Fundamental Toxicological Sciences

2018 - Vol. 5

2018 - Vol. 5

Toxicomics Report
Cyclin-dependent kinase inhibitor p21/Cip1 down-regulates the expression of proteasome activator PA28γ in normal hepatocyte-derived cells Vol.5, No.6, p.203-207
Tomofumi Fujino , Toshiyuki Oshima , Makio Hayakawa
Released: December 28, 2018
Abstract Full Text PDF[1M]

We previously reported that knockdown of the farnesoid X receptor (FXR), a bile acid-activated nuclear receptor, increases mRNA level of cyclin-dependent kinase inhibitor p21/Cip1 in normal hepatocyte-derived cell line Fa2N-4 and hepatocellular carcinoma cell line HepG2. Although p21/Cip1 protein levels of HepG2 cells are also increased by FXR knockdown, elevated levels of p21/Cip1 mRNA does not cause an increase in p21/Cip1 protein levels of Fa2N-4 cells, indicating post-transcriptional suppression of p21/Cip1 expression in Fa2N-4 cells. Given that degradation of p21/Cip1 by proteasomes is mediated by PA28γ, an activator of the 20S proteasome, we examined whether p21/Cip1 regulates the expression of PA28γ, a proteasome activator, in HepG2 and Fa2N-4 cells. In Fa2N-4 cells, ectopic expression of p21/Cip1 increased the mRNA and protein levels of PA28γ. PA28γ expression was down-regulated by knockdown of p21/Cip1. In contrast, in HepG2 cells, neither ectopic expression nor knockdown of p21/Cip1 affected the expression of PA28γ. Therefore, p21/Cip1 likely down-regulates its own expression in a post-transcriptional manner by stimulating the expression of the proteasome activator PA28γ in normal hepatocyte-derived cells, while hepatocellular carcinoma cells lack such feedback regulation of p21/Cip1 expression.

The temporal turning window for rat behavioral phenotypes by rotenone Vol.5, No.6, p.195-202
Masami Ishido
Released: December 28, 2018
Abstract Full Text PDF[13M]

Availability of animal models of human neuronal disease has been contributing to reveal their etiology. Particularly, dopaminergic neurodegeneration has been shown by exposure of rats to chemicals, such as rotenone, corresponding to the rat models of attention deficit hyperactivity disorder (ADHD) and Parkinson’s disease. A single chemical of rotenone causes two behavioral phenotypes, both of which are completely opposite; hyperactivity or hypoactivity. They were created by different timing of chemical exposure, neonatal periods or adulthood, suggesting that there would be a temporal turning point of two behavioral phenotypes. Therefore, we examine a turning point of these behavioral phenotypes by measuring the spontaneous motor activity of rotenone models. We estimate the turning window of both behavioral phenotypes would be around between three weeks and four weeks of age in the rat dopaminergic neurodegeneration. Gene set enrichment analysis extracts a cytokine network in both rat models.

Original Article
Study of metabolic activation of the UV-filter octyl methoxycinnamate for endocrine-disrupting activity Vol.5, No.6, p.185-193
Yoko Watanabe , Chihiro Emori , Naoto Uramaru , Nariaki Fujimoto , Shigeyuki Kitamura
Released: December 18, 2018
Abstract Full Text PDF[1M]

We examined whether octyl methoxycinnamate (OMC), a UV-filter, is metabolically activated by liver microsomes of rats and humans in respect to endocrine-disrupting action. OMC itself showed no agonistic activity towards estrogen receptor (ER) or aryl hydrocarbon receptor (AhR), and no antagonistic activity towards ER or androgen receptor (AR). The hydrolysis product, 4-methoxycinnamic acid (4-MCA), was also inactive in all the assays. In contrast, the desmethylated product, octyl hydroxycinnamate (OHC), exhibited agonistic activities towards ERα, ERβ and AhR. Importantly, when OMC was incubated with rat liver microsomes in the presence of NADPH, the major product was 4-MCA, and OHC was not formed at all. 4-MCA was also produced as the main metabolite of OMC by pooled human liver and small-intestinal microsomes. The OMC-hydrolyzing activity was higher in small-intestinal microsomes than in liver microsomes of both rats and humans, and was higher in humans than in rats. Therefore, OMC hydrolysis appears to be mainly catalyzed by small-intestinal carboxylesterase 2 isoforms, and partly by liver carboxylesterase 1 isoforms. We confirmed that OMC was hydrolyzed by human recombinant carboxylesterase isozymes, CES1b, CES1c and CES2. Our results indicate that OMC is not metabolically activated to OHC in humans, but is mainly hydrolyzed to inactive 4-MCA, suggesting that it is unlikely to pose a risk of human health in terms of endocrine-disrupting activity.

A zinc complex that suppresses the expression of a reactive sulfur species-producing enzyme, cystathionine γ-lyase, in cultured vascular endothelial cells Vol.5, No.6, p.181-184
Akane Takahashi , Musubu Takahashi , Tomoya Fujie , Takato Hara , Eiko Yoshida , Chika Yamamoto , Toshiyuki Kaji
Released: December 11, 2018
Abstract Full Text PDF[2M]

Persulfide species present in mammalian cells play important toxicological roles against oxidative stress and methylmercury. Cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS) are enzymes that produce persulfide species using cysteine as a substrate in the presence of vitamin B6. However, little is known about the regulatory mechanisms underlying the expression of these enzymes. In the present study, we searched for molecular probes from a library of 27 zinc complexes to analyze the mechanisms in vascular endothelial cells. We found dichloro(1,10-phenanthroline)zinc(II), termed Zn11, as a zinc complex that suppressed endothelial CSE expression without any change in CBS expression. This zinc complex can be used as molecular probes to analyze the regulation underlying endothelial CSE expression.

Original Article
Effects of fish oil-enriched Sasa-kamaboko diet in decreasing hepatic lipid levels in a mouse fatty liver disease model Vol.5, No.5, p.171-179
Masaaki Miyata , Tomoki Kinoshita , Kaho Miyahara , Mizuki Sato , Asaka Arikawa , Yoshimasa Sugiura , Yoshihisa Suzuki , Emiko Okazaki
Released: November 21, 2018
Abstract Full Text PDF[2M]

The influence of fish oil-enriched Sasa-kamaboko (a Japanese processed seafood) diet on lipid metabolism was investigated using mice lacking farnesoid X receptor (FXR) as a fatty liver disease model. Sasa-kamaboko (SK) was made from Alaska pollock surimi (fish paste), enriched with fish oil (0%, 2.5%, or 5.0%) and then freeze-dried. Fxr-null mice were fed the dried SK, mixed with AIN-93M chow in the ratio 1:1 (50% SK diet) or 1:3 (25% SK diet), for 4 weeks. Hepatic triglyceride levels, and total cholesterol levels were significantly decreased, which were dependent on the amount of added fish oil in 25% and 50% SK diets. Hepatic fatty acid synthase (Fas), acetyl-CoA carboxylase 1 (Acc1), and stearoyl CoA desaturase 1 (Scd1) mRNA levels and Fas protein levels were decreased in groups fed fish oil-enriched 50% SK diets. Brain and serum non-esterified fatty acid levels were significantly decreased in the groups fed fish oil-enriched 50% SK diet, whereas brain and serum, (but not hepatic) phospholipid levels were significantly increased in the groups. Hepatic, serum and brain n-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA; 22:6 n-3), and eicosapentaenoic acid (EPA; 20:5 n-3) levels (except for brain EPA levels) were significantly increased in groups fed fish oil-enriched 50% SK diet, whereas hepatic and serum mono-unsaturated fatty acid, oleic acid (18:1 n-9), and palmitoleic acid (16:1 n-7) levels were significantly decreased in the groups. These results suggest that a diet containing fish oil-enriched SK enhances hepatic lipid-lowering through the alteration of hepatic fatty acid composition in a fatty liver disease mouse model.

Original Article
Derivation of acceptable daily exposure value for alanine, N,N-bis(carboxymethyl)-, trisodium salt Vol.5, No.5, p.167-170
Masayuki Mishima , Dana Hoffmann , Gaku Ichihara , Satoshi Kitajima , Makoto Shibutani , Satoshi Furukawa , Akihiko Hirose
Released: November 06, 2018
Abstract Full Text PDF[794K]

Use of a non-phosphate detergent builder, alanine, N,N-bis(carboxymethyl)-trisodium salt (ABCT), has been expanded to wide range of washing and cleaning products for consumer uses and industrial applications including cleaning agents in food or pharmaceutical factories. Therefore, determination of acceptable daily exposure (ADE) of ABCT by oral, parenteral or inhalation route based on updated toxicity database could provide valuable information on the risk management for protection of consumers, patients and workers. Here, we proposed the ADEs based on the toxicological information of various in vivo and in vitro non-human studies. Because the full report of each toxicity study was not disclosed, derivation of the ADE was done based on available information mainly from ECHA database. ABCT exhibited renal toxicity as a main effect; however, ABCT did not exhibit carcinogenicity, genotoxicity, reproductive toxicity, irritation, and sensitization. Applying modification factors to the NOAEL of the animal study of longest treatment period, oral ADE was determined as 260 mg/person/day. Taking the oral bioavailability into the consideration of conversion to other routes, parenteral and inhalation ADEs were determined as 50 mg/person/day.

Protection from acetaminophen-induced hepatotoxicity by post-administration of 1O, 20O-diacetyl kamebakaurin in mice Vol.5, No.5, p.161-165
Hiroki Yoshioka , Hiroyuki Ohnishi , Masae Yoshikawa , Ming-Yu Gui , Yong-Ri Jin , Xu-Wen Li , Yoshiyuki Adachi , Naohito Ohno , Koichi Takeya , Yukio Hitotsuyanagi , Nobuhiko Miura , Yutaka Aoyagi
Released: October 19, 2018
Abstract Full Text PDF[3M]

Our previous study demonstrated that pre-administration of 1O, 20O-diacetyl kamebakaurin (Ac2KA) protected against acetaminophen (APAP)-induced hepatotoxicity. In the current study, we aimed to investigate whether post-administration of Ac2KA also protects against APAP-induced hepatotoxicity. Eight-week-old male C57BL/6J mice were fasted and then intraperitoneally injected with 450 mg/kg APAP or saline. At 60-min after the APAP injection, Ac2KA (50 mg/kg) or an ethanol/olive oil emulsion was orally administered. At 16-hr after the injection, the mice were killed, and blood samples were collected for plasma analysis. As a positive control, we used N-acetylcysteine (200 mg/kg, i.p.). Posttreatment with Ac2KA significantly attenuated APAP-induced plasma alanine aminotransferase and aspartate aminotransferase levels. Ac2KA administration also decreased the APAP-induced hepatic malondialdehyde concentration. Moreover, histological evaluation supported these observations. Our results show that Ac2KA exerts protective effects against APAP-induced hepatotoxicity when administered as both pretreatment and post-treatment.

Original Article
In utero exposure to 2,2’,4,4’,5,5’-hexachlorobiphenyl accelerates the onset of eye opening in rat offspring Vol.5, No.4, p.153-159
Kenichi Kobayashi , Muneyuki Miyagawa , Rui-Sheng Wang , Megumi Suda , Soichiro Sekiguchi , Takeshi Honma
Released: October 16, 2018
Abstract Full Text PDF[1M]

Prenatal exposure to 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and coplanar polychlorinated biphenyl (PCB) congeners has been reported to accelerate the onset of eye opening of rodents, but the effects of exposure to non-coplanar PCB congeners on the onset of eye opening remain unknown. TCDD binds to the cytosolic ligand-activated transcription factor aryl hydrocarbon receptor (AhR), leading to adverse effects through the alteration of AhR target gene expression. In contrast, non-coplanar PCB congeners show little or no binding to AhR. We examined whether in utero exposure to 2,2’,4,4’,5,5’-hexachlorobiphenyl (PCB 153), a di-ortho-substituted non-coplanar PCB congener, affects the onset of postnatal eye opening of rat offspring. Pregnant rats were given PCB 153 (0, 16, or 64 mg/kg/day) orally from gestational day 10 to 16, and somatic growth and eye opening were assessed in pups. Body weight, body length, and tail length measurements in the PCB 153 groups were lower than the values measured for the control group on postnatal days 1 to 21. However, PCB 153-exposed pups exhibited dose-dependent acceleration of eye opening. These findings suggest that in utero exposure to PCB 153 delays postnatal development but induces unexpected acceleration of eye opening that might not occur through interaction
with AhR.

Positions and numbers of hydroxyl groups in hydroxychalcone derivatives are involved in cytotoxicity against human monoblastic U937 cells Vol.5, No.4, p.149-152
Hidehiko Kikuchi , Futoshi Kuribayashi , Harishkumar Madhyastha
Released: September 29, 2018
Abstract Full Text PDF[929K]

Hydroxychalcone derivatives belonging to polyphenol group, and distributed throughout the plant kingdom, can behave as anti-cancer agents due to their cytotoxic activities. Hydroxychalcone derivatives also have the potential as chemotherapeutic drugs for cancer. In this paper, we revealed that positions and numbers of hydroxyl groups in hydroxychalcones are involved in cytotoxicity against human monoblast U937 cells. Interestingly, 2-hydroxychalcone remarkably reduced viability of U937 cells as compared to chalcone, 2’-hydroxychalcone, 4-hydroxychalcone, and 4’-hydroxychalcone. In addition, 2’, 4, 4’-trihydroxychalcone showed stronger cytotoxicity than 2’-hydroxychalcone, 2’, 4-dihydroxychalcone, and 2’, 4’-dihydroxychalcone. These results demonstrate that positions and numbers of hydroxyl groups in hydroxychalcones are involved in cytotoxicity against U937 cells. Moreover, we showed that all-trans retinoic acid-induced differentiation of U937 cells brought about an enhanced resistance against some cytotoxic hydroxychalcone derivatives. These data suggest that hydroxychalcone derivatives in combination with all-trans retinoic acid could serve as effective modifiers in therapy for leukemia.

Original Article
Anatomical and histological sex differences in the eye and its accessory tissues in Dutch belted rabbits Vol.5, No.4, p.141-147
Gakushi Kito , Ikuyo Atsumi , Yoshinori Yamagiwa , Hideyuki Sakaki , Masaaki Kurata
Released: July 18, 2018
Abstract Full Text PDF[611K]

Rabbits are commonly used as laboratory animals in ocular toxicity studies. During development of ophthalmic drugs and medical devices, knowledge regarding sex differences of ocular anatomy and histology is important in the interpretation of toxicity data. However, limited information is available regarding ocular sex differences in rabbit eyes. Information on sex differences of rabbit eyes supports the justification of using single sex in the design of toxicity studies. We therefore investigated anatomical and histological ocular sex differences in Dutch belted rabbits aged 6 to 20 weeks which are frequently used in toxicity studies. Anatomical parameters of the eyeball (axial length, diameter, weight, volume), cornea (height, diameter), lens (thickness, diameter, weight, volume), and vitreous humor (weight, volume) were measured. Paraffin blocks of eye, meibomian glands, and lacrimal glands from males and females were sectioned and stained using hematoxylin and eosin, and compared. As the results, the anatomical parameters and their growth ratios showed no difference between males and females. In the histological comparison, although the development of glandular tissues was observed in both sexes according to aging, no sex differences were found. We concluded that there is no anatomical or histological sex differences in the eyes of Dutch belted rabbits from the period of post-weaning to sexual maturation.

Original Article
Carcinogenicity study of poly-trans-[(2-carboxyethyl)germasesquioxane] (Ge-132) in F344 rats Vol.5, No.4, p.127-140
Katsuharu Iwadate , Yukari Yamaguchi , Mai Sasaki , Mikiya Nakatani , Yuko Doi , Norio Imai , Seiko Tamano , Yorifumi Nishihori
Released: July 10, 2018
Abstract Full Text PDF[423K]

Ge-132 was administered to both sexes of F344 rats at dietary levels of 0, 0.6, 1.3, and 2.5% for 2 years. There were no adverse effects on survival rate, food consumption or hematology data, although diarrhea and body-weight retardation were observed in the male and female 2.5% groups. Significant increases of kidney and adrenal weights were noted in both male and female 2.5% group. Macroscopically, dilatation of the cecum was observed in both male and female 2.5% group, and enlargement of the adrenals was observed in the male of 2.5% group. Significantly higher incidences of benign or malignant pheochromocytoma were observed in the male 1.3% group and both male and female 2.5% groups. Significantly positive trends were noted in the incidences of kidney pelvic and papilla mineralization in both sexes and cortico-medullary junction in females. To investigate possible mechanisms underlying Ge-132-associated development of pheochromocytoma, male F344 rats were administered diets containing 0, 0.6, or 2.5% Ge-132 for 4 or 13 weeks. Although loose stools and increasing water consumption were observed in treated groups, there were no body-weight retardation. Significant elevation of inorganic phosphorus in the serum was found in the 2.5% group at week 13. Dilatation of the cecum and increased cecum weight were evident macroscopically in the 2.5% group. Significant elevation of Ki-67 positive ratio in adrenal medullary cells was also found in the 2.5% group. These data indicated that Ge-132 ingestion induced disturbance in calcium/phosphorus homeostasis, and secondarily induced the development of benign or malignant pheochromocytoma in rats. Such secondary pheochromocytomas are considered to be not relevant for human risk assessment.

The impact on the activity of acetylcholinesterase of a polylysine-ApoE peptide carrier targeting the blood brain barrier Vol.5, No.4, p.123-126
Toby M. Michelena , Xuechen Tian , Xuan Zhou , Yu Meng
Released: July 03, 2018
Abstract Full Text PDF[212K]

The K16ApoE peptide has been demonstrated to deliver a supraphysiological level of protein therapeutics to the brain and further increase the life-span of mice with a lysosomal storage disorder (LSD). If successfully developed, K16ApoE would provide new treatments for LSD and many other neurological diseases. However, while the K16ApoE can cross the blood-brain barrier, data indicates a toxic response associated with it. The mechanism of toxicity must be resolved for further clinical translation. The toxic response towards the peptide was hypothesized to be induced by inhibition of acetylcholinesterase (AChE) activity at neuro-muscular junction. Here, the dose-response analysis between AChE and K16ApoE was conducted in both female and male mice. Results demonstrated that AChE activity was significantly reduced with increasing dose of K16ApoE except for the mid-dose where a dramatic increase in AChE activity was observed. Also, obvious difference in response to K16ApoE was shown when considering the influence from sex and body weight. Though the statistical analysis of the dose response and survival ratio suggested that AChE is not the primary mechanism of action for the acute toxicity of K16ApoE, the complex inhibition/stimulation response of AChE indicated that this enzyme must play a role in the toxicity of the peptide.

Chlorella supplementation decreases methylmercury concentrations of hair and blood in healthy volunteers Vol.5, No.3, p.117-122
Isao Maruyama , Takuya Uchikawa , Toshihiro Kanno , Yotaro Ando , Hisao Kitsuki , Akira Yasutake
Released: June 22, 2018
Abstract Full Text PDF[350K]

An open-label clinical trial was performed to test the effects of unicellular green alga Chlorella supplementation on mercury concentrations of hair and blood in healthy subjects. Fifty-eight healthy participants (36 male and 22 female) were assigned to Chlorella and control groups. The Chlorella group of 35 subjects received Chlorella tablets (9 g/day) for an experimental period of 3 months while the control group of 23 subjects did not. Total mercury concentrations of hair and blood were analyzed at the beginning and end of the experimental period for estimation of methylmercury (MeHg) levels in the body. The hair mercury concentration of the Chlorella group (n = 33) was significantly decreased during the experimental period (p = 0.041) while the change in the control group (n = 23) was not significant (p = 0.362). Although the decrease in blood mercury concentration in the Chlorella group (n = 19) was not significant (p = 0.084), the change of values (values at end – values at beginning) in this group was significantly greater than that in the control group (n = 20, p = 0.038). The fish intake rates remained relatively constant during the experimental period in both the Chlorella and control groups. These results suggest that supplementation with Chlorella for 3 months in healthy subjects might reduce their body MeHg levels.

In vitro metabolism of 4-methyl- and 5-methyl-2-mercaptobenzimidazole, thyrotoxic and hepatotoxic rubber antioxidants, in rat liver microsomes Vol.5, No.3, p.113-116
Atsuko Miyajima , Yukie Kuroda , Kazue Sakemi-Hoshikawa , Makoto Usami , Katsuyoshi Mitsunaga , Tomohiko Irie , Yasuo Ohno , Momoko Sunouchi
Released: June 19, 2018
Abstract Full Text PDF[728K]

The metabolism of 4-methyl-2-mercaptobenzimidazole (4-MeMBI), 5-methyl-2-mercaptobenzimidazole (5-MeMBI), and 2-mercaptobenzimidazole (MBI) was examined in vitro in rat liver microsomes. The test chemicals were incubated in the presence of liver microsomes from male Sprague-Dawley rats, and their metabolism was analyzed by HPLC. The metabolism amount increased in an incubation time-dependent manner, and was similar among the test chemicals. SKF-525A, a non-selective inhibitor of cytochrome P450 (CYP) enzymes, decreased the metabolic rate of all the test chemicals, indicating the involvement of liver microsomal CYP enzymes. When liver microsomes from rats treated with CYP-inducers (β-naphthoflavone, phenobarbital, and isoniazid) were used, 4-MeMBI was more decreased than 5-MeMBI, particularly in the phenobarbital-treated group. These results, together with the reported inducibility of the drug-metabolizing activity by the test chemicals, partly explained the counteraction in the toxic effects between 4-MeMBI and 5-MeMBI in the in vivo study.

Original Article
The Kampo formula “Juzen-taiho-to” exerts protective effects on ethanol-induced liver injury in mice Vol.5, No.3, p.105-112
Shiori Fukaya , Akito Nagatsu , Hiroki Yoshioka
Released: June 19, 2018
Abstract Full Text PDF[5M]

The aim of this study was to investigate whether the Japanese herbal medicine “Juzen-taiho-to (JTX)” has a protective effect on ethanol (EtOH)-induced liver injury. Seven-week-old male ICR mice were orally administered JTX or saline once a day for three days. Twenty-four hours after the last administration, the mice were intraperitoneally injected with EtOH (2 g/kg). The mice in each group were killed 24 hr after EtOH administration and were bled to obtain plasma. The mice injected with EtOH had high plasma levels of alanine aminotransferase and aspartate aminotransferase and lipid peroxidation. Histopathological examination of the liver of mice treated with EtOH revealed an abnormal outline around the central vein, glycogen depletion, and expression of prostaglandin-endoperoxide synthase 2. Pretreatment with JTX prevented the EtOH-induced increase in the levels of alanine aminotransferase and aspartate aminotransferase, lipid peroxidation, and histopathological changes. Our results suggest that JTX exerts protective effects against EtOH-induced liver disease by modulating oxidative stress and inflammatory response.

Original Article
Biphenyl increases the intracellular Ca2+ concentration in HL-60 cells Vol.5, No.3, p.99-103
Tomoko Inubushi , Mayumi Sugimoto , Haruka Kunimi , Haruka Hino , Atsushi Tabata , Naohiro Imura , Shin Abe , Norio Kamemura
Released: June 01, 2018
Abstract Full Text PDF[305K]

Biphenyl is a universal intermediate agent used as a protectant in various industrial activities. Biphenyl is currently used in postharvest applications as a fungicide in foreign countries to maintain the safety and quality of agricultural products. However, the risk of using biphenyl is in dispute in Japan. The toxicity of biphenyl has been studied in animals, and reportedly affects the liver and kidney especially. However, the toxic effect of biphenyl on cells is currently not well-understood, and the mechanism is unclear. We examined the toxicity of biphenyl on HL60 cells, a human promyelocytic leukemia cell line, by performing flow cytometry analyses with fluorescent probes. Biphenyl at 100 μM or greater significantly increased lethality and the intensity of side scatter on HL60 cells. Moreover, biphenyl at 30 μM or greater increased intracellular Ca2+ in a concentration-dependent manner. This increase resulted from free extracellular Ca2+ entering into the cells. These results indicate that an increase in intracellular Ca2+ may be one of several causes of the cytotoxicity induced by biphenyl. This study will contribute to the safety evaluation of biphenyl in the future.

Effect of heat shock protein gene expression on cadmium toxicity in human proximal tubular cells Vol.5, No.3, p.93-97
Jin-Yong Lee , Maki Tokumoto , Gi-Wook Hwang , Masahiko Satoh
Released: May 30, 2018
Abstract Full Text PDF[594K]

Cadmium (Cd) is an environmental pollutant which triggers toxic effects on various tissues, including the kidney. Proximal tubular cell damage is characteristic of Cd-induced renal toxicity. In our previous study, DNA microarray results showed that Cd treatment changed the expression levels of many genes in HK-2 human proximal tubular cells. Among the genes which increased their expression levels after Cd treatment, there were several genes coding for heat shock proteins. In the current study, we examined the role of heat shock protein genes in Cd-induced toxicity in HK-2 cells. Cd treatment increased the expression of the HSPA1, HSPH1, and HSPA8 genes in HK-2 cells. From these identified genes, only knockdown of HSPH1 and HSPA8 by siRNA treatment was found to decrease the viability of low-dose Cd-treated HK-2 cells compared with the control siRNA treatment group. These results suggest that several heat shock proteins are involved in the pathway which protects HK-2 cells against Cd toxicity.

Original Article
Cadmium, arsenic and lead accumulation in rice grains produced in Senegal river valley Vol.5, No.2, p.87-91
Moussa Ndong , Nathan Mise , Masaki Okunaga , Fujio Kayama
Released: May 22, 2018
Abstract Full Text PDF[206K]

Exposure to Cadmium (Cd), Arsenic (As), and Lead (Pb) in short or long term can cause health problems in humans. Rice is particularly susceptible to heavy metals contamination. Rice is the major staple food of different developing countries like Senegal leading to high exposure of the population to heavy metals if the rice is contaminated. In Senegal, two types of rice are consumed: local rice mainly produced in the Senegal river valley and imported rice from Asian countries. Thus, the objective of this study was to determine heavy metals accumulation in rice grains produced in Senegal or imported. Samples of five rice varieties produced in three different areas of the Senegal river valley and samples of imported rice from Japan, Thailand and Pakistan were analyzed for As, Cd and Pb contamination. The results showed that all samples were conform in term of contamination by As, Cd and Pb. Changes in heavy metals contamination were noticed between some rice varieties and according to localities. They were not a significant difference in the risk of exposure to heavy metals between the consumption of local produced rice and imported rice. However, the high rice intake of Senegalese could affect the safety of dietary intake of these metals by rice.

Risk assessment of reproductive and developmental toxicity in eye-drop drugs: a consideration based on threshold of toxicological concern Vol.5, No.2, p.83-85
Masaaki Kurata , Gakushi Kito , Kazuyo Sadamoto , Shingo Nemoto
Released: April 25, 2018
Abstract Full Text PDF[455K]

The systemic dose levels of eye-drop drugs are relatively low in comparison with that of systemic routes such as oral administration. We undertook overall risk assessment of eye-drop drugs for developmental and reproductive toxicity (DART) by comparing the estimated systemic dose level of eyedrop drugs with the known threshold of toxicological concern (TTC) for DART. The systemic dose level of eye-drop drugs in human were estimated to be 0.0005 to 0.05 mg/kg/day on the assumption of 0.01% to 1% of eye drop formulation, 0.04 mL/eye/time of instillation volume, and 60 kg body weight. The TTCs for DART ranged from 0.003 mg/day (0.00005 mg/kg/day; for anticacer drugs) to 7.860 mg/day (0.131 mg/kg/day). Therefore, the range of estimated systemic dose level of eye-drop drugs was almost overlapped with known TTC values for DART, excepting for that applied to the anticacer drugs. These knowledge simply indicates the safety of eye-drop drugs for DART from a view pont of absolute dosage levels, implying allowance of case by case basis planning non-clinical DART study.

Male-specific prolongation of prothombin time by industrial chemicals Vol.5, No.2, p.75-82
Takashi Yamada , Yushiro Tanaka , Ryuichi Hasegawa , Toshime Igarashi , Akihiko Hirose
Released: April 18, 2018
Abstract Full Text PDF[1M]

Prolongation of prothrombin time (PT) induced by industrial chemicals was characterized using a database of repeated dose toxicity studies, HESS DB. Of the 685 chemicals in the DB, 20 chemicals markedly prolonged the PT by more than 150% of that of vehicle control. Prolonged PTs were detected in males for 20 chemicals but no significant prolongation of PT was observed in females for 19/20 chemicals, indicating that males are apparently more susceptible to PT prolongation than females. The effective dose of the chemicals for males were relatively high, in the range of 100 to 1,000 mg·kg-1·day-1, compared to the dose range of 60 to 100 μg·kg-1·day-1 for warfarin, a typical anticoagulant. Since not all chemicals had severe hepatotoxic effects at these doses, the low protein synthesis capacity of the liver may not contribute to prolonged PT. The mechanism of PT prolongation by the chemicals was considered different from that of warfarin, which is a specific inhibitor of vitamin K epoxide reductase, because of large differences in their effective dose and lack of structural similarity between them. Herein, the possible mechanisms of PT prolongation by industrial chemicals in males are explored, with a focus on the action of estradiol and vitamin K.

Toxicomics Report
Putrescine selectively alleviates methylmercury toxicity in C17.2 mouse neural stem cells Vol.5, No.2, p.71-73
Masayuki Sato , Jin-Yong Lee , Min-Seok Kim , Tsutomu Takahashi , Akira Naganuma , Gi-Wook Hwang
Released: April 18, 2018
Abstract Full Text PDF[906K]

We previously reported fluctuating levels of many metabolites in the brain of mice administered methylmercury. In this study, addition of putrescine, a polyamine, to the medium was found to confer methylmercury resistance to C17.2 mouse neural stem cells with regard to metabolites increased by methylmercury in the mouse brain. However, putrescine had little effect on the cytotoxicity of heavy metals such as cadmium and inorganic mercury. These results suggest that putrescine may selectively alleviate methylmercury toxicity.

Assessment of bladder DNA damage due to 2-acetylaminofluorene, diuron, and terephthalic acid by the rat comet assay Vol.5, No.1, p.63-69
Takahiro Kyoya , Rika Iwamoto , Yuko Shimamura , Megumi Terada , Shuichi Masuda
Released: March 20, 2018
Abstract Full Text PDF[801K]

Chemicals that induce tumors in the urinary bladder in long-term toxicity tests often induce hyperplasia, a pre-cancerous lesion in subacute toxicity tests. Determining whether or not the mechanism by which hyperplasia induced in the urinary bladder is due to genotoxicity at an early stage is important for the extrapolation of carcinogenicity to humans. An in vivo comet assay is suitable for shortterm evaluation of urinary bladder genotoxicity. 2-Acetylaminofluorene (2-AAF), diuron, and terephthalic acid (TPA) are known to induce tumors in rat bladder by different mechanisms in long-term toxicity tests, but so far there are no reports on the bladder comet assay for the evaluation of these compounds in short-term toxicity tests. Here, a comet assay in rat urinary bladder for short-term evaluation was assessed by using rat strains in which hyperplasia induction has been reported for diuron and TPA, both of which induce hyperplasia in subacute toxicity tests, and rat strains in which tumors have been observed in longterm toxicity tests of 2-AAF. Regarding the respective DNA damaging properties of these compounds, the result was positive for 2-AAF, a typical genotoxic urinary bladder carcinogen, but negative for diuron and TPA, which induce hyperplasia through the chemical action of metabolites and through physical cytotoxicity due to urinary crystals, respectively. Thus, the comet assay of rat urinary bladder as a short term in vivo genotoxicity test is considered to be a useful and convenient method for the short-term evaluation of the genotoxicity of compounds which induce hyperplasia in the urinary bladder.

Scanning electron microscopic analysis of the bladder epithelium in terephthalic acid administered rats: a case in acute toxicity study Vol.5, No.1, p.57-62
Takahiro Kyoya , Megumi Terada
Released: March 09, 2018
Abstract Full Text PDF[3M]

Terephthalic acid (TPA) was observed to have induced tumors due to the physical cytotoxicity of urinary calculi in the urinary bladders of rats. In the acute toxicity study, the extrapolation to humans was evaluated hastily by observation of the urinary crystals and cytotoxicity on the bladder epithelium. We examined whether it was possible to observe urine crystals and corresponding alterations on the epithelium in the urinary bladders in the acute toxicity study of TPA. TPA at a dose level of 2,000 mg/kg body weight, were administered twice by oral gavage at a 21-hr interval and the bladder mucosal epithelium was observed at 3, 6 and 9 hr after the second administration using scanning electron microscope (SEM). As a result, micro crystals were observed in the bladder surface at 6 and 9 hr after the second administration, and small raised ridges on the bladder surface, which were considered to be the effects of cytotoxicity, were observed at 9 hr after the second administration. However, these were not observed in rats administered only 0.5% sodium carboxymethyl cellulose solution, which is a vehicle of TPA. In this study, in cases where the substances induce crystals in urine in a short period, it was suggested that the urinary crystals and the alterations on bladder epithelium could be detected using SEM in acute toxicity study.

Original Article
Abundant migration of midazolam and 1’-hydroxymidazolam into the fetal brain following midazolam administration to pregnant mice in the second trimester Vol.5, No.1, p.49-56
Satoshi Kitaoka , Jo Hatogai , Nanako Niikura , Miho Hirata , Nanako Fukasawa , Satoru Miyazaki , Naoto Yamaguchi , Shohei Harada , Masanaho Sasatsu , Kiyoshi Sugiyama , Wataru Ochiai
Released: March 07, 2018
Abstract Full Text PDF[513K]

Midazolam is used in pregnancy post the second trimester for the treatment of convulsive seizures or as an anesthetic during cesarean section. However, its safety has not been validated. If the migration and accumulation of midazolam into the fetal brain can be clarified, the extent of damage to the fetal brain can be predicted. Therefore, we investigated the migration of midazolam and its active metabolite 1′-hydroxymidazolam into the fetal brain. Midazolam was administered intravenously to pregnant mice in the second trimester (E14.5), and the migration of midazolam and 1′-hydroxymidazolam to the maternal brain and plasma as well as the fetal brain were analyzed over time. Fetal brain levels of midazolam and 1′-hydroxymidazolam were high at 44.7% and 44.5% of that in the maternal brain. Furthermore, both mothers and fetuses expressed 1:1 brain ratios of midazolam and 1′-hydroxymidazolam. In both mothers and fetuses, migration of midazolam into the brain was higher than the migration of 1′-hydroxymidazolam. In this study, approximately half of midazolam and 1′-hydroxymidazolam in the maternal brain was transferred to the fetal brain. During the second trimester, neural stem cells in the fetal brain differentiate into neurons and glial cells and higher brain functions are developed. In the present study, we observed increased migration of midazolam and 1′-hydroxymidazolam into the fetal brain during this period. Therefore, damage to neuronal development may still be observed in the fetus even with post-second trimester midazolam use.

Original Article
Subacute toxicity evaluation of KMRC011, a Toll-like receptor-5 agonist administered by intramuscular injection to rats Vol.5, No.1, p.33-48
Min-Sung Kang , Min-Seok Kim , Sung-Hwan Kim , Doin Jeon , Hyeon-Young Kim , Kwang-Hyun Lim , Su-Cheol Han , Kyuhong Lee , Woo-Jong Lee , Chi-Min Choi , Hong-Soo Lee , Jong-Choon Kim
Released: March 07, 2018
Abstract Full Text PDF[3M]

The present study investigated the potential subacute toxicity of KMRC011, a Tolllike receptor-5 agonist, by a 4-week repeated intramuscular injection in Sprague-Dawley rats. The test article was administered once daily by intramuscular injection to rats at doses of 0, 0.06, 0.13, and 0.25 mg/kg/day for 4 weeks. At the end of the treatment period, 10 rats/sex/group were sacrificed. The study was continued for the remaining 5 rats/sex in the vehicle control and high dose groups without treatment for 2 weeks (recovery period). During the test period, clinical signs, mortality, body weight, food consumption, ophthalmoscopy, urinalysis, hematology, serum biochemistry, gross findings, organ weight, and histopathology were examined. Hematological investigations revealed a decrease in the hemoglobin, hematocrit, and mean corpuscular hemoglobin values and an increase in the absolute and relative reticulocyte counts. Histopathological evaluation indicated an increase in the incidence of inflammatory cell infiltration in the cecum, lymphocytes infiltration in the duodenum, and hemopoiesis in the femoro-tibial joint/marrow and sternum/marrow in male and female rats. These changes decreased or were no longer observed after the 2-week recovery period, indicating these were reversible changes. Otherwise, no adverse effects were observed in any treatment group. Based on these results, the no-observed-adverse-effect level was considered to be greater than 0.25 mg/kg/day in the rats

Original Article
Comparison of stabilities of nitrenium ions and in vitro and in vivo genotoxic potential, between four aniline derivatives Vol.5, No.1, p.21-32
Hidekazu Suzuki , Takuya Ninoseki , Ayumi Hayashi , Yasunori Hase , Takuya Matsui , Masaru Naito , Shoichiro Sugai
Released: February 21, 2018
Abstract Full Text PDF[255K]

Stabilities of nitrenium ions estimated by in silico analyses and in vitro and in vivo genotoxicity were compared for four aniline derivatives, 2-chloro-4-methylaniline (2C4MA), 4-chloro-2-methylaniline (4C2MA), 2-chloro-4,5-difluoroaniline (2C4,5DFA) and 4-trifluoromethylaniline (4TFMA). The AM1 values as an index of stability of the nitrenium ions of 2C4MA, 4C2MA, 2C4,5DFA and 4TFMA were -5.38, -4.67, 8.36 and 16.6 kcal/mol, respectively, indicating that the potential of mutagenicity is high for 2C4MA and 4C2MA and low for 2C4,5DFA and 4-TFMA. The specific mutagenicity determined in Ames tests with S9 mix for 2C4MA and 4C2MA was 4,067 and 12,500 revertants/mg/plate, respectively. The specific mutagenicity could not be determined for 2C4,5DFA because the results of the Ames tests were equivocal. Among the four aniline derivatives, only 4TFMA showed positive results with and without S9 mix in the Ames tests and the specific mutagenicity of 4TFMA were 1,590 and 1,910 revertants/mg/plate for TA100 with and without S9 mix, respectively. These results indicated that the mutagenic potential is high for 2C4MA and 4C2MA and is low for 2C4,5DFA and 4TFMA. In vivo genotoxicity is positive for 2C4MA, 4C2MA and 2C4,5DFA and is negative for 4TFMA. The results of in silico analyses and in vitro and in vivo genotoxicity tests were consistent for aniline derivatives with strong mutagenicity (2C4MA and 4C2MA) but were not for those with weak mutagenicity (2C4,5DFA and 4TFMA). Careful assessment for the risk of carcinogenicity is necessary for aniline derivatives with weak mutagenicity by combining in silico analyses and in vitro and in vivo genotoxicity tests.

Original Article
Increased hepatic triglyceride level induced by a glucokinase activator in mice Vol.5, No.1, p.13-20
Junichi Namekawa , Mari Yasui , Atsuko Katayanagi , Mitsuyuki Shirai , Fumitoshi Asai
Released: February 06, 2018
Abstract Full Text PDF[1M]

Glucokinase (GK) is an enzyme that catalyzes the phosphorylation of glucose to glucose-6-phosphate, and plays an important role in maintaining glucose homeostasis by regulating secretion of insulin from pancreatic β-cells and glucose metabolism in the liver. Recently, GK activators are expected as a novel therapeutic agent for Type 2 diabetes mellitus (T2DM). However, the increase in plasma triglyceride (TG) levels is one of the major issues for the development of GK activators. In this study, we evaluated the effects of the GK activator GKA50 on the plasma and hepatic TG in mice. Male CD-1 mice received a single oral dose of vehicle or GKA50 (15, 30, or 60 mg/kg), and plasma glucose and insulin levels were measured. Next, CD-1 mice received oral doses of vehicle or GKA50 (20 or 60 mg/kg) once daily for 4 days, and clinical signs, body weight, food consumption, blood chemistry, and hepatic TG were evaluated. In the single oral dose study, dose-dependent decrease in plasma glucose levels and increase in plasma insulin levels were observed. In the 4-day repeated dose study, there were no treatment-related changes in clinical signs, body weight, food consumption, or plasma TG levels. In the 60 mg/kg group, a significant increase in the hepatic TG level was observed. Additionally, the detailed analysis of TG species composition revealed marked increases in TGs mainly composed of 18:1 fatty acids. This study revealed that GKA50 enhanced insulin secretion and hepatic glucose utilization, and increased hepatic TGs, which were mainly composed of 18:1 fatty acids.

Original Article
A 28-day repeated oral-dose toxicity study of insecticide synergist N-(2-ethylhexyl)-1-isopropyl-4-methylbicyclo[2.2.2] oct-5-ene-2,3-dicarboximide in rats Vol.5, No.1, p.1-11
Mariko Matsumoto , Masatoshi Furukawa , Katsumi Kobayashi , Takako Iso , Toshime Igarashi , Takashi Yamada , Akihiko Hirose
Released: January 19, 2018
Abstract Full Text PDF[254K]

N-(2-Ethylhexyl)-1-isopropyl-4-methylbicyclo[2.2.2]oct-5-ene-2,3-dicarboximide (Synepirin 500; CAS: 13358-11-7) is used as a synergist, a chemical that makes pesticide ingredients more effective. People can be exposed to Synepirin 500 by using insecticides containing this chemical or from residues in food. The Japanese government chose this chemical as a target substance in its existing chemical testing program. Crl:CD(SD) rats were administered 0, 40, 200, and 1000 mg/kg/day Synepirin 500 by gavage for 28 days, followed by a 14 day recovery period. Diarrhea or soft feces were observed in both sexes at 1000 mg/kg/day. Absolute and/or relative liver weights significantly increased at ≥ 40 mg/kg/day in females and at ≥ 200 mg/kg/day in males. Absolute and/or relative thyroid weights significantly increased in both sexes at 1000 mg/kg/day. These changes were still significant at the end of the recovery period in females. Significantly prolonged prothrombin time and activated partial thromboplastin time were observed in males receiving ≥ 40 mg/kg/day. Histopathological changes in the liver and thyroid were observed in both sexes at 1000 mg/kg/day. On the basis of the effects on the liver, the level of the lowest observed adverse effect from repeated doses of Synepirin 500 was judged to be 40 mg/kg/day for rats.