Fundamental Toxicological Sciences

2020 - Vol. 7

2020 - Vol. 7

Letter
Effects of estrogen on fatty-acid-induced cytotoxicity in mouse Neuro-2a neural cells Vol.7, No.2, p.115-121
Masahiro Ogawa , Takahiro Kyoya , Takahiro Kimura , Megumi Terada
Released: March 27, 2020
Abstract Full Text PDF[1M]

The accumulation of free fatty acids induces lipotoxicity in neural cells. Estrogen, 17β-estradiol (E2) protects against the damage of cells in various organs and tissues. However, the role of E2 on lipotoxicity in neural cells remains unclear. In this study, we investigated the effects of E2 on stearic acid (saturated fatty acid)- and oleic acid (unsaturated fatty acid)-induced cytotoxicity in retinoic acid-induced mouse neuroblastoma Neuro-2a differentiated into neural cells. Cell viability was evaluated by lactate dehydrogenase release from Neuro-2a neural cells. Stearic acid and oleic acids suppressed the cell viability in a dose-dependent manner. E2 prevented oleic acid-induced cytotoxicity but had no effect on stearic acid-induced cytotoxicity. ERα-selective agonist prevented cytotoxicity in Neuro-2a neural cells. In contrast, ERβ-selective agonist slightly significantly enhanced the cytotoxicity in the presence of oleic acid. Oleic acid, but not stearic acid, increased the mRNA level of p62/Sqstm1. E2 treatment statistically significantly, but slightly, enhanced the stearic acid-induced Bax expression. In contrast, E2 and ERα-selective agonist inhibited the oleic acid-induced the p62/Sqstm1 expression. Our results suggested that fatty acids induced cytotoxicity in Neuro-2a neural cells, and estrogen prevented the oleic acid-induced cytotoxicity via ERα, but not ERβ. Further studies are needed to understand the role of ERβ in neuron injury under normal conditions.

Original Article
Evaluation of cytokine storms in a disseminated intravascular coagulation monkey model Vol.7, No.2, p.105-114
Yoshitaka Hirasawa , Atsushi Fujiwara , Kazuya Tabata , Kenji Yoshida , Tsutomu Negama , Takayuki Anzai , Shin-ichi Sato
Released: March 27, 2020
Abstract Full Text PDF[2M]

The purpose of this study was to profile cytokine storms (cytokine release syndrome) in the LPS-induced disseminated intravascular coagulation (DIC)-cynomolgus monkey model by measuring changes in 22 cytokines using Luminex. In this study, increases were noted in 20 cytokines, excluding IL-4 and IL-17A. Specifically, IL-6, IL-8, G-CSF and TNF-α, pro-inflammatory cytokines, and IL-10, an anti-inflammatory cytokine, as well as MCP-1, markedly increased by 10,000 pg/mL or more. In addition to the marked increases in the pro-inflammatory cytokines IL-6 and G-CSF, the concentrations of IL-5, IL-18, IFN-γ, VEGF and IL-15 increased continuously. Also, in addition to the marked increases in the pro-inflammatory cytokine IL-8 as well as in MCP-1, the concentrations of IL-1ra, IL-2, IL-1β, IL-12/23 (p40), GM-CSF and TGF-α gradually decreased after initially increasing. On the other hand, in addition to the marked increases in the pro-inflammatory cytokine TNF-α and anti-inflammatory cytokine IL-10, MIP-1β and MIP-1α transiently increased and then rapidly disappeared from serum. IL-13 increased at 6 hr after administration only. Since the behavior of cytokines in this monkey model was similar to those noted in DIC in humans, this model will be useful for evaluating the efficacy of anti-DIC drugs. In addition, this model will also be useful for assessing the risk of cytokine storm development, which is a serious adverse effect of certain types of antibody drugs and CAR-T cell-based therapies.

Original Article
5-Fluorocytosine induces fetal skeletal malformations in rats by altering expression of Homeobox genes Vol.7, No.2, p.97-103
Takayuki Kumamoto , Mika Senuma , Mai Todoroki , Fumiaki Kumagai , Hajime Imai , Reiko Suzuki , Tetsuo Ogawa , Makiko Kuwagata
Released: March 27, 2020
Abstract Full Text PDF[2M]

5-Fluorocytosine (5-FC) is an antimycotic and teratogenic compound. Oral administration of 5-FC to pregnant rats on gestation days (GD) 9 and 13 was shown to induce thoracolumbar supernumerary ribs (TSR, 14th rib) and abnormal digits, respectively, in fetuses. This study investigated the effects of 5-FC on homeobox genes, which control the anterior-posterior-axis. 5-FC (75 mg/kg) was administered orally on GD9 and GD13, and tissues collected from cranial and caudal regions of TSR sites were analyzed. Following 5-FC administration on GD9, the levels of expression of Hoxa10, which determine the position of the thoracic and lumbar vertebrae, were decreased at GD13. Analysis of hindlimbs 6 hours after administration on GD13 showed decreases in expression of Hoxa11, Hoxd12, and Hoxd13, the Hox genes responsible for limb formation from the proximal to distal, and from the anterior to posterior directions. The present findings showed that altered expression of Hox genes contributes to 5-FC teratogenicity.

Original Article
Utility of measuring long bone length in toxicity studies: results of a 14-day repeated dose oral toxicity study of dexamethasone in young, periadolescent and adult rats Vol.7, No.2, p.85-96
Chihiro Noguchi , Satoshi Tsuji , Yutaka Nakanishi , Minoru Sasaki
Released: March 03, 2020
Abstract Full Text PDF[6M]

Measurement of the bone length is not used routinely in toxicity studies but used to examine growth and bone toxicity. To investigate the utility of measuring the bone length in toxicity studies and to identify the appropriate site, we evaluated femur, tibia, humerus, and sternum, in a 14-day repeated dose oral toxicity study of Dexamethasone (DEX), which is known to cause growth retardation and osteoporosis, in young, periadolescent, and adult rats. To observe the effect of decreased food consumption, we also evaluated the changes in each diet-restricted group in which the food intake restricted to levels corresponding to that consumed by the DEX-treated periadolescent and adult rats. Significant decreases of the bone length at all the measured sites and histopathological findings in growth plates and/or trabecular bone were observed in the DEX-treated young and periadolescent rats. Significant decreases of the femoral length and decreased trabecular bone were observed in the DEX-treated adult rats. No histopathological changes were observed in any of the diet-restricted groups, while decreases of the femoral length, similar to that in the DEX-treated adult rats were observed in the diet-restricted adult rats. The results suggested that measurement of the bone length in femur, tibia, humerus, and sternum was useful in young and periadolescent rats, and measurement of the femoral length was useful in adult rats. Moreover, our results showed that the decreases in the femoral length in the DEX-treated adult rats were not only related to the DEX-treatment, but were also influenced by the decreased food consumption.

Original Article
Arsenite inhibits gene expression of perlecan, syndecan-1, -2, -3 and biglycan in cultured vascular endothelial cells Vol.7, No.2, p.77-83
Dong-pan Wu , Tsuyoshi Nakano , Yayoi Tsuneoka , Tsutomu Takahashi , Yo Shinoda , Toshiyuki Kaji , Yasuyuki Fujiwara
Released: March 03, 2020
Abstract Full Text PDF[1M]

Arsenic is an environmental pollutant and is a possible risk factor for vascular diseases such as atherosclerosis. Vascular proteoglycans (PGs) are key molecules in the initiation and progression of atherosclerosis. We previously demonstrated that arsenite, but not arsenate, decreases the synthesis of both heparan sulfate proteoglycans (HSPGs) and chondroitin/dermatan sulfate proteoglycans (CS/DSPGs) in cultured vascular endothelial cells. In the present study, we aimed to identify the PG molecules whose expression is decreased by arsenite, using a culture system of bovine aortic endothelial cells. The results indicate that a 24-hr treatment of arsenite significantly decreases the mRNA levels of a large HSPG perlecan, small HSPGs—syndecan-1, -2 and -3—, and a small CS/DSPG biglycan in vascular endothelial cells without nonspecific cell damage; the expression of syndecan-4 mRNA was unaffected by arsenite. The decreased expression of perlecan, syndecan-1 and biglycan genes began after 3 hr of arsenite treatment. However, arsenate did not change the mRNA expression levels of perlecan and biglycan in the cells. These results suggest that the inhibition of synthesis by arsenite occurs in particular types of proteoglycans, i.e. perlecan, syndecan-1, -2, -3, and biglycan in vascular endothelial cells.

Original Article
Toxicological evaluation of DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) Vol.7, No.2, p.55-76
Osamu Ohgoda , Ian N. Robinson
Released: March 03, 2020
Abstract Full Text PDF[1009K]

In the inhalation field, lipids such as egg phosphatidylcholine (PC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and dipalmitoylphosphatidylcholine (DPPC), are considered to be generally recognized as safe (GRAS), comprising materials that are endogenous to the lungs and locally present in large quantities. Indeed, PC, DSPC and DPPC may be used to form liposomes which are known to promote an increase in drug retention time and reduce the toxicity of drugs after administration. Unfortunately, published literature guidance about the safety evaluation of these lipids as pharmaceutical excipients for use in inhaled products and about application for marketing authorization, is very limited. The purpose of this article is to review the potential toxicity of DSPC for pulmonary administration. Given the use of air and vehicle controls in a range of inhalation toxicology studies as well as negative genotoxicity and also reproductive toxicity results, it is thought that the use of DSPC is shown to be safe for pulmonary administration.

Original Article
Comparison of the liver findings after simvastatin-treatment between Spontaneously Diabetic Torii-Leprfa (SDT fatty) rats and Sprague-Dawley rats Vol.7, No.1, p.41-54
Tadakazu Takahashi , Yusuke Suzuki , Naohito Yamada , Kaoru Toyoda , Keisuke Goda , Katsunori Ryoke , Chizuru Matsuura , Akio Kobayashi , Shoichiro Sugai , Kayoko Shimoi
Released: January 28, 2020
Abstract Full Text PDF[3M]

One of the risk factors for drug-induced liver injury (DILI) is the diabetic state. Our previous investigation showed that liver injury after repeated oral dosing with allyl alcohol and carbamazepine was enhanced more in the Spontaneously Diabetic Torii-Leprfa (SDT fatty) rats than in the Sprague-Dawley (SD) rats. It was caused by lower hepatic detoxification due to depleted hepatic glutathione synthesis. This is because simvastatin, frequently used in diabetic patients, shows a positive high reaction in a GSH adduct assay in vitro although the GSH adduction is considered not to be a major metabolic pathway of simvastatin. Therefore, in the present study, effects of simvastatin-treatment on the liver were compared between the Sprague-Dawley (SD) rats and SDT fatty rats in order to obtain additional information to estimate the potential risk of DILI in the diabetic state. There were no effects with simvastatin on the liver in the SD or SDT fatty rats after 13-week oral dosing of simvastatin. These results indicate that simvastatin does not have potential to induce liver injury in diabetic state and are consistent with the reports for the clinical use of simvastatin in the diabetic patients.

Original Article
Triphenyltin inhibits GA-binding protein α nuclear translocation Vol.7, No.1, p.33-40
Naohiro Kidoguchi , Keishi Ishida , Seigo Sanoh , Masatsugu Miyara , Yaichiro Kotake
Released: January 23, 2020
Abstract Full Text PDF[2M]

Organotin compounds such as triphenyltin (TPT), which are common environmental pollutants, had been widely used as antifouling agents for ship bottoms. Although toxic effects of organotins through nuclear receptors such as retinoid X receptor (RXR) and peroxisome proliferator-activated receptor (PPAR) γ have been well demonstrated, other mechanisms underlying organotin-induced toxicity have hardly been reported. In the present study, we focused on the transcription factor GA-binding protein (GABP), which regulates the expression of various housekeeping genes, as a novel target of TPT toxicity. We investigated the change of GABPα subunit protein expression induced by TPT. Although 100-500 nM concentration of TPT was not found to affect the total protein expression of GABPα, TPT significantly decreased nuclear translocation of GABPα in human embryonic kidney (HEK) 293T cells. In addition, TPT increased intracellular reactive oxygen species (ROS) levels. Both inhibition of GABPα nuclear translocation and the increase in ROS levels were observed in menadione (an ROS inducer)-treated HEK293T cells. Our results indicate that TPT causes inhibition of GABPα nuclear translocation, which may be triggered by ROS production. This might have serious implications in cellular physiology, thereby affecting cell survival.

Original Article
Safety and efficacy of a 48-week long-term ingestion of D-allulose in subjects with high LDL cholesterol levels Vol.7, No.1, p.15-31
Misuzu Tanaka , Akane Kanasaki , Noriko Hayashi , Tetsuo Iida , Koji Murao
Released: January 17, 2020
Abstract Full Text PDF[894K]

D-allulose is one of the rare sugars with almost zero calories and several health benefits. Previous studies have reported the safety of D-allulose in normal, overweight/obese, and diabetic humans. However, one study reported significant increases in T-Cho and LDL-C after 12 weeks of D-allulose intake; this report was not a randomized controlled trial and these changes were considered to be due to seasonal variations. We, therefore, conducted a randomized, double-blind, placebo-controlled trial in 90 subjects with high LDL-C levels for 48 weeks to clarify the influence of long-term D-allulose consumption on cholesterol metabolism and efficacy. Subjects were randomly divided into 3 groups: high-dose D-allulose (15 g D-allulose/day), low-dose D-allulose (5 g D-allulose/day), and placebo group (0 g D-allulose/day); each subject consumed a daily test beverage for 48 weeks. Clinical examinations were performed every eight weeks, beginning from initial consumption until week 52. No significant increases in T-Cho and LDL-C between test groups were observed, and 48 weeks of D-allulose consumption did not change risk factors for atherosclerotic cardiovascular disease. Furthermore, no clinical problems were recognized for other parameters. Additionally, significant improvements in hepatic enzyme activities, fatty liver score, and glucose metabolism after long-term D-allulose consumption were observed. The results from our study revealed that 1) D-allulose consumption is considered safe for long-term intake up to a year, and 2) D-allulose may be effective for improving hepatic functions and glucose metabolism.

Letter
Usefulness and limitations of mRNA measurement in HepaRG cells for evaluation of cytochrome P450 induction Vol.7, No.1, p.9-14
Kenta Mizoi , Yuuki Fukai , Eiko Matsumoto , Satoshi Koyama , Seiichi Ishida , Hajime Kojima , Takuo Ogihara
Released: January 15, 2020
Abstract Full Text PDF[1M]

Cytochrome P450s (CYPs) are involved in the metabolism of various drugs, and may generate toxic metabolites or intermediates that result in drug-induced liver injury (DILI). Consequently, inducers of CYPs may promote DILI. In a draft test guideline, the Organisation for Economic Co-operation and Development (OECD) recommends measurement of the metabolic activity of CYP as an index for assessing CYP-inducing activity. However, change of mRNA level has also been used as a simple parameter to evaluate CYP induction. In this study, therefore, we examined the usefulness and limitations of mRNA expression measurement for evaluation of the induction of CYP1A2, CYP2B6, and CYP3A4 by omeprazole, phenobarbital, and rifampicin (RIF), respectively, in HepaRG cells, a well-established cell line derived from human hepatocellular carcinoma. The results of mRNA measurement correlated well with the results of metabolic activity measurement in the lower concentration ranges for all inducers, even though we observed significant decreases in albumin and urea secretion in the presence of 10 µM RIF, reflecting its known hepatotoxicity. Our results indicate that mRNA measurements and metabolic activity measurements in HepaRG cells generally give comparable results for fold-induction of CYPs.

Original Article
Azoxystrobin at sub-cytotoxic concentrations disrupts intracellular zinc homeostasis: A flow cytometric analysis with rat thymic lymphocytes and fluorescent probes Vol.7, No.1, p.1-7
Mai Shoji , Masaki Asada , Akihiko Matsumoto , Haruki Nishino , Ao Yi Xiang , Mizuki Mizobuchi , Naoki Kanematsu , Hajime Miura , Norio Kamemura
Released: January 10, 2020
Abstract Full Text PDF[3M]

Azoxystrobin is a broad-spectrum fungicide having a wide usage. However, the toxic effect of azoxystrobin in humans is not reported. In Japan, azoxystrobin was detected at a five-fold higher concentration than the normal upper limit (2.5 mg/kg) in a shipment of an Australian barley used in different food products. Thus, there is a chance of azoxystrobin exposure through food to humans, and hence it is imperative to study the toxic effects of this compound. In this study, the toxic effect of azoxystrobin was evaluated to predict its adverse effects on human. Azoxystrobin at 3-30 µM (approximately 1.2-12.1 mg/L) raised the intracellular Zn2+ concentration of rat thymic lymphocytes. This increase was due to an influx of extracellular Zn2+ and a release of intracellular Zn2+. Azoxystrobin partially inhibited the temperature-dependent Zn2+ influx, thus jeopardizing the cellular Zn2+ homeostasis. Because Zn2+ is an important intracellular messenger in lymphocytes, this altered Zn2+ homeostasis might lead to adverse effects if the blood concentration of azoxystrobin reaches 3 µM or more in humans. However, by extrapolating the azoxystrobin pharmacokinetics data of rats to human, it can be predicted that such high blood concentration may be unlikely in humans.