Fundamental Toxicological Sciences

2024 - Vol. 11 No. 1

2024 - Vol. 11

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.