Fundamental Toxicological Sciences

2024 - Vol. 11

2024 - Vol. 11

Toxicomics Report
Alteration of gene levels in fetal brain by prenatal exposure to methylmercury, copper, and their combination Vol.11, No.3, p.131-139
Kensuke Sato , Ryota Nakano , Yoshitaka Yamazaki , Hikaru Isobe , Yun-Gi Kim , Masahiro Hosonuma , Masahiro Akiyama , Yoshito Kumagai
Released: June 19, 2024
Abstract Full Text PDF[1M]

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.

Minireview
Understanding the effects of food restriction on toxicological parameters: A comparative analysis in rats, dogs, and monkeys Vol.11, No.3, p.123-130
Nozomi Fujisawa
Released: June 19, 2024
Abstract Full Text PDF[763K]

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.

Original Article
Effect of differently coated titanium dioxide nanoparticles on the lung in wild-type and Nrf2 null mice Vol.11, No.3, p.109-121
Ryoya Takizawa , Akihiko Ikegami , Cai Zong , Syun Nemoto , Yuki Kitamura , Nathan Mise , Gaku Ichihara , Sahoko Ichihara
Released: June 11, 2024
Abstract Full Text PDF[1M]

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.

Original Article
The health risk assessment of mercury in rice from paddy fields around Nam Son landfill, Hanoi, Vietnam Vol.11, No.3, p.99-108
Nguyen Thi Quynh , Huiho Jeong , Ahmed Elwaleed , Yasuhiro Ishibashi , Koji Arizono
Released: June 11, 2024
Abstract Full Text PDF[2M]

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.

Original Article
Glial differentiation of mouse embryonic-stem-cell-derived neuronal precursors is a sensitive marker for assessing developmental neurotoxicity of insecticides Vol.11, No.3, p.87-98
Tomohiro Ito , Xian-Yang Qin , Yoshika Kurokawa , Yang Zeng , Satoshi Otsuka , Wenlong Wang , Hideko Sone
Released: June 11, 2024
Abstract Full Text PDF[2M]

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.

Original Article
Activation of Akt–cAMP response element-binding protein (CREB) signaling as an adaptive response to an electrophilic metabolite of morphine Vol.11, No.2, p.79-85
Kohei Matsuo , Shigeru Yamano , Akira Toriba , Kimihiko Matsusue , Yoshito Kumagai , Yumi Abiko
Released: May 21, 2024
Abstract Full Text PDF[1M]

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.

Letter
Investigation of solvents that can be used as vehicles to evaluate poorly soluble compounds in short-term oral toxicity studies in rats Vol.11, No.2, p.69-78
Keigo Ikeda , Mami Kochi , Tomoaki Tochitani , Naohisa Umeya , Izumi Matsumoto , Yuta Fujii , Toru Usui , Izuru Miyawaki
Released: May 14, 2024
Abstract Full Text PDF[2M]

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.

Original Article
Effects of food restriction for 3 or 7 days on toxicity-related parameters in rats Vol.11, No.2, p.57-67
Naohisa Umeya , Kumiyo Okada , Naoe Nishimura , Izumi Matsumoto , Toru Usui , Izuru Miyawaki
Released: April 09, 2024
Abstract Full Text PDF[4M]

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.

Original Article
Extracellular vesicle small RNAs secreted from mouse amniotic fluid induced by repeated oral administration of VPA to pregnant mice Vol.11, No.1, p.37-56
Ryuichi Ono , Makiko Kuwagata , Mie Naruse , Akihito Watanabe , Masao Takano , Takuro Hasegawa , Hiromasa Takashima , Yusuke Yoshioka , Takahiro Ochiya , Yoko Hirabayashi , Satoshi Kitajima
Released: March 22, 2024
Abstract Full Text PDF[3M]

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.

Original Article
Development of an in vivo pain assessment method for exposure to intermediate-frequency magnetic fields Vol.11, No.1, p.27-35
Shin Ohtani , Akira Ushiyama , Wasoontarajaroen Siriwat , Keiji Wada , Yukihisa Suzuki , Kenji Hattori
Released: March 19, 2024
Abstract Full Text PDF[2M]

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.

Original Article
Comparative analysis of bile canaliculi formation in fresh and flask-delivered human hepatocytes from humanized mouse livers under sufficient oxygen supply Vol.11, No.1, p.17-25
Fumiya Tokito , Ya Gong , Dhimas Agung Kurniawan , Shohei Kaneko , Hiroki Shioda , Sangho Lee , Atsuhito Kushima , Mutsumi Inamatsu , Chise Tateno , Hyunjin Choi , Masaki Nishikawa , Yasuyuki Sakai
Released: February 21, 2024
Abstract Full Text PDF[3M]

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.

Toxicomics Report
Indeterminate alteration of TRPM8 and p21/Cip1 levels in normal human keratinocytes by incubating with medium including substances released by squamous carcinoma cells Vol.11, No.1, p.11-15
Tomofumi Fujino , Saki Ohkawa
Released: February 09, 2024
Abstract Full Text PDF[1M]

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.

Original Article
Repeated dose administration toxicity studies - Use of t-tests in multiplicity data analysis Vol.11, No.1, p.1-10
Katsumi Kobayashi , Kalathil Sadasivan Pillai
Released: January 24, 2024
Abstract Full Text PDF[806K]

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.