Various chemicals, including pesticides, heavy metals, and metabolites of tobacco, have been detected in fetal environment. Fetuses are exposed to these chemicals at relatively low concentrations; however, their risk of developing neurological and behavioral disorders increases after birth. We aimed to evaluate the effects of five chemicals (diethylphosphate, cotinine, octachlorodipropyl ether, mercury, and selenium) detected in the serum of pregnant mothers on neural development using human neurospheres (NSphs) differentiated from induced pluripotent stem cells. Exposure to each chemical at serum concentrations revealed no effects on NSph development. However, combined exposure to the five chemicals caused a significant decrease in NSph size and altered gene expression and neural differentiation. Thus, we next focused on DNA methylation to investigate changes in NSph properties caused by chemical exposure. Combined exposure to chemicals had extremely small effects on the DNA methylation status of NSphs at individual gene loci. However, stochastic changes in methylation status caused by chemical exposure were significantly accumulated throughout the entire genome. These results suggest that the five chemicals acted as epimutagens that alter the epigenetic status during human neural development at the biological level. Taken together, we showed for the first time, the epimutagen-induced alterations in neural differentiation at serum concentrations using an in vitro human neuronal model.

Objective. Lidocaine has been reported to induce neurotoxicity, which is further enhanced by high glucose levels. This study is aimed to explore the underlying mechanisms of lidocaine neurotoxicity in spinal cord neurons of diabetes. Methods. Take thirty specific pathogen-free (SPF) healthy Sprague-Dawley (SD) rats and thirty Goto-Kakizaki (GK) rats, aged 12 weeks, weighing 180-200 g. The spinal cord neurons of rats were isolated and cultured in vitro. Cell Counting Kit-8 was used to detect cell proliferation to determine the appropriate concentration and duration of lidocaine. Mitochondrial function was assessed using ATP content, cellular oxygen consumption rate, mitochondrial membrane potential, ROS production, and mitochondrial ultrastructure. Western blot was applied to detect the expression of autophagy- and mitophagy-related molecules PINK1, p-AMPK, LC-3II/LC3-I ratio and mTORC1. Immunofluorescent staining was used to detect the expression of PINK1 and LC3. Results. Lidocaine decreased cell viability of spinal cord neurons in concentration- and time-dependent manners. And lidocaine treatment aggravated mitochondrial dysfunction in GK rats. Furthermore, mitophagy was activated in diabetes, and lidocaine exposure up-regulated mitophagy. AMPK activator MK8722 aggravated mitochondrial damage, increased the expression of PINK1, p-AMPK, LC-3II/LC3-I ratio, and decreased the expression of mTORC1, while AMPK inhibitor Compound C and autophagy inhibitor Bafilomycin A1 reduced mitochondrial damage and decreased the expression of PINK1, p-AMPK, LC-3II/LC3-I ratio, and increased the expression of mTORC1. Conclusions. Lidocaine induced neurotoxicity of spinal cord neurons in GK rats via AMPK-mediated mitophagy.

Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease (ESRD). Although current therapeutic strategies for DKD, including sodium-glucose cotransporter-2 inhibitors and mineralocorticoid receptor antagonists, have shown some degree of efficacy, they have failed to completely halt the progression of DKD to ESRD owing to the complexity of DKD pathogenesis. Elucidating the pathophysiological mechanism of DKD is essential for the development of novel therapeutic strategies. In this study, we investigated the pathophysiological characteristics of uninephrectomized (UNx) KK-Ay mice and examined the effects of salt supplementation on the acceleration of renal injury in these mice. UNx KK-Ay mice exhibited pathophysiological renal abnormalities with glomerular and tubulointerstitial fibrosis. Additionally, salt supplementation exacerbated renal injury, particularly tubular injury. These results suggest that UNx KK-Ay mice are useful models for advanced DKD and that salt exacerbates tubular damage in DKD.

ICH S3A Q&A focused on microsampling (MS) was published to help accelerate the use of MS and states that MS is useful because toxicokinetic (TK) evaluation with conventional blood sampling volume requires many animals for TK satellite groups; however, there are few reports of MS application in mice. We investigated the influence of MS on toxicity evaluation in mice by comparing the toxicity parameters with and without MS after a single oral administration of 1-naphthylisothiocyanate (ANIT), a hepatotoxic substance. Blood samples (50 µL/point) were collected from the tail vein of 3 mice per group at 2 or 3 time points during a 24-hr period, and toxicity was evaluated 2 days after administration. ANIT-related changes suggesting liver or gallbladder injury were noted in blood chemistry and histopathology. Some of these changes such as increases in focal hepatocyte necrosis and inflammatory cell infiltration in the liver as well as mucosal epithelium necrosis in the gallbladder were apparently influenced by MS. A tendency to anemia was noted in animals with MS but not without MS, which was also noted in the vehicle-treated controls, suggesting influence of blood loss. The current results indicate that ANIT hepatotoxicity could be evaluated in mice in which blood samples were collected by MS for most parameters; however, parameters in anemia and pathology in the liver and gallbladder were influenced by MS in this study condition with ANIT. Therefore, MS application in mice should be carefully considered.