Tetramethylammonium hydroxide (TMAH) and substances that release tetramethylammonium (TMA) are classified as Priority Assessment Chemical Substances (PACSs) under registration number 17 of the Japan Chemical Substances Control Law (CSCL, 1973). This classification requires a thorough human health hazard assessment and derivation of Hazard Assessment Value (HAVs) for the oral and inhalation exposure at the Assessment II stage. We analyzed their general, developmental, reproductive toxicity, genotoxicity, and carcinogenicity using hazard data from both domestic and international risk assessment agencies and subsequently proposed an HAV. For oral exposure, a no-observed-adverse-effect-level (NOAEL) of 1 mg/kg/day, based on transient or persistent salivation in parent rats from a TMAH developmental and reproductive toxicology (DART) screening study, was chosen as the point of departure (POD). The POD was then divided by uncertainty factors (UFs) totaling 1,000 (interspecies variation: 10, intraspecies variation: 10, short study duration: 10), resulting in an oral HAV of 0.001 mg/kg/day for TMAH. Due to a lack of hazard data for humans and animals via inhalation, an HAV for the inhalation route was not established.

In the development of drugs that affect the central nervous system (CNS), it is important to determine whether they have dependence potential and if so, to establish an effective dose within a range that does not manifest dependency. Methods for evaluating drug dependence include self-administration tests using experimental animals while assessment of the minimum effective dose (MED) involves gross behavioral observation. This study aims to examine the relationship between the most frequently self-administered dose (peak self-administered dose, PSAD) and the MED amongst different types of CNS depressants in order to optimize dose range selection for the evaluation of drug reinforcing effects. PSAD was investigated by intravenous self-administration in rhesus monkeys conducted as daily 2 hr-sessions under a fixed ratio 5 schedule with a 1-min time-out after each administration. MED was investigated by gross behavioral observation following cumulative dosing. For opiates, the PSAD was 0.016 mg/kg/infusion for morphine, 0.06 mg/kg/infusion for codeine, 0.25 mg/kg/infusion for butorphanol and 0.063 mg/kg/infusion for pentazocine. For anesthetics and sedatives, the PSAD was 0.5 mg/kg/infusion for pentobarbital, 0.25 mg/kg/infusion for thiopental, 0.06 mg/kg/infusion for ketamine and 0.063 mg/kg/infusion for midazolam. The PSAD/MED ratio was 1/63-1/32 for opiates and 1/8-1/2 for anesthetics and hypnosedatives. While previous research by Fujiwara et al. (2016) suggested that a dose range lower than the MED for gross behavioral effects should be used for intravenous self-administration in the evaluation of drug reinforcing effects, this study further indicates that the optimal dose range may vary depending on drug type.

Cadmium is an environmental contaminant that accumulates in the human kidneys. Non-rice grains, vegetables, and potatoes are major sources of cadmium in the U.S. However, the daily cadmium intake levels reported in studies conducted in the U.S. vary widely, ranging from 4.63 to 51.3 µg/day/person. Most studies in the U.S. have not measured the actual cadmium concentrations in the collected foods but utilized the database of the average cadmium concentrations provided by the U.S. Department of Agriculture (USDA). In the present study, food and beverages were collected extensively in the U.S. from 1979 to 1980, and the actual cadmium concentrations were determined. Individual food intake data were obtained from a total diet study conducted in 1980 by the USDA. In accordance with previous reports, vegetables, grains, and potatoes were the primary sources of dietary cadmium intake. The estimated total cadmium intake was approximately 15 µg/day/person. We also found a 17-fold difference in cadmium concentrations in carrots between the production areas, which was greater than the variation reported for carrots in Japan. The factors affecting the variation in cadmium concentrations in vegetables, including carrots, need to be clarified. As the relative importance of vegetables as a source of cadmium intake is increasing in Japan due to decreased rice consumption, more attention should be paid to variations in cadmium concentrations in vegetables in Japan.

A major health outbreak in March 2024 with renal impairments occurred by ingested food supplements made from beni-koji (red yeast rice) in Japan, which has been considered as major health issue. A troponoid compound puberulic acid (PA) that was produced by contaminated Penicillium spp. is attracting attention as a causative agent of this health disaster occurred by beni-koji (red yeast rice). Regarding toxicity, it was reported that PA showed weak cytotoxicity against fetal human lung fibroblast-like MRC-5 cells with an IC₅₀ value of about 289 μM (Iwatsuki et al., 2011). However, understandings about the physiological effects of PA against human tissues and cells still remain poor and insufficiently studied. Therefore, in this study, we investigated the effect of PA on the viability and the all-trans retinoic acid (ATRA)-induced superoxide anion (O₂^-)-generating ability of human leukemia U937 cells. PA remarkably showed a strong cytotoxicity accompanied by apoptosis, which was enhanced by ATRA. Furthermore, PA dramatically down-regulated the ATRA-induced O₂^--generating activity in a dose-dependent manner. Quantitative RT-PCR and immunoblot analyses showed that PA significantly reduces the ATRA-induced O₂^--generating activity via down-regulating gene expression levels of gp91-phox, which is an essential factor for the O₂^--generating activity of leukocytes. These findings revealed that PA has not only the strong ATRA-enhancible cytotoxic effect but also the drastic reducing effect on the ATRA-induced O₂^--generating activity through down-regulating transcription of gp91-phox gene. We expect that our findings will contribute to resolve the large-scale health disaster caused by beni-koji (red yeast rice).

Seborrheic dermatitis (SD) is a prevalent condition that results in dandruff, itching, and discomfort, and affect approximately 3–10% of the general population. Proliferation of the genus Malassezia, a microorganism inhabiting the scalp, is considered a contributing factor. Despite reducing the Malassezia population, other diseases, including SD, may still develop due to an increase in Staphylococcus aureus, which is associated with atopic dermatitis (AD) and SD, or a decrease in Staphylococcus epidermidis, which produces glycerol (moisturizer) and inhibits S. aureus growth. Therefore, we investigated the concentrations of anti-microbial reagents (pyrrolidone carboxylate-zinc [PCA-Zn] and Hinokitiol) and malt oligosaccharides (MT: corn-derived oligosaccharide mainly containing maltotetraose) that inhibited or promoted the growth of three types of scalp microorganisms. Individually, 0.50–1.00 mM PCA-Zn or 0.05–0.20 mM Hinokitiol displayed a marked growth-inhibitory effect on Malassezia furfur without a decline in S. epidermidis or an increase in S. aureus. Conversely, 0.02% MT individually exerted a growth effect on S. epidermidis but not on M. furfur. We then examined the effects of a mixture of the above-mentioned reagents on scalp-resident microorganisms. Our results indicated that 0.10 mM or 0.20 mM Hinokitiol combined with 0.02% MT markedly inhibited M. furfur growth and were the most effective at increasing S. epidermidis or decreasing S. aureus, compared to the single or combined effects of other reagents. Overall, our study provides valuable information on Hinokitiol and oligosaccharides concentrations in mixtures for use in shampoo-type cosmetics, and quasi-drugs, to prevent and treat SD.

Exomaltotetraohydrolase (G4ase) catalyzes the hydrolysis of (1->4)-α-_D-glucosidic linkages in amylaceous polysaccharides from the non-reducing ends removing successive maltotetraose residues. A safety assessment was conducted for G4ase produced by the non-genetically modified strain of Pseudomonas stutzeri, MO-19. Two standardized acute oral toxicity studies using female rats were performed on G4ase having a TOS not determined for the first study and 7.13% TOS for the second. The 50% lethal dose (LD₅₀) of G4ase was determined to be more than 2000 mg/kg, corresponding to more than 143 mg-TOS/kg. A 2-week oral repeated toxicity study in rats at 1000 mg/kg/day (highest dose; TOS not determined) demonstrated no treatment related toxicity and was used to identify the appropriate dose for a 90-day study. Results of a standardized 90-day oral repeated toxicity study (gavage) of G4ase (7.13%-TOS) using rats demonstrated that the No Observed Adverse Effect Level (NOAEL) of G4ase was 1000 mg/kg/day (the highest dose), corresponding to 71.3 mg-TOS/kg. Four standardized genotoxicity studies of bacterial reverse mutation, chromosomal aberration, and in vivo and in vitro micronucleus tests were performed on G4ase (5.19, 5.19, 7.13 and 6.65%-TOS, respectively). It was concluded that G4ase did not induce gene mutation in Salmonella typhimurium and Escherichia coli, did not induce chromosomal aberrations in cultured mammalian cells, and did not induce micronucleated erythrocytes in rat bone marrow cells or human spleen cell line lymphoblasts. Taken together these data indicate that G4ase from P. stutzeri strain MO-19 is safe for use as a processing aid in manufacturing food for human consumption.

Cadmium is a known contributing factor for cardiovascular diseases, such as atherosclerosis and hypertension. Although zinc protects against cadmium cytotoxicity in vascular endothelial cells, the detailed mechanisms, especially the differences in the roles of a metal transporter Zrt- and Irt-like protein 8 (ZIP8) and metallothionein (MT), remain unclear. ZIP8 is involved in the uptake of cadmium, whereas MT is induced by and sequesters it. Zinc protects bovine aortic endothelial cells from cadmium cytotoxicity in a culture system in a concentration-dependent manner. Zinc significantly decreased intracellular cadmium accumulation. Cadmium elevated the expression of ZIP8 mRNA, and zinc significantly suppressed this increase in a concentration-dependent manner. However, the expression of MT was not induced by zinc alone but by cadmium; the induction of MT by cadmium was suppressed by zinc at lower concentrations, but intensified by zinc at higher concentrations. However, even when MT induction was strongly suppressed by siRNA-mediated knockdown, cadmium cytotoxicity was reduced by zinc at both lower and higher concentrations. In the absence of zinc, cadmium cytotoxicity was increased by metal response element-binding transcription factor-1 (MTF-1) siRNA-mediated knockdown of MT-1/2. Consequently, it has been suggested that zinc protects the vascular endothelial cells through a concentration-dependent mechanism. Specifically, the decrease in ZIP8 expression is crucially important for the protective effect of zinc at low concentrations against cadmium cytotoxicity in vascular endothelial cells, whereas both the decrease in ZIP8 and the induction of MT contribute to the protection by zinc at high concentrations.

Aristolochic acids (AAs) with strong bio-toxicity are the natural compounds that consist in Aristolochiaceae plants. There are the governing health issues regarding toxicities of AAs although Aristolochiaceae plants have been used as herbal medicines. For example, AAs are known as significant risk factors for nephropathy, urological cancer, liver cancer and so on. However, the understandings about the molecular mechanisms of toxicity of each AA derivative have been still poor and insufficiently studied. In this study, we investigated the effects of four AA derivatives (AA-A, AA-B, AA-C and AA-D) on the viability and the all-trans retinoic acid (ATRA)-induced superoxide anion (O₂^-)-generating ability of human leukemia U937 cells. AA-A and AA-C remarkably reduced cell viability when co-treated with ATRA while AA-B and AA-D had little effect on viability of U937 cells. On the other hand, only AA-C among the four AAs dramatically up-regulated the ATRA-induced O₂^--generating ability. Quantitative RT-PCR and immunoblotting analyses showed that AA-C significantly enhances the ATRA-induced O₂^--generating ability via up-regulating gene expression levels of gp91-phox, which is an essential factor for the O₂^--generating ability of phagocytes. These findings revealed that AA-C has not only the ATRA-enhanced cytotoxic effect but also the remarkable enhancing effect on the ATRA-induced O₂^--generating ability via up-regulating transcription of gp91-phox gene.

Tomatidine is an aglycone of α-tomatine, a glycoalkaloid present in tomato plants, and has muscle atrophy inhibitory effect and anticancer activity. Tomatidine-Rich Tomato Leaf Extract powder (TRTLE) contains 60% tomatidine, which is converted to tomatidine by acid hydrolysis after extracting α-tomatine from tomato leaves. The purpose of this study was to evaluate the safety of TRTLE by conducting a series of toxicity studies in mice and bacteria to support its safe food use. Single-dose and 90-day repeated-dose toxicity study were conducted in 6-week-old ICR male and female mice to calculate the LD₅₀ and non-toxic dose (NOAEL) of TRTLE. In the single-dose toxicity study, a single oral dose of 667, 2,000, and 5,000 mg TRTLE/kg body weight (bw) was administered, and in the 90-day repeated-dose toxicity study, 133 mg TRTLE/kg bw was administered orally daily. In addition, the Ames test was performed with Salmonella Typhimurium and Escherichia coli to determine the genotoxic activity. The single-dose toxicity study indicated the LD₅₀ was 833 mg TRTLE/kg bw (tomatidine equivalent: 500 mg/kg bw). In the 90-day repeated-dose toxicity study, no abnormalities due to TRTLE were observed in each laboratory test, including general symptoms, body weight changes, hematology, urinalysis, and histopathological examination. In the Ames test, TRTLE was confirmed not to be mutagenic with or without metabolic activation. Based on these data, the NOAEL in mice was determined to be 133 mg TRTLE/kg bw (tomatidine equivalent: 80 mg/kg bw).

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.