Toxicological Research最新文献

Environmental pollutants, whether naturally occurring or manufactured for commercial use, are pervasive in modern ecosystems. Endocrine disruptors, particulate matter, and micro- and nanoplastics are especially concerning because of their ubiquity, persistence, resistance to degradation, and bioaccumulation. Their global dispersal enables widespread human exposure and poses systemic health risks. The brain, with limited antioxidant capacity and a lipid-rich composition, is highly vulnerable to pollutant-induced injury. It exhibits sex-dependent differences in neurotransmission, neuroanatomy, glial populations, and neuroinflammatory responses, which can shape susceptibility to environmental insults. Yet the direct effects of environmental toxicants on the brain remain incompletely defined, partly because many toxicology studies have used only male animals, lacked sex-stratified analyses, or omitted sex altogether. Converging evidence from epidemiological, animal, and cellular studies links pollutant exposure to sex-dependent neurotoxic outcomes spanning neurodevelopmental and neurodegenerative disorders and functional impairments, reflecting intrinsic differences between male and female brains. This review synthesizes current knowledge on sources and exposure routes of key pollutants-endocrine disruptors, particulate matter, and micro- and nanoplastics-along with their sex-dependent neurotoxic effects and underlying mechanisms. Recognizing sex-dependent vulnerabilities is essential to inform public health policies, targeted interventions, and regulatory strategies to prevent pollutant-associated brain diseases.

The 3Rs are guiding principles that must be followed when designing studies and conducting toxicity research. There is ongoing controversy regarding the use of animals in research. Moreover, the European Union outlawed animal experimentation in cosmetic products on 11 March 2013. More recently, the FDA Modernization Act 2.0 removed the requirement to use animal studies as part of the process for obtaining a licence for a biological product. These moral, ethical, and legal constraints have increased the need for alternative testing procedures. 3D cell cultures have gained popularity in recent years. Small microfluidic platforms known as "organs-on-chips" are dynamic cell cultures that mimic specific microenvironments. These microchips allow scientists to collect in vivo-like data. Organs-on-chips are considered a promising replacement for animal testing, and toxicity research is rapidly adopting this novel approach, much like other scientific fields, such as neuroscience, stem cell research, and cancer investigations. Therefore, the purpose of this review is to discuss the areas of toxicology where these platforms are currently being used, summarise the most recent toxicological applications of the aforementioned platforms, discuss the opportunities and challenges they present for toxicological research, and explore the interdisciplinary approaches applied within the field of toxicology.

Particulate matter 2.5 (PM_2.5) is an air pollutant that causes skin inflammation and aging. Phloroglucinol (PG) is a naturally occurring polyphenol with strong antioxidant potential that is widely used in pharmaceutical production. Information regarding the protective effect of PG against PM_2.5-induced cellular damage is lacking; therefore, this study evaluated the protective effect of PG against the detrimental effects of PM_2.5. PG reduced cellular reactive oxygen species levels in a dose-dependent manner. Furthermore, PG reduced PM_2.5-induced 8-hydroxydeoxyguanosine, protein carbonylation, 8-isoprostane, interleukin (IL)-6, and IL-1β levels. PG reduced PM_2.5-induced apoptosis by inhibiting the expression of pro-apoptotic proteins and mitochondrial membrane depolarization. PG restored PM_2.5-impaired nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (Ho-1) protein expression. Furthermore, PG reduced PM_2.5-induced mitogen-activated protein kinase pathway-related protein expression. Assessment of mouse skin tissue further confirmed that PG could restore PM_2.5-reduced Nrf2 and Ho-1 expression and reduce PM_2.5-induced thickening of the mouse epidermis. Overall, PG exhibited a strong potential to reduce PM_2.5-mediated skin cell damage by activating the antioxidant defense system.

Liver fibrosis, a major histopathological indicator of chronic liver injury, is also a key feature of metabolic dysfunction-associated steatohepatitis. Its quantitative assessment in preclinical toxicology is frequently inconsistent and subjective. This study aimed to develop and validate multi-scale, patch-based convolutional neural network classification algorithms for automated fibrosis quantification in a carbon tetrachloride (CCl₄)-induced mouse model. We sought to determine the optimal patch size for accurate predictions. Accordingly, male C57BL/6 mice (n = 19) were categorized into the following three groups: vehicle control (n = 5), high-fat diet (HFD) and CCl₄ positive control (n = 9), and HFD and CCl₄ with elafibranor (ELA) treatment (n = 5). Liver tissues were stained with Sirius-red, digitized as whole slide images, and cropped into patches of 32 × 32, 64 × 64, or 128 × 128 pixels. Each algorithm was trained, validated, and tested in an 8:1:1 ratio over 40 epochs with a batch size of 32 to classify fibrotic, normal, and background regions. All models performed robustly, with validation accuracies exceeding 98% and F1-scores above 0.96. Particularly, the 32 × 32 model exhibited the highest correlation with pathologist's measurements (Spearman's r = 0.9609; p < 0.05) and the most accurate estimation of absolute fibrotic area compared to expert assessments. This model also accurately detected the antifibrotic effects of ELA. These findings establish that the 32 × 32 patch-based classification approach provides a rapid, reproducible, and objective method for liver fibrosis quantification in preclinical toxicology, with strong potential for integration into digital pathology workflows.

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Supplementary information: The online version contains supplementary material available at 10.1007/s43188-025-00326-8.

Toxic substances can cause serious harm to aquatic organisms and humans who consume them. Rapid ecotoxicity assessment and genotoxicity assessment should be performed simultaneously to detect potential harm caused by toxic substances. In a previous study, an ecotoxicity and genotoxicity assessment system was established by treating fish cells derived from Cyprinus carpio (C. carpio) with toxic substances in a medium containing 1% fetal bovine serum (FBS) for 6 h. In this study, these conditions (1% FBS/6 h) were applied to fish cells derived from Macropodus ocellatus (M. ocellatus). Surprisingly, the new assessment tool using M. ocellatus cells provided ecotoxicity and genotoxicity data similar to those of C. carpio cells. In addition, the new assessment tool demonstrated its suitability as an assessment platform by demonstrating ecotoxicity and genotoxicity for substances known to be genotoxic (fluxapyroxad, fipronil, clarithromycin, 2,4-di-tert-butylphenol, perfluorooctanoic acid, prochloraz, abamectin, and climbazole). In conclusion, this study established an ecotoxicity and genotoxicity assessment system that can rapidly generate data. This assessment platform can be used as a tool to analyze a large number of toxins within a given period of time.

Supplementary information: The online version contains supplementary material available at 10.1007/s43188-025-00325-9.

Phototoxicity assessment is a critical component of preclinical safety assessment of pharmaceuticals with potential photosensitivity. 3T3 Neutral Red Uptake (NRU) Phototoxicity test, OECD Test Guideline 432, has been widely used as a reference method but concerns about oversensitivity to UVA, insufficient cell attachment and resultant difficulties in achieving the acceptance criteria is raised, which is mainly attributable to aged and over-sensitized BALB/c 3T3 cell line. Here, we compared BALB/c 3T3, NIH 3T3, and HaCaT cell lines for in vitro NRU phototoxicity test using 3 phototoxic pharmaceuticals and non-phototoxic chemicals with well-established photo-reactivity profiles. BALB/c 3T3 cells showed oversensitivity to UVA, which often showed decreased viability below 80% of non-irradiated control. Cell counts were increased to 2 × 10⁴ cells to meet the viability criteria. With established experimental condition, in vitro phototoxicity tests were done with three cell lines. While all three cell lines succeeded in distinguishing phototoxic pharmaceuticals, significant differences were noted in sensitivity metrics: BALB/c 3T3 displayed the highest responsiveness, whereas NIH 3T3 exhibited moderate sensitivity followed by HaCaT. In addition, NIH 3T3 and HaCaT showed higher tolerance to UV-induced damages, uniform adherence to culture surface, and minimal well-to-well variability, thereby improving assay reproducibility. Collectively, these findings demonstrated that NIH 3T3 and HaCaT cells can be used as better alternatives to BALB/c 3T3 in the in vitro phototoxicity test.

Atp8a2 is a type of phospholipid flippases, highly expressed in the cerebellum, functioning to maintain the stability and normal function of the cytomembrane by transporting phosphatidylserine into the cytoplasmic membrane. Atp8a2 mutations and knockout can cause neuronal PS externalization and cerebellar ataxia. The cerebellar damage caused by acrylamide (ACR) exposure has similar pathological features to the symptoms caused by Atp8a2 mutations and knockout. However, the expression of Atp8a2 in the cerebellum and whether Atp8a2 alterations are involved in the pathogenesis of ACR neurotoxicity remain unclear. Here, we find that gavage of 0.5 mg/kg and higher doses of ACR decreased Atp8a2 expression in male SD rats' Purkinje cells, while the loss of Purkinje cells was observed only at 20 mg/kg. The upregulation of Atp8a2 blocks phosphatidylserine externalization and the loss of Purkinje cells and mitigates the increase in the number of microglia in SD rats exposed to 20 mg/kg ACR. These suggest that Atp8a2 expression is sensitive to the neurotoxicity of ACR, and decreased Atp8a2 expression is involved in the mechanism of ACR-induced cerebellar injury. This study provides a new important clue for understanding the mechanism of ACR-induced cerebellar lesions and evaluating dose standards for ACR neurotoxicity.

Supplementary information: The online version contains supplementary material available at 10.1007/s43188-025-00322-y.

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.