increased cleaved Beclin-1 levels, suggesting a transition from autophagy to apoptosis and implicating Beclin-1 cleavage as key modulator of apoptosis. Furthermore, the ROS scavenger N-acetyl cysteine (NAC) reduced BPAF-induced ROS production, suppressed Beclin-1 cleavage, and partially restored GC-1 spg proliferation. Collectively, these findings demonstrate that BPAF-induced spermatogonia toxicity is mediated by ROS and regulated through Beclin-1 cleavage, underscoring the need for further investigation of BPAF's reproductive toxicity and the development of strategies to protect male reproductive health.

Black carbon has attracted significant attention because of its severe health hazards. Carbon black (CB), a commercially available standardized particulate material, is widely used as a surrogate model in toxicological studies. The voltage-gated proton channel Hv1, encoded by the Hvcn1 gene, is specifically expressed in lung epithelial cells and regulates the generation of reactive oxygen species. However, the role of Hv1 in lung homeostasis remains unclear. In this study, we constructed an Hv1 knockout (KO) mouse model via CRISPR/Cas9 technology to investigate the impact of channel deficiency on lung injury induced by exposure to CB particles. Our findings revealed that Hv1 deficiency significantly exacerbated lung injury caused by CB particles compared with that in wild-type (WT) mice. Specifically, Hv1 knockout mice presented significantly elevated levels of inflammatory and cytokine factors in bronchoalveolar lavage fluid (BALF). Further analysis demonstrated that Hv1 deficiency led to increased malondialdehyde content and decreased superoxide dismutase activity in the BALF of mice exposed to CB particles, indicating increased oxidative stress. Histopathological staining and immunohistochemical experiments confirmed that the absence of the proton channel resulted in thickened alveolar walls, exacerbated inflammatory cell infiltration, and increased fibrous protein deposition in lung tissues. Further immunohistochemical analysis revealed that, compared with WT mice, Hv1 KO mice presented significantly decreased E-cadherin expression and increased vimentin and α-SMA expression in lung tissue after CB particle exposure. Furthermore, exposure to CB particles significantly elevated transforming growth factor-beta 1 levels in the BALF of Hv1 KO mice relative to WT controls. Collectively, these findings demonstrate that Hv1 deficiency potentiates particulate matter-induced lung injury by exacerbating pulmonary inflammation, oxidative stress, and epithelial‒mesenchymal transition. This study establishes Hv1 as a critical protective factor against particulate matter-induced lung damage and highlights its potential as a therapeutic target for preventing and treating particulate matter-associated pulmonary disorders.

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Supplementary information: The online version contains supplementary material available at 10.1007/s43188-025-00319-7.

β-Alanine is a non-essential β-amino acid used as a dietary supplement for improvement of exercise performance. The hepatic influence of β-alanine has been controversial in previous studies and its effects on nonalcoholic fatty liver disease (NAFLD) are uncertain. In the present study, we examined the role of β-alanine on diet-induced NAFLD in mice and determined the hepatic S-amino acid (SAA) metabolism to identify the cellular mechanisms. Male C57BL/6 mice were provided with a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) for 2 weeks to induce NAFLD, and simultaneously supplemented with β-alanine (3%, w/v) in drinking water. CDAHFD-induced liver injury, steatosis, and oxidative stress as shown by the increased serum alanine aminotransferase and aspartate aminotransferase activities, and hepatic triglyceride and lipid peroxide levels, respectively, but β-alanine alleviated these changes. The CDAHFD promoted significant changes in SAA metabolites, including reduced S-adenosylmethionine (SAM) and hypotaurine levels, and elevated homocysteine and taurine levels in the liver. β-Alanine attenuated the decrement of the SAM induced by the CDAHFD via the restoration of methionine adenosyltransferase activity, which appeared to ameliorate fat accumulation by stimulating hepatic lipid export via very-low-density lipoprotein secretion. Moreover, the induction of fatty acid β-oxidation, as shown by the elevations of peroxisome proliferator-activated receptor-gamma coactivator 1-α, carnitine palmitoyltransferase 1A, and acyl-CoA dehydrogenase medium chain proteins, may contribute to the anti-steatogenic effect of β-alanine. Normalizations of hepatic homocysteine and hypotaurine levels due to the restorations of cystathionine β-synthase and cysteine sulfinic acid decarboxylase, respectively, along with increased glutathione levels may be the mechanisms of inhibition of CDAHFD-induced oxidative stress by β-alanine. These results suggest that β-alanine can improve NAFLD via its antioxidant and anti-steatotic effects by restoring hepatic SAA metabolism.

Flame retardants are integral components in numerous consumer and industrial products. Accumulating research has shown that retardants disrupt the endocrine system via the modulation of thyroid hormone receptors (THRs). To investigate the mechanisms underlying this effect, we established a luciferase reporter assay system using HEK293 cells expressing the human THR isomers THRα and THRβ, and screened six flame-retardant compounds with agonistic or antagonistic activity. We examined THR agonism or antagonism in these compounds, which included organophosphate (tris(3-chloropropyl) phosphate), bisphenol-type (tetrabromobisphenol A), and brominated compounds (decabromodiphenyl ethane [DBDPEthane], decabromodiphenyl ether [DBDPEther], 1,2-bis(2,4,6-tribromophenoxy) ethane, and DC604). Among these, DBDPEthane, a widely used flame retardant, has emerged as a potential endocrine-disrupting chemical. The structurally related compounds DBDPEther and DBDPEthane were found to exert antagonistic effects on both THRα and THRβ. To elucidate the molecular basis of this antagonism, molecular docking analysis was performed using the ligand-binding domains of THRα and THRβ. The results indicated binding of DBDPEthane within ligand-binding pockets of both THRα and THRβ, forming specific hydrogen bonds and hydrophobic interactions that may support its antagonistic effects. To further characterize the dynamic interactions between DBDPEthane and THRα or THRβ, we conducted molecular dynamics simulations, using the root mean square deviation (RMSD), root mean square fluctuation (RMSF), and solvent-accessible surface area (SASA) as metrics. The results revealed distinct binding stability and conformational flexibility between DBDPEthane and THRβ, supported by RMSD, RMSF and SASA. These findings highlight the potential of DBDPEthane to antagonize both THRα and THRβ, providing functional and structural insights into its thyroid-disrupting properties in the context of receptor subtype selectivity.

Supplementary information: The online version contains supplementary material available at 10.1007/s43188-025-00316-w.

Pesticides, biocides, and disinfectants used in veterinary medicine are frequently applied in spray form, necessitating inhalation toxicity testing. This study aimed to cultivate cells in an air-liquid interface (ALI) system for in vitro inhalation toxicity screening as an alternative to animal testing and to evaluate the effects of benzalkonium chloride (BKC) exposure on cells. A549 and Calu-3 cells were cultured under ALI conditions for three weeks, with transepithelial electrical resistance (TEER) and hematoxylin and eosin (H&E) staining confirming cell proliferation, integrity, and multilayered epithelial formation. The cells were exposed to aerosolized BKC (2-16.5 mg/mL) using the VITROCELL® cloud system. Toxicity was assessed through WST-1, lactate dehydrogenase (LDH), and reactive oxygen species (ROS) assays, revealing concentration-dependent cytotoxicity. IC₅₀ values ranged from 4.5 to 19 mg/mL in A549 cells and 3.6 to 6.7 mg/mL in Calu-3 cells, indicating the latter's higher sensitivity to BKC exposure. LDH and ROS assays further demonstrated significant cell damage following exposure, supporting the use of Calu-3 cells for detecting toxicity indicators in inhalation studies. The results highlight the importance of minimizing aerosol exposure to protect respiratory health and demonstrate the potential of ALI systems for reliable inhalation toxicity assessments. However, additional studies are needed to evaluate the reproducibility of the cell models and incorporate more comprehensive toxicity mechanism indicators to enhance accuracy and reliability in inhalation toxicity evaluation.

Mercuric chloride (HgCl₂), a common environmental neurotoxin, induces neuronal injury through incompletely characterized mechanisms. Recent findings suggest a regulatory role for microRNAs (miRNAs) in mercury-induced neurotoxicity, with miR-143-3p significantly enriched in the brain and implicated in neuronal viability. This study investigated the functional role and underlying mechanisms of miR-143-3p in HgCl₂-induced neurotoxicity using PC12 cells as a model system. Cells were treated with HgCl₂ for 48 h, followed by evaluation of cell viability and apoptosis via MTT assay and flow cytometry, respectively. Neuronal morphology was assessed using inverted phase-contrast microscopy, while reactive oxygen species (ROS) levels were quantified using DCFH-DA staining. The expression levels of miR-143-3p and its downstream targets were determined by RT-qPCR, and protein expression was analyzed through western blotting. A luciferase reporter assay was employed to confirm the interaction between miR-143-3p and LMO4. Results revealed that silencing miR-143-3p alleviated HgCl₂-induced neurotoxicity in PC12 cells. Mechanistically, miR-143-3p was found to directly bind the 3' untranslated region (3'UTR) of LMO4. Overexpression of LMO4 conferred protection against HgCl₂-induced neuronal damage. Further analysis showed that miR-143-3p suppresses the Akt/GSK3β/mTOR signaling cascade by targeting LMO4. Either silencing LMO4 or pharmacologically inhibiting Akt diminished the neuroprotective effects observed upon miR-143-3p knockdown. These findings suggest that miR-143-3p exacerbates HgCl₂-induced neurotoxicity eby downregulating LMO4 and suppressing the Akt/GSK3β/mTOR pathway.

Puberty is a critical period for skeletal growth. Although previous studies have demonstrated detrimental effects of caffeine on the skeletal system, most have used male rats to avoid the individual variability associated with rising estrogen in females. Hence, the effects of pubertal caffeine exposure on bone growth in females remain largely unknown. To investigate this, immature female Sprague-Dawley rats were assigned two experimental groups, which received caffeine doses of 120 and 180 mg/kg/day, respectively for 4 or 8 weeks, and a control group. At the end of the experiment, leg bone lengths and weights were measured, serum estradiol levels were determined, and histomorphometric analyses of the growth plates (GPs) were performed. Body weight was significantly lower in the two caffeine-fed groups than in the control group at both 4 and 8 weeks, and caffeine exposure significantly reduced leg bone length and weight, especially after 4 weeks of exposure. Bone mineral content was also reduced at both time points, whereas bone mineral density was affected only after 4 weeks. Notably, serum estradiol levels were significantly elevated in the caffeine-treated groups after 4 weeks of exposure. Histomorphometric analysis of the proximal tibial GP revealed that the caffeine-fed groups consistently had greater GP heights, lower bone formation parameters, and reduced spongiosa heights compared to controls. These findings suggest that caffeine directly or indirectly impairs the mineralization and osteoblastic activity required for proper bone maturation. Further research is needed to determine whether these adverse effects on long bone development in females persist following the cessation of caffeine exposure.

Supplementary information: The online version contains supplementary material available at 10.1007/s43188-025-00314-y.

Salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, SAL) is a dopamine-derived tetrahydroisoquinoline, first identified in the urine of a Parkinson's disease patient treated with L-DOPA. SAL is generated endogenously via both enzymatic and non-enzymatic Pictet-Spengler condensation of dopamine with acetaldehyde. It is also found in dietary sources such as bananas, mushrooms, and cocoa, as well as in certain microbes. SAL has been detected in cerebrospinal fluid and various brain regions. SAL acts as a neuroactive compound implicated in ethanol's reinforcing effects and has been proposed as an endogenous prolactin-releasing factor. Its structural similarity to the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) has long provoked suspicions that SAL may induce dopaminergic neurodegeneration through generation of reactive oxygen species and subsequent activation of cell death pathways. However, recent in vitro and in vivo studies reveal a biphasic profile: at low concentrations, SAL exerts neuroprotective effects, whereas at higher concentrations, it becomes neurotoxic. This review therefore summarizes differential pathophysiological roles of SAL based on experimental and clinical findings, highlighting its context-specific, dose-dependent actions in the central nervous system.

This study explored the integration of advanced deep learning with key pharmaceutical biomarkers to enhance early diabetes prediction. We developed a multimodal ensemble approach that leverages transformer architectures to capture complex dependencies in heterogeneous healthcare data and Diffusion Models to address class imbalances by generating synthetic samples. Our research utilized diverse data sources, including electronic health records, medical imaging, and wearable device time-series data, supplemented with synthetic samples to better represent minority populations such as patients with type 1 and gestational diabetes. Critical biomarkers, including C-peptide, insulin, and hemoglobin A1c, were incorporated to improve model interpretability. The methodology involved extensive evaluation using accuracy, area under the receiver operating characteristic (ROC) curve (AUC), precision, recall, and F1-score, with cross-validation to mitigate overfitting. We also implemented interpretability features to provide clinicians with insight into the significance of biomarkers. Results showed a 6.2% improvement in minority class recall when pharmaceutical biomarkers were combined with diffusion-based augmentation. The model demonstrated enhanced classification stability and provided clear insights into clinical decision-making, highlighting the influence of biomarkers on disease progression and treatment outcomes. Future work will focus on multicenter validation, integration of additional omics data, and specialized validation across diverse populations. These findings underscore the potential of AI-driven biomarker analysis for advancing early diagnosis and personalized diabetes management, with broader implications for chronic disease prediction.

Semaglutide is an anti-obesity and anti-diabetic drug used in long-term weight management and for the treatment of type 2 diabetes. A toxicology study was conducted on a generic synthetic version of Semaglutide Injection (2 mg/3 mL) produced by Biocon Pharma, using New Zealand White Rabbits. In this study, Rabbits (4/sex/group) were administered subcutaneously, once weekly, with Semaglutide Injection at dose levels of 0.062, 0.124, and 0.248 mg/kg for a period of 90 days. Across all dose groups, treatment-related significant decreases in food consumption, weight loss, fewer fecal pellets, limited urine pools, and decreased activity were observed. These reported changes are known class effects of GLP-1 receptor agonists. Additionally, all changes noted in hematology, clinical, and anatomic pathology parameters were linked to reduced food intake and weight loss. Results showed one mortality each in treatment groups with Semaglutide administered at 0.124 mg/kg (mid dose) and 0.248 mg/kg (high dose). Mortality was related to the exaggerated pharmacological effect on body weight and feed consumption. Based on the findings from this study, the No-Observed-Adverse-Effect-Level (NOAEL) for Semaglutide Injection 2 mg/3 mL (0.67 mg/mL) was found to be 0.062 mg/kg in New Zealand White Rabbits.