Sexual dimorphism is recognized in nephrotoxic acute kidney injury, with females being less susceptible than males. Thallium (Tl), a highly toxic heavy metal, induces severe systemic disorders following exposure, including gastrointestinal and neurological disorders and renal failure. However, sex-related differences in Tl-induced nephrotoxicity remain poorly understood. Previous studies have shown that Tl preferentially accumulates in the outer medulla of the kidney of male rats, resulting in mitochondrial dysfunction and medullary thick ascending limb (mTAL) injury, characterized by calcium deposits and impaired renal function. This study investigated the effects of Tl on the kidneys of female rats and the underlying mechanisms. Tl₂SO₄ (30 mg/kg) was administered to rats. Nephrotoxicity was measured 2, 5, and 14 days after Tl-administration using biochemical assays of blood and urine samples, histopathology of the kidney, and transcriptome analysis using microarrays. As in male rats, female Tl-loaded rats developed severe renal dysfunction with calcium deposits in the outer medulla within 5 days after Tl administration. The pathological features were similar to those of male rats; however, the mitochondrial oxidative stress and calcium deposits in the medulla were less extensive in female rats than in male rats. These preliminary findings suggest sex-dependent differences, which might be derived from the differences in sex-hormones, in Tl-induced renal injury and suggest potential involvement of differential oxidative stress handling, mitochondrial responses, and transporter activity. These new insights could assist with the development of therapeutic strategies for treating Tl intoxication in humans.

Colistin is a cationic cyclic lipopeptide antibiotic used against multidrug-resistant Gram-negative bacteria; however, its clinical application is limited by nephrotoxicity. Although oxidative stress, mitochondrial dysfunction, and endoplasmic reticulum stress are implicated in the primary cytotoxic mechanisms, the secondary inflammatory pathways remain poorly understood. In this study, we aimed to elucidate the mechanisms underlying colistin-induced nephrotoxicity by analyzing the molecular responses of injured renal tubules isolated by laser microdissection (LMD) in a rat model. Six-week-old male Sprague–Dawley rats were subcutaneously administered colistin (0, 15, or 30 mg/kg/day) for 28 days. Increases in serum creatinine and blood urea nitrogen were associated with tubular vacuolation, single-cell necrosis, and regenerative changes in proximal tubules. Immunohistochemistry revealed increased expression of kidney injury molecule-1 (KIM-1) and presence of cleaved caspase-3 in the injured tubules, indicating epithelial regeneration and apoptosis, respectively. LMD-based microarray analysis identified 486 upregulated and 472 downregulated genes in vacuolated/regenerative tubules compared with normal tubules. Pathway analysis indicated activation of immune-related processes, particularly associated with macrophage activation and trafficking, including interleukin-34 (IL-34). In situ hybridization confirmed Il34 mRNA expression in the cytoplasm of the injured tubules, and accumulation of CD68-positive macrophages around KIM-1-positive tubules. Conversely, no significant change was observed in CD163-positive macrophages following colistin treatment, suggesting proinflammatory M1 macrophage predominance. These findings indicate that tubular IL-34 induction and subsequent macrophage recruitment amplify colistin-induced nephrotoxicity at the secondary level, suggesting that proximal tubules function as both primary targets and effectors of inflammation. Targeting the IL-34-related signaling could thus serve as a potential approach to alleviate colistin-induced renal injury.

Zopiclone (ZOP) and eszopiclone (EZOP) are hypnotics belonging to the cyclopyrrolone class, which are metabolized to zopiclone-N-oxide (ZOPNO) and N-desmethylzopiclone (NDZOP), respectively, and finally to 2-amino-5-chloropyridine (2A5C) after long-term storage, freezing, and conservation. This study aimed to examine the stability of ZOP and EZOP using a commercially available immunoassay urine screening kit for ZOP. ZOP and EZOP, as well as their metabolites and 2A5C, were analyzed in urine using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). Spiked urine samples were used to confirm cross-reactivity for the immunoassay test and to compare the quantitative results of the clinical urine samples. The results indicate that urine samples stored for an extended period or altered postmortem may lead to false-negative results for ZOP, providing important information for interpreting results in cases involving ZOP.

Neurotoxicity of acrylamide has been demonstrated both in humans and animals, while the mechanisms remain largely unknown. We recently reported TNF-α deletion suppressed acrylamide-induced neurotoxicity in mice at low dose. Here we further investigated expression of antioxidant and proinflammatory cytokines to explore roles of TNF-α. Wild type and TNF-α KO mice were exposed to acrylamide at 0/12.5/25 mg/kg bw for 28 days. The results showed that acrylamide significantly decreased body weight at 12.5 and 25 mg/kg bw, but decreased brain weight only at 25 mg/kg bw. TNF-α deletion didn’t alleviate the above effects. Also, TNF-α deletion didn’t alleviate decrease of grip strength at 25 mg/kg bw. Immunohistochemical results showed that TNF-α deletion alleviated noradrenergic axon degeneration in cortex S1FL and S1HL regions at 12.5 mg/kg bw, but not 25 mg/kg bw. Moreover, TNF-α deletion suppressed acrylamide-induced upregulation of TGF-β and NF-κB, but didn’t suppress upregulation of IL-6, suggesting possible roles of IL-6 in acrylamide-induced neurotoxicity, particularly at high concentration. Moreover, this study showed a dipolar effect of TNF-α deletion on oxidative stress pathways, i.e., at 25 mg/kg bw, TNF-α deletion suppressed upregulation of oxidative stress (Keap1/HO-1/Gclc/Gclm/Sod/Cat/Gstm/MT-1); however, at 12.5 mg/kg bw, TNF-α deletion accelerated upregulation of Nqo1/Gclm/Sod1/Cat/Gsr. Taken together, genetic TNF-α ablation, at least partially, alleviated acrylamide neurotoxicity at low concentration. Limited alleviation effects at high concentration generated a hypothesis that this may be due to IL-6 signaling and dipolar regulating effects of TNF-α deletion on oxidative stress pathway. This study provided new insights into acrylamide neurotoxicity and TNF-α-targeting strategy.

Non-clinical safety evaluations, including those of impurities, are important for vaccine development to ensure safety in humans. However, information on the mechanisms of impurity-induced adverse effects remains limited. In a repeated-dose toxicity study of our lipid nanoparticle formulated mRNA vaccine (mRNA-LNP vaccine) candidate, severe anemia was observed in rats after multiple administrations. In this study, we conducted hematological analyses and bone marrow examinations in vivo to investigate the cause and mechanism of test article-related delayed anemia. In addition, we performed in vitro mechanistic studies including antibody titer measurements and colony-forming unit assays. We found that test article-related anemia was caused by the inhibition of erythroid differentiation in the bone marrow, mediated by antibodies against erythropoietin (EPO). Furthermore, the test article was found to contain human EPO mRNA as an impurity. Lastly, the spike study showed that a minute quantity of human EPO mRNA present in mRNA-LNP vaccines as an impurity induced anemia in rats. Taken together, our data demonstrate that immune-mediated delayed anemia can be induced by impurity-oriented anti-EPO antibodies that neutralize endogenous EPO and inhibit erythroid differentiation. Our presented approach of determining the mechanism of delayed toxicity caused by impurities may be helpful in future safety evaluations.

Triphenyl phosphate (TPhP) is a typical organophosphorus flame retardant (OPFR). Due to its high production and widespread use, exposure to TPhP has been shown to induce nephrotoxicity in animal models. Endoplasmic reticulum (ER) stress is found to be correlated with kidney disease caused by exogenous environmental pollutants. Nevertheless, the connection between ER stress and the nephrotoxic effects caused by TPhP is limited. In this study, human renal tubular epithelial cells (HKC) were chosen to explore the effects of TPhP on cell viability, cell apoptosis, and ER stress. Our study indicated that cell viability was dramatically inhibited in a dose-dependent manner. The half lethal concentration (LC₅₀) value of TPhP after 48 hr exposure is 126.4 µM. A concentration-related Caspase-3 activation and apoptosis occurrence were observed in HKC cells following TPhP treatment. Additionally, the induction of ER stress was demonstrated by the up-regulated expression of ER stress-related genes. To elucidate the role of ER stress in cell damage, sodium 4-phenylbutyrate (4-PBA), an ER stress inhibitor, was used in the co-treatment with TPhP. Results revealed that 4-PBA treatment effectively alleviated TPhP-induced ER stress and cytotoxicity in HKC cells. Taken together, these results indicated that ER stress plays a primary role in TPhP-induced nephrocyte damage and 4-PBA could attenuate these effects.

OE₂imC₃C, a zwitterionic liquid, has attracted considerable attention as an emerging solvent with low toxicity. However, the safety data in research reports are limited, and its applicability to toxicity tests remains unclear. Therefore, we investigated the detailed safety profile of OE₂imC₃C and found that it was negative in both the bacterial reverse mutation and chromosome aberration tests, indicating no concern for genotoxicity. The hepatocellular toxicity of OE₂imC₃C stock solution (OS) was lower than that of dimethyl sulfoxide (DMSO). OS was widely applied and gave correct hepatocellular toxicity of ellagic acid (a functional food molecule with anticancer, antioxidant, and other properties), based on the high solubility. In contrast, DMSO revealed false toxicity in the test because the undissolved ellagic acid crystals damaged the cells. The general applicability of OS to food-related compounds was confirmed using Hansen solubility parameters. OS is a unique solvent capable to prevent artifacts in toxicity assays involving poorly soluble compounds such as food and drug molecules.