Journal of Applied Toxicology最新文献

Three-dimensional, self-organizing structures derived from stem cells, known as organoids, represent a groundbreaking advancement in preclinical drug development. Organoid-based platforms advance preclinical testing by providing an accurate representation of human tissue architecture and genetics, surpassing traditional two-dimensional cultures and animal models in testing both drug safety and efficacy. Researchers are shifting toward organoid-based systems as primary components of new approach methodologies, as global regulatory bodies increasingly acknowledge animal testing limitations. This review delivers an exhaustive examination of organoid technologies and their applications in drug testing. Our study explores current methods used to model toxic responses in different organs-such as the liver, kidney, and heart-while highlighting how personalized and disease-specific organoids can enhance the accuracy of efficacy testing. Our investigation also examines regulatory frameworks and outlines the path toward organoid platform standardization and validation before their integration into drug development processes. Complex neural organoids show great promise but continue to face significant challenges, including biological variability, a lack of universal standards, and ethical concerns. The combination of organoid technology with microengineering techniques, artificial intelligence-based analysis, and high-throughput screening methods represents a transformative change in translational medicine. Organoid-based systems represent both scientific breakthroughs and ethical necessities, as they provide human-specific data while reducing dependence on animal testing. If organoid development progresses with regulatory approval, it could fundamentally transform drug discovery and safety evaluation methods.

This study investigates the in vitro effects of florasulam, a widely used herbicide with known environmental impact, on bull epididymal sperm and primary testicular cells. Epididymal spermatozoa were collected from the cauda epididymis attached to one testis of a paired set obtained from a local abattoir and diluted to a concentration of 1 × 10⁸ spermatozoa/mL. The other testis was used to isolate testicular cells, which were then seeded onto 12-well and 96-well plates at the concentration of 5 × 10⁵ and 5 × 10⁴ cells per well, respectively. Sperm samples were exposed to various concentrations of florasulam (0-1000 μg/mL) for 2 h and evaluated for motility (M), plasma membrane integrity (PMI), acrosome integrity (AI), and mitochondrial membrane potential (MMP). Likewise, testicular cells were treated with different concentrations of florasulam for 48 h and assessed for cytotoxicity, apoptosis, steroidogenesis, and MMP. Statistical analyses were performed using ANOVA followed by Duncan's multiple range test. The results showed that florasulam exposure significantly reduced sperm motility and MMP at concentrations of 100-1000 μg/mL. Additionally, 10 μg/mL florasulam stimulated cell proliferation, whereas 10, 100, and 500 μg/mL inhibited steroid secretion in testicular cells. Apoptosis was significantly increased at 500 and 1000 μg/mL, and MMP was negatively affected at 1000 μg/mL (p ≤ 0.05). These findings provide the first evidence that florasulam, even at sub-toxic concentrations, can impair male reproductive function by reducing sperm motility and mitochondrial activity, and by inducing apoptosis and hormonal disruption in testicular cells. This highlights its potential risk to cattle fertility and broader environmental reproductive health.

Acute inhalation toxicity studies provide crucial information regarding short-term exposure to volatile chemicals and help to ensure the safe handling and use of these chemicals. However, acute inhalation toxicity assessment requires specialized equipment, and most animal facilities are not able to carry out such studies. Consequently, an extremely large number of chemicals remain unassessed. Therefore, we investigated intra-Tracheal Intra-Pulmonary Spraying (TIPS) as a method for testing the acute inhalation toxicity of volatile chemicals. TIPS does not require specialized equipment and consequently is an approach that can be used by essentially all animal facilities. We diluted 12 chemicals in physiological saline and administered the chemicals to test animals using TIPS. When the acute LD50 values obtained by TIPS were converted to LC50 values, there was good agreement between the TIPS LC50 values and the reported inhalation LC50 values of nine of 11 water-soluble chemicals. Two water-soluble chemicals had TIPS LC50 values that were more than 2.5-fold higher than the published inhalation LC50 values, and water-insoluble xylene had an LC50 value that was more than 2.5-fold lower than the published inhalation LC50 value. This strong agreement between TIPS LC50 values and published LC50 values suggests that TIPS has the potential to be used for acute inhalation toxicity testing. However, further studies are needed to establish testing systems that will identify solutes that can and cannot be used for TIPS testing of different water-soluble chemicals and for TIPS testing of water-insoluble chemicals.

Reactive oxygen species (ROS) act as essential signalling mediators but become damaging when their generation exceeds the capacity of antioxidant defences. Predicting ROS-driven toxicity requires models that account for both the physicochemical drivers of radical formation and the biological pathways affected by oxidative stress. This review examines recent advances in mechanistic quantitative structure-activity relationship (QSAR) approaches for ROS prediction, highlighting descriptor systems that capture electrochemical reactivity, redox cycling behaviour, DNA alkylation potential and mitochondrial electron-leakage risk. Monte Carlo-optimised fragment descriptors developed using the CORAL platform are discussed for their ability to reveal substructural triggers of redox imbalance while retaining mechanistic transparency. Emerging multitarget nano-QSAR frameworks are considered for cases in which oxidative stress arises from converging mechanistic routes. The review also evaluates the strengths and limitations of current datasets, including inconsistencies between commonly used oxidative-stress assays, and outlines practical strategies for harmonising heterogeneous experimental outputs. Finally, it considers how mechanistic QSAR models can support regulatory evaluation through transparent hazard identification, grouping and read-across justification, as well as alignment with adverse outcome pathway frameworks, thereby moving the field toward more mechanistically grounded and regulatory-defensible tools for assessing ROS-mediated toxicity.

Hydroxychloroquine sulfate (HCQ), widely prescribed for autoimmune disorders, carries unresolved concerns regarding potential genotoxic risks. This integrated in vitro assessment comprehensively evaluated HCQ's cytogenotoxic profile using bacterial reverse mutation (Ames test in TA98/TA100 strains, 5-80 μg/plate), DNA strand break detection (alkaline Comet assay), chromosomal instability assessment (cytokinesis-block micronucleus test in human lymphocytes, 10-40 μg/mL), oxidative stress biomarkers (TOS/TAR/OSI), plasmid DNA protection (pBR322 under UV/H₂O₂), and molecular docking targeting DNA polymerase δ. Results demonstrated no mutagenicity in TA98. While a statistically significant (p ≤ 0.001) increase in revertants was observed in TA100 at a single concentration, this was transient, nondose-dependent, and biologically insignificant as it remained below the two-fold threshold defined by OECD criteria. No significant DNA damage occurred in mammalian systems, with Genetic Damage Index ≤ 0.14 and micronucleus frequency consistently below 9.75%. Molecular docking revealed weak binding affinity to DNA polymerase δ (ΔG = -5.6 kcal/mol). HCQ induced pronounced dose-dependent cytostasis, evidenced by a 20.5% reduction in Nuclear Division Index at 40 μg/mL, without accompanying genotoxicity. Redox modulation was confirmed through a 15.4% decrease in oxidative stress index. Crucially, HCQ exhibited a complex, biphasic effect on plasmid DNA, paradoxically exacerbating damage at a low concentration (10 μg/mL) while offering significant protection at higher concentrations under combined oxidative stress. These data establish HCQ as a cytostatic agent devoid of genotoxic risk, reinforcing its clinical safety profile while highlighting the necessity for environmental risk validation through in vivo models.

LIN-24, an aerolysin-like pore-forming protein in Caenorhabditis elegans, exemplifies how ancient cytolytic mechanisms have evolved into regulated cellular processes. Initially identified for inducing nonapoptotic, engulfment-dependent cell death in vulval precursor cells, LIN-24 has emerged as a multifunctional regulator of metabolism, stress resilience, and immune defense. Its expression increases during starvation and bacterial infection, promoting lipid mobilization, mitochondrial remodeling, and activation of DAF-16, MAPK, and SKN-1 pathways, thereby enhancing survival and pathogen resistance. Conversely, gain-of-function mutations trigger cytotoxic membrane disruption, illustrating LIN-24's dual role in cytotoxicity and cytoprotection. Despite these advances, its precise structure, regulatory mechanisms, and interaction networks remain undefined. Understanding how LIN-24's pore-forming activity is contextually controlled will clarify how eukaryotes repurpose toxic domains for adaptive functions and may provide translational insights into human pore-forming proteins such as perforins and gasdermins involved in immune defense and programmed cell death.

Phycocyanin is a pigment-binding protein extracted from different algae for its great antioxidant and protecting properties. Uranium is a naturally radioactive metal showing many hazards effects on living organisms when exposed to it. The present study aims to show the effect of phycocyanin to decrease the ability of uranium to cross, accumulate, and distribute in six brain areas (cortex, cerebellum, hippocampus, striatum, midbrain, and hypothalamus) and demonstrated the expected neuroprotective role of phycocyanin against uranium intoxication in adult male albino rats. One hundred twelve rats are grouped as control, phycocyanin (PC), uranium (U), and PC+U group. Results showed that the daily administration of phycocyanin could decrease the uranium accumulation in the different brain areas. The daily intraperitoneal injection with uranium caused a significant decrease in norepinephrine (NE), dopamine (DA), serotonin (5-HT), and adenosine triphosphate (ATP), and reduced glutathione (GSH) accompanied by a significant increase in disulfide glutathione (GSSH), gamma aminobutyric acid (GABA), and glucose level. On the other hand, phycocyanin administration showed a significant change in malondialdehyde (MDA) and GSH level. The coadministration of phycocyanin in parallel with uranium injection showed that phycocyanin has a neuro-mitigation effect on uranium's adverse effect in all the tested parameters. The ameliorative effect of phycocyanin may be regarded as its high activity of scavenging the free radicals and its highly antioxidant effect.

The transport of pharmaceutical compounds into aquatic ecosystems poses a significant environmental threat, particularly due to the presence of drugs that cannot be completely removed during wastewater treatment processes. Diclofenac (DCF), one of the most widely used nonsteroidal anti-inflammatory drugs worldwide, is among the pharmaceuticals frequently detected in aquatic environments due to its high consumption levels and persistence in the environment. It is known that this compound causes neurotoxicity, behavioral disorders, and physiological stress responses in aquatic organisms even at low concentrations. This study aimed to determine the effects of diclofenac exposure on oxidative stress, circadian rhythm, and behavioral parameters in zebrafish larvae. For this purpose, zebrafish embryos and early-stage larvae were exposed to DCF at concentrations of 0.5, 2.5, and 12.5 μg/L for 120 h. Subsequently, to investigate the effect of DCF on oxidative stress, SOD, CAT, GPX, and AChE enzyme activities and gene expression levels were analyzed. To examine its effects on behavior and circadian rhythm, thigmotaxis and locomotor activity analyses were performed. Additionally, to determine the molecular-level effects of behavioral changes, the expression levels of the bdnf, 5ht4, crhr, bmal1, per, and gnat2 genes were analyzed. Overall, our findings indicate that DCF affects behavioral activity, neurotransmitter metabolism, oxidative stress response, circadian rhythm, and retina-related molecular regulators in zebrafish larvae in a multilevel manner. These results highlight the potential risks of pharmaceutical contaminants on neurodevelopmental processes in aquatic ecosystems and demonstrate that even environmental doses can produce complex responses in biological systems.

tert-Butyl hydroperoxide (t-BHP), a common oxidative stress inducer in cellular models, exhibits differential cytotoxic effects on AC16 and H9c2 cardiac cell lines. This study investigated these effects by treating both cell lines with varying t-BHP concentrations, resulting in a dose-dependent increase in cell mortality for both. The half-maximal inhibitory concentration (IC₅₀) was 108.4 μmol/L for H9c2 cells and 419.3 μmol/L for AC16 cells. Under identical t-BHP exposure, H9c2 cells demonstrated lower survival rates than AC16 cells, suggesting greater t-BHP tolerance in AC16 cells. While increasing cell mortality in H9c2 cells correlated with a gradual rise in reactive oxygen species (ROS) levels, AC16 cells displayed an "inverted U-shaped" trend. This implicates ROS as a critical factor in t-BHP-induced H9c2 cell mortality, a conclusion applicable to AC16 cells only below 350 μmol/L t-BHP, above which other non-ROS mechanisms may accelerate cell mortality in AC16 cells. Seahorse metabolic analysis indicated that t-BHP exposure significantly reduced basal respiration, adenosine triphosphate (ATP) production, nonmitochondrial respiration, maximal respiration, and respiratory reserve capacity in both cell lines. Metabolically, H9c2 cells depend primarily on mitochondrial oxidative phosphorylation, while AC16 cells favor glycolysis. Moreover, proton leak in H9c2 mitochondria progressively decreased with increasing t-BHP, whereas AC16 cells exhibited significantly increased mitochondrial proton leak (p < 0.01) from the initial t-BHP exposure, independent of concentration. The aforementioned results indicate that, establishing a model of cardiomyocyte death induced by increased oxidative stress with t-BHP, H9c2 cells robustly simulate this pathological process. However, when selecting AC16 cells, great attention should be given to the t-BHP concentration.