p-Cymene, is a monocyclic monoterpene hydrocarbon, commonly used as a flavoring agent in food. A 90-day repeated oral toxicological study of p-cymene was conducted to examine the toxicological properties and determine the no-observed-adverse-effect level (NOAEL) of p-cymene in Crl:CD (SD) rats at the following doses: 0 (corn oil), 2.4, 12, and 60 mg/kg/day. No mortality or abnormal clinical signs were observed in the treatment groups. The body weight, food consumption, ophthalmoscopy, and gross pathology of the rats were also not affected by p-cymene treatment. However, in the 60 mg/kg group, certain parameters decreased in males, including hemoglobin and hematocrit, red blood cell count, triglyceride, total protein, and albumin. In females, urine volume and total potassium excretion increased, whereas specific gravity, and sodium, potassium, and chlorine concentrations decreased. Increased liver weight was observed in both males and females. Histopathological observations revealed centrilobular hepatocellular hypertrophy. In the 12 mg/kg group, no adverse effects of p-cymene treatment were observed in both sexes. In conclusion, the NOAEL of p-cymene was 12 mg/kg/day for both sexes under the present experimental conditions, considering the alterations in urinalysis, hematology, clinical biochemistry, and histopathology.

Titin is a giant protein that is specifically expressed in striated muscle and essential for the maintenance of sarcomere structure and function. Recently, the N-terminal fragment of the Titin (N-titin) has been reported to show high levels in human urine in patients with muscular diseases and is expected to serve as a diagnostic biomarker for these diseases. In this study, we examined the utility of N-titin as a biomarker to detect muscle atrophy in mice. Male BALB/c mice (6 weeks of age, n=5 per group) were given 10 mg/L dexamethasone (DEX) dissolved in drinking water for 4 weeks. The gastrocnemius muscle (GAS) weight was significantly decreased and mRNA levels of muscle atrophy-related genes (Atrogin-1 and MuRF-1) were increased in the GAS after 4 weeks of DEX treatment. Although there were no degenerative/necrotic changes in the histopathological examination, the muscle fiber cross-sectional area significantly decreased in the GAS. On the other hand, there were no DEX treatment-related changes in the muscle weights and the muscle fiber cross-sectional area in the soleus muscle. These results suggest that 4-week of DEX treatment preferentially caused atrophy of fast-dominant muscle. Under the condition of this study, urinary N-titin/CRN ratio markedly increased from Week 2 of the DEX treatment. From the above results, the urinary N-titin/CRN ratio could be a biomarker for monitoring skeletal muscle atrophy in mice.

We investigated the characteristics of pharmaceutical concentrations and antimicrobial activities in river water from the Tone River system in Gunma Prefecture. The mean concentrations of diphenhydramine, clarithromycin, carbamazepine, and bezafibrate in the midstream of the Tone River were 8.6, 29, 3.8, and 8.1 ng/L, respectively. Their concentrations were nearly half of those in the midstream of the Ayase River, the main water source of which is wastewater. Seasonal variations in pharmaceutical concentrations were high in winter and low in late spring and autumn. This variation depended on the flow rate of the river water, which in turn depended on the rainfall in the upstream area. Except for bezafibrate, the pharmaceutical concentrations in river water did not change after 5 days of incubation at 30°C, indicating that biochemical degradation during the hot summer season was minimal. A comparison of the concentrations between the sampling locations revealed that the pharmaceutical load was proportional to basin population, and the annual fluxes of pharmaceuticals from Gunma Prefecture were estimated to be 98, 210, 28, and 53 kg/year, respectively. Disc diffusion assay of some samples of Tone River water extracts revealed inhibition zones owing to their antimicrobial activity. However, no relationship was observed between the diameter of the inhibition zone and clarithromycin concentration in the river water. These results suggest that the antimicrobial activities of the river samples were not dependent on clarithromycin. We are currently investigating the pollution and drug-resistant bacteria present in the Tone River in detail.

Intratracheal instillation is a useful method for evaluating airway toxicity of various substances. However, there is limited information on this method in repeated dose toxicity studies. This study aimed to supplement existing background data by intratracheally administering distilled water for injection (DW) or phosphate-buffered saline (PBS) to four groups of six male SD rats each under anesthesia by inhalation of sevoflurane or isoflurane. Additionally, a non-intratracheal instillation group with inhalation anesthesia and a non-treatment control group with neither intratracheal instillation nor inhalation anesthesia were established. DW or PBS droplets were instilled via intratracheal intubation once a day, five days a week, for four weeks following inhalation anesthesia. The examination included hematology, blood chemistry, biochemical and cytological analysis of bronchoalveolar lavage fluid (BALF), organ weight measurement, gross necropsy, and histopathological examination of the lungs. No apparent abnormalities were observed in hematology, blood chemistry, or biochemical and cytological analysis of BALF. However, histopathological examination revealed perivascular/peribronchiolar eosinophil infiltration in the lungs induced by sevoflurane and isoflurane. The change was more pronounced with DW or PBS dosing, and was most severe in the DW groups, accompanied by focal inflammation. This study provides useful background data for conducting repeated dose toxicity studies via intratracheal instillation in rats.

Methylmercury (MeHg), a potent neurotoxin, poses substantial risks to prenatal brain development by crossing the placental barrier. In our daily lives, we are exposed to various environmental metals simultaneously with MeHg. Therefore, the combined exposure effects of these metals and MeHg should be investigated. Hence, this study examined the combined fetal exposure effects of MeHg and copper (Cu), an essential element. Gene expression changes in the fetal brains of mice exposed to MeHg, Cu, or both were examined through RNA-seq analysis. Our results showed that the number of variable genes exposed to combined MeHg and Cu increased compared with that in single exposure. Most of them were gene variations specific to combined exposure. Gene Ontology biological process analysis revealed the amplified effects on GABAergic interneurons in the cerebral cortex under combined exposure. IPA pathway analysis indicated considerable variations in pathways related to oxidative stress, neuronal development, and energy metabolism, including the activation of NRF2-mediated oxidative stress response and the suppression of mitochondrial fatty acid beta-oxidation. These findings highlighted the complexity and enhanced risks of combined MeHg and Cu exposure. Therefore, neurodevelopmental effects were more severe and multifaceted than those caused by individual exposures. This research highlighted the importance of understanding the mechanisms of the combined exposure effects of MeHg.

In non-clinical toxicity studies for drug development, reduced food intake in experimental animals can lead to fluctuations in various toxicological parameters, complicating the distinction between drug toxicity and secondary effects of reduced food intake. This review examines the parameters that change due to food restriction in rats, dogs, and monkeys, and discusses the presumed mechanisms behind each parameter change. The parameters include standard toxicological evaluation parameters, such as body weight, blood chemistry, hematology, urinalysis, bone marrow cell analysis, organ weight, and histopathology. This review also highlights the differences in parameter changes across animal species and food restriction conditions, providing crucial insights for improving the quality of non-clinical toxicity studies and enhancing human translatability. The review underscores the need for a comprehensive analysis of these parameters to understand animal nutritional status within toxicity studies. This information can improve the reliability of toxicity evaluations.

Nanoparticles (NPs) are used in a variety of fields, including industry, medicine, and food production. Predicting the potential toxicity and biological effects of NPs is challenging due to the influence of their physicochemical properties, such as particle shape and coating constituents. This study investigated the pulmonary effects of differently coated titanium dioxide (TiO₂) NPs in wild-type and nuclear factor erythroid 2-related factor 2 (Nrf2) null mice. C57BL6/J wild-type and Nrf2 null mice were exposed to uncoated TiO₂ NPs, or NPs coated with either hydroxide aluminum, or with aluminum hydroxide/stearic acid. After a two-week exposure period, no significant changes were observed in lung weight, cell counts of bronchoalveolar lavage fluid, mRNA levels of proinflammatory cytokines, and antioxidant gene expression in either mouse strain. In addition, human lung carcinoma A549 cells exposed to three types of TiO₂ NPs showed no significant changes in cell viability, cytotoxicity, or intracellular reactive oxygen species production. These findings suggest that the toxicity of the TiO₂ NPs, regardless of surface modification, is minimal to the respiratory system of mice and human alveolar epithelial cells.

Mercury (Hg) accumulation in rice is a health concern due to the consumption of rice as the staple food. This study evaluated the mercury contamination in rice plants, which are typical foods cultivated in the Red River Delta. During the harvest season, rice samples were collected and separated into husk and brown rice, together with polished white rice and bran rice from mill shop. For brown rice, the Hg concentration ranges from 7.18 ± 0.73 to 16.32 ± 2.57 µg/kg. Additionally, brown rice samples near landfill or highway tend to have higher Hg concentrations than sites farther away. Hazard quotient (HQ) was used to measure the health risk of Hg in this study. HQ values of male and female all were less than one, indicating that consuming rice from Nam Son and Bac Son might not cause potential human health risk of Hg exposure.

Many epidemiological and animal exposure studies have suggested that exposure to environmental substances is a major risk factor for developmental neurotoxicity (DNT), such as in autism, and is related to the increasing relevance of neurodevelopmental disorders. Recent efforts have led to the development of various in vitro approaches that use cell lines and pluripotent stem cells to assess numerous environmental substances. In this study, we developed a method for assessing DNT using a mouse embryonic stem (mES) cell model that focuses on differentiation into neuronal cells (neural cells and astrocytes). Using this model system, we found that six insecticides inhibited the differentiation of neural precursor cells into astrocytes. Our data indicated that the effects of insecticides on glial differentiation were more sensitive than those of several DNT markers reported in previous studies. This mES cell model can make a quick assessment of DNT potential and may be a useful tool for screening substances with potential to induce DNT.

Morphinone (MO) is an electrophilic metabolite of morphine. Electrophiles can modify thiol groups of proteins, resulting in the activation of redox signaling pathways and toxicity. We have previously reported that the atmospheric electrophile, 1,4-naphthoquinone, and electrophilic organometallic compound, methylmercury, activate protein kinase B (Akt) signaling through modification of phosphatase and tensin homolog deleted from chromosome 10 (PTEN), which is a negative regulator of Akt. In the present study, we examined whether MO activates Akt signaling. Exposure of HepG2 cells to MO enhanced translocation of Akt to the nucleus in a concentration-dependent manner. MO phosphorylated Akt and its downstream protein, cAMP response element-binding protein (CREB), and upregulated B-cell lymphoma 2 (Bcl-2), an anti-apoptotic protein. An analogue of MO dihydromorphinone that was not electrophilic did not enhance the phosphorylation of Akt and CREB or expression of Bcl-2, suggesting the importance of electrophilicity of MO in activation of the cascade. Pretreatment of the cells with wortmannin suppressed MO-mediated phosphorylation of Akt and CREB and expression of Bcl-2, and enhanced MO-induced cytotoxicity, indicating that MO activates Akt–CREB–Bcl-2 signaling in HepG2 cells. This signaling pathway might be capable of modulating MO-mediated toxicity in cells.

In non-clinical toxicity studies of orally dosed small molecule drugs, methyl cellulose (MC) is commonly used as the vehicle. It is well tolerated and easy to prepare. However, it is not suitable as the vehicle for all poorly soluble compounds. The objective of this study was to evaluate the no-observed-effect levels (NOELs) of solvents that are possible alternative vehicles for 2-week oral administration of poorly soluble drugs. Five animals/group were dosed once daily with 25 mg/kg/day MC 400 (0.5% MC, control group), up to 5,000 mg/kg/day of polyethylene glycol 400 (PEG 400), 5,500 mg/kg/day of dimethyl sulfoxide (DMSO), 1,000 mg/kg/day of hydroxypropyl-β-cyclodextrin (HP-β-CD), 250 mg/kg/day of polysorbate 80 (Tween 80), 600 mg/kg/day of sodium dodecyl sulfate (SDS), 9,000 mg/kg/day of olive oil and sesame oil, and 600 mg/kg/day of lactic acid. Parameters evaluated included clinical signs, clinical pathology, organ weight, gross pathology, and histopathology. The NOELs were considered to be 1,250 mg/kg/day for PEG 400, 1,000 mg/kg/day for HP-β-CD, 250 mg/kg/day for Tween 80, 4,500 mg/kg/day for olive oil, 4,500 mg/kg/day for sesame oil, and 600 mg/kg/day for lactic acid. The NOELs of DMSO and SDS could not be determined, because rats dosed with DMSO or SDS showed DMSO-specific offensive odor or SDS-related significant irritant effects even at the lowest dose levels (DMSO, 1,100 mg/kg/day; SDS, 150 mg/kg/day). This study provides the NOELs of several solvents that could be used as vehicles in 2-week oral toxicity studies in rats.

In toxicity studies performed during drug development, the secondary effects of decreases in food consumption that are not direct toxic effects may cause incorrect interpretations of toxicologic profiles. Although previous studies have characterized the effects of reductions in food intake on toxicological parameters in rats, these were conducted for ≥2 weeks, making it difficult to determine whether changes in toxicity-related parameters are secondary to a reduction in food intake or compound effects in the short-term studies conducted in the early stages of drug discovery. Therefore, we evaluated the effects of low food intake for 3 or 7 days on toxicity-related parameters. Male and female rats were fed ad libitum (control group) or at 80%, 60%, or 40% of the mean pre-measured food intake for each group for 3 or 7 days, and their general condition, body weights, water consumption, quantitative urinalysis parameter, hematology, blood chemistry, myelogram, organ weights, histopathology were evaluated. Similar to the previous studies of food restriction of ≥2 weeks, there were decreases in the reticulocytes and leukocytes on hematology and in erythroblasts and myelocytes on a myelogram, especially in their later stages of differentiation, after 7 days of food restriction. Furthermore, natriuresis, which develops in fasted humans, was also identified after 7 days of food restriction. The present study is the first to identify changes in toxicity-related parameters during short-term food restriction. The perspective obtained from this study should aid in the future interpretation of the toxic effects of compounds that cause reductions in food intake.

Extracellular vesicles (EVs) are particles released not only from blood cells but also from various organs. EVs, which are lipid bilayer vesicles, contain proteins, DNAs, and RNAs. The RNA and proteins within EVs display cell-specific characteristics. EVs derived from tumor cells are identified as biomarkers with diagnostic accuracy exceeding 90% for early cancer detection. Furthermore, EV RNA in serum has serves as a biomarker for toxicity. EVs have been found in various body fluids, including saliva, tears, urine, and amniotic fluid. In this study, we aimed to investigate the potential use of EV RNA in amniotic fluid as an indicator of developmental toxicity. Pregnant mice were exposed to valproic acid (VPA), a developmental toxicant, at concentrations of 0, 300, or 600 mg/kg/day on gestational days (GDs) 9–11. The study involved measuring VPA concentration in maternal plasma and fetuses on GD11, fetal weight on GD15 and 18, and assessing external morphological abnormalities on GDs11, 15 and 18. Additionally, EVs were collected from fetal amniotic fluid, and a comprehensive gene expression analysis of EV RNA was conducted on GD15. As a result, the concentration of VPA in the fetuses was not associated with the implantation location. Additionally, the VPA-treated group exhibited intrauterine growth retardation and teratogenic effects, including neural tube defects and digit malformations. EV RNA analysis identified differentially expressed EV small RNAs, both suppressed and induced, in the VPA-treated group compared with the control (vehicle, 0.5% Methylcellulose) group. These findings suggest that EV RNA in amniotic fluid serve as an indicator of developmental toxicity.

Research data on the biological effects of intermediate-frequency magnetic fields (IF-MF) remain inadequate, and there are no protocols currently exists that can assess the biological effects of electromagnetic fields similar to those used with the OECD guidelines for chemicals. IF-MF <100 kHz have a dominant stimulatory effect, which has raised concerns about their effects on neurological disorders. The purpose of this study was to investigate methods for detecting pain in response to IF-MF exposure and to establish a standardized protocol for electromagnetic field pain assessment for use in various environments. The von Frey test, which can assess foot pain, was performed using the partial sciatic nerve ligation (PSL) model, which is a nerve hypersensitivity and allodynia model, together with IF-MF-exposed, sham-exposed, and no-treatment (control) groups. Significant changes were observed at all postoperative measurement points in the PSL group, whereas no significant differences were present among the other groups. In addition, gene expression analysis for four inflammation-related factors (P2rx4, Ccl2, Irf8, and Iba1) was performed using real-time quantitative PCR in the sciatic nerve on postoperative day 15 after exposure. The expression of these genes was significantly upregulated in the PSL group but was unchanged in the remaining three groups. These results confirm that IF-MF exposure (1 hr/day), which is 2.3 times higher than the basic restriction for occupational exposure according to the ICNIRP guidelines, does not cause pain and that these detection methods with positive controls are effective as pain assessment methods for IF-MF exposure.

Functional bile canaliculus formation in cultured human hepatocytes is crucial for in vitro studies of hepatobiliary disposition and drug-induced cholestasis. Human hepatocytes isolated from humanized mouse livers (PXB-cells) are promising cell sources for these studies. PXB-cells available in tissue culture flasks allow users to recover and reseed in different cell culture formats, thereby enhancing their adaptability to various in vitro culture systems. However, the reseeding process may induce cellular stress, affecting subsequent cultures, and its specific effect on bile canaliculus formation is yet to be explored. Furthermore, the role of sufficient oxygen supply in bile canaliculus formation in PXB-cells remains incompletely understood. In this study, we compared hepatic function and bile canaliculi formation in freshly seeded PXB-cells (Fresh PXB-cells) and reseeded PXB-cells (Flask-delivered PXB-cells) under sufficient oxygen supply through oxygen-permeable plates. The flask-delivered PXB-cells recovered their levels of albumin production and cytochrome gene expression to those of fresh PXB-cells after seven days of culture. On days seven and 14 of culture, bile canaliculus formation was similar in both fresh and flask-delivered PXB-cells, as confirmed by fluorescein-labeled bile acid excretion and immunostaining for the bile canaliculi marker MRP2. Notably, analysis of bile canalicular length revealed a significant increase in bile canalicular length with adequate oxygenation, whereas no significant difference was detected between the conditions under the same oxygen supply on days seven and 14. The findings of this study provide valuable insights into the use of PXB-cells for in vitro assessments in drug discovery and toxicological research.

TRPM8, sensor of cold temperatures, regulates epidermal cell proliferation through CDK inhibitor p21/Cip1 and downregulation of TRPM8 causes p21/Cip1 decrease, associated with carcinogenesis. Given that lipids-activated nuclear receptor PPAR gamma negatively regulates the expression of TRPM8 in normal epidermal cells and that carcinoma cells generally secrete exosomes including various lipids, we examined whether TRPM8 and p21/Cip1 expressions in normal human keratinocytes are altered by incubating with medium including substances secreted by squamous carcinoma cells. TRPM8 and p21/Cip1 expressions in normal human keratinocytes HaCaT cells are altered by incubating with medium including substances secreted by squamous carcinoma SAS cells (“SAS medium”), however, the alteration of TRPM8 and p21/Cip1 expressions is indeterminate. In some case, “SAS medium” increased p21/Cip1 in TRPM8-independent manner whereas it decreased p21/Cip1 through both of TRPM8-dependent and independent pathway in other case. We also obtained the data showing that “SAS medium”-induced TRPM8 increase did not result in p21/Cip1 increase, probably from offset by TRPM8-independent downregulation of p21/Cip1. In all cases PPAR gamma level was not altered and “SAS medium” decreased TRPM8 level of HaCaT cells even when PPAR gamma was knocked down, indicating PPAR gamma-independent regulation of TRPM8 by “SAS medium”. These results suggest that squamous carcinoma cells secrete various substances which increase and decrease p21/Cip1 level in nearby normal epithelial cells. Ratio of amounts of substances secreted by squamous carcinoma cells may vary depending on the cell condition and increasing ratio of substances which downregulates p21/Cip1 expression results in increased risk of carcinogenesis.

In conducting repeated dose administration toxicity (RDAT) studies with rats and mice, a minimum of three dose groups and one control group are normally set for determing NOEL/NOAEL (no-observed effect level/no-observed adverse effect level) of the test item. For comparison of data among the groups, initially, the data are analysed by analysis of variance (ANOVA). If ANOVA shows a significant difference, then groups means are compared by a multiple comparison range test (MCRT). However, in RDAT studies, at the end of the long duration of the test substance administration, the distribution of the data obtained varies considerably among the groups and the number of animals decreases due to mortality/morbidity, especially in the high-dose groups. Increased variance in the distribution of the data and decreased animals in one or more groups may result in an insignificant ANOVA, though the low-dose group may show a marked difference compared to the control. Dunnett's multiple comparison test is commonly used to compare each treatment group with the control group. However, Dunnett's test has a lower ability to detect significant differences than the t-test, and its detection power decreases with the increase in the number of groups. Therefore, we recommend the t-test, by-passing ANOVA, which has a high detectable significant difference in the two-group test. In addition, the application of the t-test eliminates the need to select an MCRT. However, the final judgment of the adverse effects may be made based on the toxicological relevance in consideration of the statistical analysis results.