We previously reported that 3-acetyl-5-methyltetronic acid (AMT) had inhibitory effects on rat renal vitamin K₁ 2,3-epoxide reductase (VKOR), as well as anti-fibrotic effects on Thy-1 glomerulonephritis and cisplatin-induced renal fibrosis in rats. In the present study, we investigated the general toxicity of AMT in male Crl:CD (SD) rats following a single or 2-week oral administration. After a single oral dose up to 1,500 mg/kg, no death or bleeding tendency was observed in any animal. In the 2-week repeated toxicity study, we performed clinical observations, body weight measurements, a urinalysis, hematology, blood chemistry, gross autopsy, organ weight measurements, and histopathology. The result obtained showed significant decreases in the red blood cell count, hematocrit value, hemoglobin concentration, and urinary calcium. However, no bleeding tendency was observed, even at the highest dose of 400 mg/kg. We also confirmed that the oral bioavailability of AMT was 56.7% in a pharmacokinetic study, and the area under the blood concentration (AUC) at 400 mg/kg of the 2-week oral toxicity study in rats was markedly larger than that in renal fibrosis model rats at 30 mg/kg intravenously. We concluded that AMT does not cause systemic bleeding in rats at the dose levels which AMT showed anti-fibrotic effects.

Glucose toxicity and lipotoxicity are important states in obesity and diabetes. We previously reported that thiamine supplementation decreases body weight and visceral fat mass in rats with obesity-related diabetes. Glucose-dependent insulinotropic polypeptide (GIP) acts on pancreatic β cells to promote insulin secretion. According to established theory, GIP is derived from the gastrointestinal tract. We previously discovered increased expression of the GIP gene in the livers of obese rats with diabetes receiving high-dose thiamine. We referred to our previous dataset of gene expression analysis using a microarray for livers, which led to the new idea for the present study. We focused on “liver-derived GIP” to demonstrate GIP protein expression in the liver and visually present localization of GIP in the liver. Four-week-old male Otsuka Long-Evans Tokushima Fatty (OLETF) rats were randomly divided into two groups: an unsupplemented control group and a thiamine-supplemented group receiving 2 g of thiamine/L in drinking water for 51 weeks. GIP protein expression in the livers of OLETF rats at 55 weeks of age were determined by western blotting and immunohistochemical analysis. GIP protein expression in the liver was increased in thiamine-supplemented rats compared with that in controls, suggesting that it is involved in preventing and controlling obesity-related diabetic complications. The novel findings of this study that GIP is expressed in the liver, is likely to be added to the story regarding GIP modification of the obese diabetic state.

Cadmium (Cd) is a toxic metal that can cause renal proximal tubular cell damage. Our previous research demonstrated that Cd induces apoptosis by suppressing the BIRC3 gene expression in human proximal tubular cells (HK-2 cells). BIRC3, a member of BIRC family, inhibits apoptosis by suppressing caspase activity. Cd has been shown to induce caspase-3 activation through the suppression of BIRC3 expression in HK-2 cells. In this study, we examined Birc family gene expression in the kidney and liver of mice exposed to Cd for 67 weeks. Cd exposure decreased the expression of Birc3 in the kidney but increased Birc3 expression in the liver of mice. In our previous in vitro study, Cd decreased BIRC3 expression predominantly in proximal tubular cells. The present findings strongly indicate that the decrease in BIRC3 gene expression is implicated in the induction of apoptosis by Cd in proximal tubules.

Metallothionein (MT) is a small, metal-binding protein that can act as a scavenger of free radicals. To determine whether MT is involved in ethanol-induced hepatotoxicity, which is known to occur through oxidative stress, we studied sensitivity to hepatotoxicity caused by ethanol in MT-null mice genetically deleted for MT-I and MT-II. MT-null mice and wild-type mice were i.p. administrated with ethanol (99.5%, 2.0 g/kg). The increase in GPT, GOT, and LDH activities in the serum of MT-null mice was significantly higher than in the wild-type mice 24 hr after ethanol treatment. Histopathological examination in the liver of ethanol-treated MT-null mice demonstrated vacuolar degeneration. In contrast, histopathologic change was less prominent in the liver of ethanol-treated wild-type mice. Moreover, ethanol increased lipid peroxidation levels only in the liver of MT-null mice. These results indicate that deletion of MT is associated with ethanol-induced severe hepatotoxicity through oxidative stress.

Obesity is a major health problem worldwide that is associated with the increased risk of type 2 diabetes and other chronic diseases. Sasa veitchii, which belongs to the Gramineae family, has various properties including anti-obesity properties. However, detailed mechanism of anti-obesity was not reported. This study aimed to investigate the therapeutic effect of Sasa veitchii leaf extract (SE) on obesity characteristics induced by a high-fat diet (HFD) such as hyperglycemia, insulin resistance, and inflammatory response. Four-week-old male ddY mice were freely fed a HFD or a normal diet (control) for 12 weeks. During the experimental 12-week period, treatment with saline or SE, 0.2 mL twice per day by oral gavage, was conducted, and body weight was measured weekly. At the end of the experiment, the mice were euthanized after a 16-hr fasting period, and their plasma was collected. Liver and epididymal adipose tissue samples were collected and weighed. Moreover, after 10 weeks of feeding, oral glucose tolerance test was performed. Treatment with SE significantly decreased body weight, adipose tissue weight, plasma glucose, insulin, leptin, and pro-inflammatory cytokines compared with HFD groups, and markedly reduced the impairment of glucose tolerance in obese mice. Furthermore, HFD-induced adipocyte hypertrophy was improved by treatment with SE. Moreover, adipocyte differentiation marker such as proliferator-activated receptor γ was activated by SE treatment. Our findings demonstrate that SE may reduce obesity-induced glucose and insulin tolerance, presumably by the induction of the proliferator-activated receptor γ.

To assess the toxicity of acenaphthylene, Sprague-Dawley rats were repeatedly administered the chemical via oral gavage at daily doses of 0, 4, 20, or 100 mg/kg/day for 28 days, followed by a 14-day recovery period. Decreases in body weight, food consumption, and body weight gain were observed in males and females in the 100 mg/kg/day group. Additionally, increases in water consumption and urine volume, and decreases in osmolality were observed in both males and females in this group. Moreover, this highest dose was linked to decreases in the reticulocyte percentage and increases in platelet counts in males and females, and females additionally exhibited increases in the hemoglobin concentration, mean corpuscular hemoglobin concentration, and activated partial thromboplastin time. Meanwhile, total cholesterol and phospholipid levels were elevated in males and females treated with 100 mg/kg/day acenaphthylene, with males additionally displaying increased total protein and albumin levels. Increased relative liver weights and changes in liver histopathology were observed in males and females treated with 20 or 100 mg/kg/day acenaphthylene. Additionally, organ weight and/or histopathological changes were observed in the thymus, heart, femoral and sternal bones including bone marrow, urinary bladder, kidneys, spleen, and adrenal gland in both sexes, in the stomach in males, and in the uterus, ovaries, and mesenteric lymph nodes in females in the 100 mg/kg/day group. Some changes exhibited plasticity in the recovery period. Based on these results, the no-observed-effect-level of acenaphthylene after repeated 28-day oral administration was 4 mg/kg/day.

The Ames test is used for the mutagenic assessment of drugs; however, it may not provide an accurate genotoxic profile for bactericidal compounds. This study was performed to clarify 1) whether the total number of genotoxicity assays performed (#Assays) was greater during antibiotic development than during the development of other drugs, particularly antivirals, possibly due to the requirement for additional assessments, 2) whether the maximum doses of the Ames test were less when an alternative assay had been performed for antibiotics, and 3) whether some particular alternative assay had an advantage to minimize #Assays in the last decade. Genotoxicity data submitted to the Pharmaceuticals and Medical Devices Agency in Japan during 2004 –2015 were used. The #Assays was greater and the maximum doses of the Ames tests were lower for antibiotics, which was more obvious when alternative mutagenic assays had been performed. The mouse lymphoma assay or hypoxanthine-guanine phosphoribosyl transferase gene mutation assay was performed preferentially as an alternative. For antibiotic development, preferred genotoxicity test packages should be discussed in the future for a better understanding of the genotoxic potential of antibiotics.

CYP3A4 is an important drug-metabolizing enzyme induced by various compounds causing drug-drug interactions. However, the molecular mechanism of CYP3A4 induction is not completely understood. CYP3A4 induction is caused by pregnane X receptor (PXR) through binding to some PXR binding elements. These elements comprise an everted repeat separated by six nucleotides in the promoter region and distal nuclear receptor binding element 1 (dNR-1) as well as the essential distal nuclear receptor binding element for CYP3A4 induction (eNR3A4) in the enhancer region of the CYP3A4 gene. Recently, we found that polycyclic aromatic hydrocarbons including anthracene induce CYP3A4 in HepG2 cells with a different induction profile from that of rifampicin (RF), a typical PXR ligand. When a CYP3A4 reporter plasmid in which the eNR3A4 DNA fragment binds directly to the CYP3A4 promoter (-362 bases) was evaluated in a reporter assay, dibenz[a,h]anthracene (DBA) induced reporter activity, while RF did not. To be induced reporter activity by RF, more 14 nucleotides 5′ upstream of the eNR3A4 (rifampicin eNR3A4: reNR3A4) DNA fragment were required. However, eNR3A4 and reNR3A4 did not respond to recombinant PXR without dNR-1. These results suggest that eNR3A4 and reNR3A4 are necessary for CYP3A4 induction by DBA and RF, respectively, and that dNR-1 is indispensable for full induction through PXR.

The safety of L-citrulline was investigated in male and female Sprague-Dawley rats by oral gavage administration for 4 weeks at a dose level of 2,000 mg/kg/day. Animals were sacrificed following the administration period. In the results, there were no toxicologically significant changes in general condition, clinical observations, body weight, food consumption, ophthalmology, urinalysis, hematology, blood chemistry, organ weights, or necropsy. In histopathological evaluation, squamous cell hyperplasia in the limiting ridge in the stomach was observed in some males and females in the test group. However, the lesion was limited and that tissue is specific to rodents. Thus it was considered to be less toxicologically significant. Our results indicate that repeated oral administration with L-citrulline under the present experimental conditions is well tolerated in male and female rats.

To assess the toxicity of N-phenyl-1-naphthylamine, Sprague Dawley rats were repeatedly administered with the chemical by oral gavage daily at doses of 0, 4, 20, 100, and 500 mg/kg/day for 28 days, followed by a 14-day recovery period. A significant decrease or decreasing trend of red blood cell counts, hemoglobin concentration, hematocrit, and mean corpuscular hemoglobin concentration and a significant increase in reticulocyte counts were observed at a dose of 500 mg/kg in both male and female rats. Increase in blood urea nitrogen and sodium levels was observed in male rats that received 500 mg/kg; increase in serum total protein, albumin, and calcium levels and in albumin/globulin ratio were observed in female rats that received 500 mg/kg. Increase in relative liver weight in female rats that received 100 mg/kg and increase in the absolute and relative liver weights in both male and female rats that received 500 mg/kg were observed; increases in the absolute and relative spleen weights and absolute kidney weight in female rats that received 500 mg/kg were observed. Hypertrophy of centrilobular hepatocyte and extramedullary hematopoiesis in the spleen were observed in both male and female rats at doses of 100 and 500 mg/kg. Renal tubular dilatation and papillary necrosis were observed in both male and female rats that received 500 mg/kg. These changes had the reversible trend in the recovery period. Based on these results, the no-observed-effect-level of N-phenyl-1-naphthylamine after a repeated daily oral administration for 28 days was determined to be 20 mg/kg/day for both sexes.

Organophosphorus (OP) insecticides are used worldwide to protect agricultural crops and dwellings. These chemicals phosphorylate diverse serine hydrolases, including acetylcholinesterase. Among them, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), components of the endocannabinoid signaling system (ECS) in male reproductive organs, are candidate targets of OP insecticide-induced spermatotoxicity. The effects of OP insecticides on the ECS in male reproductive accessory organs have not yet been investigated. In the present study, we examined the potential inhibitory effect of dichlorvos (DDVP) against FAAH and MAGL in male reproductive organs. In vitro screening assays were conducted by activity-based protein profiling with a fluorophosphonate chemical probe using samples of the testis, epididymis, prostate, and seminal vesicle of Wistar rats. Ex vivo assays were then performed using organs from rats orally administered 0, 5, or 10 mg/kg DDVP, 6 days per week for 9 weeks. In vitro assays showed that DDVP inhibited FAAH in the proteomes of rat testis, epididymis, and prostate, but scarcely inhibited MAGL. DDVP failed to inhibit FAAH and MAGL in the seminal vesicles. Ex vivo assays confirmed inhibition of FAAH in the proteomes of the prostate, testis, and epididymis of DDVP-treated rats, which exhibited morphologically abnormal sperm and decreased sperm motility. In conclusion, DDVP reduced the activity of FAAH but not of MAGL in male reproductive organs excluding the seminal vesicle; prostate involvement was demonstrated for the first time. Endocannabinoid signaling inhibition in these organs might contribute to sperm abnormality via deterioration in seminal plasma quality.

An analysis of mRNA levels of 39 Cyp enzymes in the mouse brain and neuroblastoma cell lines, C-1300N18 and NB2a was performed using a real-time reverse transcriptase-polymerase chain reaction. Relative expression levels were quantified by normalized β-actin levels, and compared to the mRNA expression level of the cannabinoid receptor (CB1R), which is abundantly expressed in the mouse brain. Mean 2^-ddCts of CB1R in mouse brain, C-1300N18, and NB2a cells were 1.043, 1.003, and 1.005, respectively. Among Cyp mRNAs in the mouse brain, Cyp1b1 mRNA was the most abundantly expressed (2^-ddCt = 0.310), followed by Cyp46a1 mRNA (0.246). The other Cyp mRNAs moderately expressed (0.011 ~ 0.117) were Cyp1a1, 1a2, 2b10, 2c29, 2c50, 2d9, 2d10, 2d12, 2d22, 2d26, 3a11, 3a41, 4f14, 4f15, 4f16, and 4x1. On the other hand, 10 out of 39 Cyp mRNAs (Cyp 2b9, 2b13, 2b19, 2c37, 2c38, 2c55, 3a44, 4a12, 4a14, and 4f18) were not detectable (2^-ddCt < 0.001). In the neuroblastoma cell lines, C-1300N18 and NB2a, Cyp1b1 mRNA was also the most abundant and preferentially expressed, and relative expression levels to CB1R were 4.674 and 5.084, respectively. Thirteen other Cyp mRNAs (Cyp1a1, 1a2, 2a5, 2b10, 2c44, 2c50, 2c55, 2d10, 2d22, 3a11, 4f13, 4f15, and 4f16) were detected in the neuroblastoma cell lines, whereas 17 Cyp mRNAs (Cyp2c29, 2c37, 2c39, 2c40, 2d12, 2d34, 2e1, 3a16, 3a25, 3a41, 3a44, 4a10, 4a12, 4a14, 4f14, 4x1, and 46a1) were not under the current conditions. The pattern of Cyp mRNA expression was similar for both neuroblastoma cell lines. The present results provide fundamental and useful information on the significance of particular Cyp enzymes in the mouse brain and neuroblastoma cell lines, C-1300N18 and NB2a, which may be valuable tools for investigations on the neural expression and function of Cyp1b1.

There are many subclones of human breast cancer MCF-7 cells that respond to different degrees of estrogen and that have been used for testing the estrogenic activity of environmental chemicals such as bisphenol A. Here, we examined the estrogenicity of bisphenol A in an MCF-7 subclone. It was not able to detect the increment of BrdU incorporation by neither 10^-7 M bisphenol A alone nor 10 ng/mL epidermal growth factor (EGF) alone in the cells (1 x 10⁴ cells) up to 50 hr. However, in the presence of 10 ng/mL EGF, bisphenol A dramatically increased cell proliferation (ED₅₀ = 10 pM). The synergistic responses were the chemical concentration-dependent manner. Thus, in this study, we demonstrate the new insight into the action of bisphenol A in the MCF-7 subclonal cell.

Cadmium alters the temporal dynamics of various cellular proteins, including heat shock proteins and protooncogenes. To reveal the molecular dynamics during cadmium cytotoxicity, we investigated the conventional nucleocytoplasmic transport pathways. Western blot analyses revealed that exposure of porcine kidney LLC-PK₁ cells to cadmium (10 μM) temporally induced HSP 70 and Ran in the cytoplasm and Ran and RCC1 in the nucleus. RCC1 could be detected in the nucleus as early as 1 hr after exposure of the cells to the metal. Thus, HSP70-Ran-RCC1 transport system appears to be involved in early phase cadmium cytotoxicity.

The disposition of perfluorododecanoic acid (PFDoA), a perfluorocarboxylic acid with 12 carbon atoms, was studied in male rats. Rats received an oral administration of PFDoA at a dose of 50 mg/kg. The body weights of PFDoA-treated rats were slightly less than those of vehicle-treated control rats. PFDoA administration resulted in an increase in liver weight; it was highest at 5 days after the treatment and gradually decreased thereafter. Higher liver weight was observed until 70 days after the treatment. Concentrations of PFDoA in plasma and various tissues were estimated up to 70 days after dosing. A large amount of PFDoA was found in the liver. The PFDoA concentration was 263.94 ± 32.94 μg/g in the liver; the value was 7.93 times higher than that of serum 5 days after treatment. The hepatic PFDoA amount was found to be 29.63% of the dose. A certain amount of PFDoA was found in the brain and adipose tissues where perfluorocarboxylic acids with less than 11 carbon atoms were sparsely distributed. The half-life of PFDoA was 55.3, 49.3, 52.4, 57.1, and 49.8 days for serum, liver, kidneys, brain, and adipose tissue, respectively. PFDoA increased hepatic levels of mRNA for Cyp4A10, Acot1, and Acox1, target genes of PPARα, suggesting that PFDoA can activate PPARα, as was observed with other PFCAs. Elevated levels of these 3 genes were observed 70 days after treatment, and the levels were less than those at 7 days. The differences between PFDoA and PFCAs with less than 11 carbon atoms were discussed.

4,4’-Methylenediphenyl diisocyanate (MDI) is a cross-linking agent. Chemical reactivity of MDI with endogenous substances such as albumin and glutathione (GSH) is assumed to be responsible for MDI toxicity. We examined the cytotoxic effect of MDI on rat thymocytes, under the condition that endogenous biological substances except for cells were nominally absent, in order to study chemico-biological interactions between MDI and cells. The treatment of 10-50 μM MDI for 3 hr significantly increased the side scatter signal intensity of cytograms, without affecting forward scatter intensity. The increase in side scatter signal intensity by MDI was associated with an increase in cell lethality. The treatment of cells with 50 μM MDI for 3 hr increased cell lethality without increasing the population of preapoptotic annexin V-positive living cells. In contrast, H₂O₂ at 100 μM significantly increased the population of annexin V positive living cells prior to cell death. MDI at 30-50 μM did not affect the increase in cell lethality induced by H₂O₂ or A23187. Simultaneous application of 50 μM GSH did not affect the cytotoxicity of 50 μM MDI. It was therefore concluded that the process of cell death induced by MDI could not be attributed to oxidative stress and intracellular Ca²⁺ overload, and that MDI possesses cytotoxic actions that are not significantly related to its chemical reactivity with GSH.

In addition to low molecular weight drugs, many antibody drugs have been developed in recent years. The safety of these drugs is mainly evaluated in animal toxicity experiments and it is difficult to detect all the toxicities that may occur in humans. Although trastuzumab (Herceptin®), an antibody drug that is used for the treatment of breast cancer, as well as E-8010, a low molecular inhibitor of phosphodiesterase-5, did not cause cardiotoxicity (e.g. dysfunction, QT prolongation, and arrhythmias) in monkeys, they caused cardiotoxicity in humans. The present study examined, whether or not the human cardiotoxicity of these drugs could be predicted using cardiomyocytes derived from human-induced pluripotent stem cells (hiPS-CMs) using an MED64-multielectrode array and assessed the usefulness of hiPS-CMs in antibody drug testing. At 1 and 3 mg/mL, trastuzumab prolonged the field potential duration (QT interval on electrocardiography) by ≥ 10% and induced arrest, respectively. At 0.1 and 1 μmol/L, E-8010 prolonged the field potential duration by ≥ 10% and induced early after-depolarization (proarrhythmia), respectively. The human cardiotoxicity induced by trastuzumab and E-8010 could be predicted using hiPS-CMs. It was thought that a multielectrode array using hiPS-CMs would be a useful tool for predicting the clinical cardiotoxicity of antibody drug candidates in addition to small molecular drug candidates in the preclinical setting.

We examined whether the nuclear localization of caspase-activated DNase (CAD) are involved in cadmium-induced apoptosis. Exposure of porcine kidney LLC-PK₁ cells to cadmium cleaved poly (ADP-ribose) polymerase (PRAP) as sufficiently as TNF-α did in the renal cells. However, nuclear localization of CAD was not seen during cadmium-induced renal apoptosis, but done during apoptosis by TNF-α. On other hands, in rat splenocytes cadmium could induce nuclear localization of CAD. Thus, our data suggest that activation of caspase is not always to allow the translocation of CAD into the nuclei for the internucleosomal cleavage of DNA, and suggest that the same apoptotic stimulus by cadmium may utilize different sets of caspase cascades or unknown death pathways which are coupled to the caspase proteolysis in different cell types.

A previous study showed that single intracisternal administration of p-nitrotoluene into neonatal rats caused hyperactivity. To evaluate the neural risk assessment of p-nitrotoluene, it is crucial to test the potential of the chemical via environmental exposure route. In this study, we tested the hypothesis that oral exposure to p-nitrotoluene would exhibit the effects observed previously with direct instillation. Oral administration of 600 μg/day p-nitrotoluene into male Wistar rat pups, 5 days to 3 weeks of age, caused significant hyperactivity at 4-5 weeks of age. Treated rats were about 1.3 times active in the nocturnal phase than the vehicle-treated control rats (p < 0.05). The long-term effects of the chemical caused a large reduction in tyrosine hydroxylase (TH) immunoreactivity in the midbrain at 7 weeks of age, at which terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling-positive cells were detected. Immunohistochemical analysis of glial fibrillary acidic protein also revealed p-nitrotoluene-induced gliosis in the substantia nigra, suggesting neurodegeneration. Furthermore, neonatal p-nitrotoluene-induced lesion decreased the gene expression levels of dopamine transporter in adult rats. Thus, we conclude that p-nitrotoluene via oral exposure route caused rat hyperactivity, concomitant with gliosis and impairment of TH immunoreactivity, most likely due to degeneration of dopaminergic neurons.

Poly-trans-[(2-carboxyethyl) germasesquioxane] (Ge-132) was administered at dietary levels of 0 (control), 0.3, 0.8 and 2.5% to groups of 20 male and 20 female CB6F1-Tg rasH2 (rasH2) mice for 26 weeks. As a positive control, 10 rasH2 mice of each sex received a single intraperitoneal injection of 75 mg/kg N-methyl-N-nitrosourea (MNU). There were no differences in survival between Ge-132-treated groups and the control. Loose stool, increase water intake, and dilatation of the cecum were evident in both male and female 2.5% groups; however, there were no histopathological abnormality found in the cecums of these mice. There was no significant Ge-132 treatment-related increase in the incidence of any neoplastic lesions compared to negative control. In the positive control MNU groups, malignant lymphomas and squamous cell papillomas of the forestomach frequently occurred. Thus, the experimental system employed showed clear negative results for induction of tumors due to Ge-132 administration, indicating the absence of Ge-132 carcinogenicity in mice.

Drug candidates sometimes cause a prolongation of the electrocardiogram QT-interval (QT) and torsades de pointes in humans, despite the fact that they do not cause them in non-rodent animals. Recent studies suggest that the cardiomyocytes derived from human induced-pluripotent stem cells (hiPS-CMs) are of sufficient quality to assess the cardiotoxicity of drugs in the preclinical setting. Thus, the usefulness of hiPS-CMs in correctly predicting the cardiotoxicity of drug candidates in the clinical setting, was examined using conventional drugs in the calcium transient analysis system FDSS/μCELL and the multielectrode array system MED64. The selection of the test drugs was based on previously reported studies. E-4031 and cisapride prolong the QT in humans, dogs and monkeys. Both drugs prolonged the calcium fluorescence peak width (PWD) in the FDSS/μCELL system and the field potential duration (FPD) in the MED64 system, both of which are thought to be surrogates of the QT. Diphenhydramine, famotidine and E-8010 prolong the QT in humans but not in dogs or monkeys. These drugs prolonged the PWD and FPD. On the other hand, verapamil and nifedipine prolong the QT in dogs or monkeys but not in humans. Both drugs shortened the PWD and FPD. These results suggest that the hiPS-CMs assay could correctly predict the QT effects in humans. The hiPS-CMs would be useful for predicting the effects of drug candidates on the QT of humans in preclinical in vitro studies.

Animal tests, such as the local lymph node assay (LLNA), are the gold standard for assaying skin-sensitizing potential. However, because of concerns about animal welfare, extensive research has been conducted on the use of various cell lines, such as human leukemia cells, for in vitro assays of skin-sensitizing potential, but such assays have not replaced animal tests as stand-alone assays. Because Langerhans cells—a type of dendritic cell—are the main antigen-presenting cells in the epidermis and because they play a central role in the induction of allergic skin disorders, these cells may be useful for skin-sensitizing-potential assays. Here, we investigated the utility of the murine dendritic cell line DC2.4 for in vitro assay of the skin-sensitization potential of 2,4-dinitrochlorobenzene (DNCB), 2-mercaptobenzothiazole (MBT), and α-hexyl cinnamaldehyde (HCA), which are categorized as extremely, moderately, and weakly sensitizing, respectively, on the basis of LLNA results. DC2.4 cell viability decreased dose-dependently with increasing concentration upon treatment with each of the compounds for 24 hr; the DNCB, MBT, and HCA concentrations that resulted in 75% cell viability were 6.07, 120.14, and 118.70 μg/mL, respectively. At nontoxic concentrations (concentrations less than the 75% cell viability concentrations), these compounds dose-dependently upregulated the expression of both CD86 and CD54 on the surface of DC2.4 cells. Their potency decreased in the order DNCB > MBT > HCA, which agrees with the order indicated by the LLNA. These results suggest that DC2.4 cells may be a viable replacement for human leukemia cells in in vitro assays of skin-sensitization potential.

To analyze the permeability of rat skin to silver nanoparticles, the dorsal skin of Sprague-Dawley rats was exposed to 5 nm Ag nanoparticles or silver nitrate (Ag⁺ ions) percutaneously for 24 hr after disruption of the epidermal barrier by tape stripping (TS) or acetone wiping (AC). Systemic toxicity was examined hematologically and histopathologically, and by assessing blood biochemistry. Although parakeratosis, decrease in keratohyaline granule, and thickening in the epidermis occurred following exposure to both 5 nm Ag nanoparticles and Ag⁺ ions after TS or AC, no Ag-specific changes were observed. Inductively coupled plasma mass spectrometry (ICP-MS) showed silver in the skin of rats exposed to both 5 nm Ag nanoparticles and Ag⁺ ions after TS or AC. Silver was only detected in the liver of rats exposed to Ag⁺ ions after TS, but not exposed to 5 nm Ag nanoparticles after TS or AC. No abnormal histopathological changes in the liver were observed in all rats. In the blood, silver was below detectable levels in all rats and had no adverse effects on hematology or blood biochemistry. These results indicate that silver ions released from 5 nm Ag nanoparticles can percutaneously infiltrate the body only when the skin barrier is disrupted, but does not induce any acute toxicity.

It is known that phototoxicity is related to chemical structure. We have previously reported a prediction method of chemical structure-based in silico phototoxicity for in vitro 3T3 NRU-PT assay. To improve the concordance of the method, here we added new descriptors related to another mechanism of phototoxicity. We focused on potential energy, which certainly contributes to chemical reactivity regardless of photo-irradiation. As a result, stretch-bend, a form of potential energy, was found as a new descriptor of phototoxicity. Our analysis strongly indicates that chemical reactivity is an important factor for phototoxic outcome.

The effects of Δ⁹-tetrahydricannabinol (Δ⁹-THC), cannabidiol (CBD), cannabidiolic acid (CBDA), and 2-methyl-2’-fluoro-anandamide (MF-AEA, a stable analog of anandamide) on the enzymatic activity of purified human calpain-1 (CAPN1) were investigated in the present study. Although leupeptin, a calpain inhibitor, reduced calpain-1 activity in a dose-dependent manner (1, 5, and 25 μM), among the four cannabinoids tested, Δ⁹-THC and CBD exerted stimulatory effects on calpain-1 enzymatic activity in the same concentration range as leupeptin. CBDA and MF-AEA did not modulate calpain-1 activity, indicating that the phenol structure in Δ⁹-THC/CBD is a key point, and the carboxylic acid moiety appears to be negatively involved. This is the first study to show that the phytocannabinoids, Δ⁹-THC and CBD have the ability to activate the enzymatic activity of human calpain-1.

A repeated-dose ocular instillation toxicity study is a type of general toxicity study having unique design characteristics of species selection and administration methods, because the test article, an eye drop formulation, is instilled in the eyes. The present survey was conducted to reveal the current status of the design of repeated-dose ocular instillation toxicity studies. Information on study design was collected from the common technical documents of 21 eye-drop drugs approved in the last decade in Japan. The species most frequently employed was rabbits, followed by monkeys, then dogs. The most frequently used breed of rabbit was New Zealand white, followed by Dutch-belted. Both sexes were used in almost all the studies. In most cases, the maximum concentration of test articles was set as 3- to 10-fold higher than the clinical doses, and dosing frequency per day was set as 1.5 to 2 times the clinical usages. In many cases, a single eye of each animal was instilled with one or two drops or a fixed volume (e.g., 0.050 mL/eye in rabbits, 0.030 mL/eye in monkeys, and 0.030 to 0.100 mL/eye in dogs) of the test article. As optional ophthalmological examinations, measurements of intraocular pressure and corneal thickness were integrated frequently. In conclusion, this survey revealed design characteristics of repeated-dose ocular instillation toxicity studies, which were different in some respects from systemic dose toxicity studies. The results can be used as a baseline when considering the study design of such studies.

The intermediate filament of mature Sertoli cells is vimentin (Vim). One of the toxicological consequences of phthalate exposure is a selective decrease in Vim, an intermediate-sized (10 nm) cytoplasmic microfilament, in Sertoli cells. Vim in Sertoli cells of rats exposed in utero to 100 mg/kg/day di(n-butyl) phthalate (DBP) on gestation days 12-21 was quantified. Immunohistochemical analysis revealed that Vim aggregated in Sertoli cells, but desmin filaments did not. Vim images were extracted from electron microscopic images using the computer program Imaris (Bitplane Scientific, Zeiss) and analyzed using Image-Pro plus (Media Cybernetics, USA). The amount of perinuclear Vim located within 0.5 μm of the nuclear membrane, where most Vim is aggregated, and the Vim volume ratios of the DBP group were similar to those of the vehicle group at 7 and 9 weeks, but those of the DBP group had decreased 0.63-times at 14 weeks and 0.48-times at 17 weeks compared to those of the vehicle groups. The present study showed that the testicular toxicity of in utero exposure to DBP seemed to be delayed type toxicity, and showed that improved morphometric methods cold be used widely for quantitative analysis of cellular cytoplasmic filaments.

We aimed to identify novel biomarkers using toxicoproteomics to profile the urinary proteome of renal papillary necrosis (RPN) in experimental model rats. RPN was induced in rats by a single intraperitoneal injection of 2-bromoethylamine hydrobromide (BEA). Urinary proteins from four groups (control group; BEA-treated but no RPN; BEA-treated with RPN but no increase in blood urea nitrogen [BUN]; and BEA-treated with RPN and increase in BUN) were pooled. Relative quantitation of pooled urinary proteins was then performed by two-dimensional liquid chromatography-tandem mass spectrometry with the isobaric labeling technique. We found 77 proteins were changed in RPN model rat urine.

A series of safety studies were conducted using isomaltodextrin (IMD), a new dietary fiber that is produced from starch using enzymes. IMD consists of only alpha linkaged glucose molecules, has an average molecular weight of approximately 5,000, is freely soluble in water, and contains greater than 80% fiber (AOAC 2001.03). No genotoxicity was observed when IMD was ssayed in standardized bacterial reverse mutation, micronucleus, and chromosome aberration tests. The LD₅₀ of IMD was found to be more than 2,000 mg/kg in an acute toxicity study in rats, and the no observed adverse effect level (NOAEL) was determined to be 1,000 mg/kg/day in a 90-day subacute gavage toxicity study in rats. No animals died, and no abnormal findings due to consumption of IMD were observed in either of these studies. Both NOAEL values were the highest doses tested. The NOAEL for loose stools was examined in humans in two separate studies. Based on these results, the NOAEL for IMD-related loose stools was considered to be 0.8 g/kg-BW. In a 4-week high-dose ingestion study in humans and a 12-week low-dose ingestion study in humans, laboratory values were found to be within the normal range of variation. The results of the current safety assessment studies suggest that isomaltodextrin is safe for human consumption.

β, β-dimethylacrylalkannin is a major active chemical component extracted from Lithospermum erythrorhizon, a traditional Chinese medicine that exhibits strong antimicrobial, anti-cancer and anti-inflammatory activities. However, its potential toxicity has not been rigorously studied. To confirm its safety, the oral toxicity of β, β-dimethylacrylalkannin was evaluated in vivo. An acute oral toxicity study in mice demonstrated that β, β-dimethylacrylalkannin was practically nontoxic based on its high median lethal dose (LD₅₀ > 10 g/kg). No deaths or abnormal responses were observed in the acute toxicity test using Wistar rats, suggesting that the maximum tolerated dose of β, β-dimethylacrylalkannin was greater than 10 g/kg. Chronic toxicity studies also revealed an absence of mortality and clinical symptoms, and no treatment-related adverse effects were detected by hematology, blood biochemistry and urinalysis examinations in all rats treated with 10-160 mg/kg/day β, β-dimethylacrylalkannin during a 6-month period. Increases in the relative organ weight of the lungs of females and the liver of males were observed at 160 mg/kg. Histopathological analyses revealed brown pigmentation in renal tubular epithelial cells at the middle and high doses (40-160 mg/kg/day). The no-observed-adverse-effect level (NOAEL) of β, β-dimethylacrylalkannin is 10 mg/kg/day. These results suggest that β, β-dimethylacrylalkannin is potentially safe for further development as a therapeutic agent in humans.

We have reported that four transcription factors (PAX4, PAX6, TCF3, and HMGA1) were markedly activated in the cerebellum of mice treated with methylmercury. In this study, to clarify the relationship between these transcription factors and methylmercury toxicity, siRNA targeting each of the four transcription factors were introduced individually into C17.2 mouse neural stem cells, and the sensitivity of the cells to methylmercury was investigated. Among the four transcription factors, knockdown of the gene for TCF3 increased the methylmercury sensitivity of C17.2 cells. Therefore, we suggest that TCF3 may have a protective effect against methylmercury toxicity.

Methylmercury is a harmful heavy metal that selectively disrupts the central nervous system. To clarify the transcription induction mechanism that operates in the brain in response to methylmercury exposure, we used a protein/DNA binding assay to identify transcription factors activated in the cerebella of mice administered methylmercury. We identified PAX4, PAX6, TCF3 and HMGA1 as transcription factors activated by methylmercury.

Protein expression changes were examined in day 10.5 rat embryos cultured for 24 hr in the presence of valproic acid (VPA), using two-dimensional electrophoresis and mass spectrometry. Exposure to VPA at an embryotoxic concentration of 1.2 mM resulted in quantitative changes in many embryonic protein spots (22 decreased and 29 increased). For the increased protein spots, 10 proteins were identified, including alpha-fetoprotein, phosphorylated cofilin-1, and serum albumin. These proteins are candidate protein biomarkers that may be involved in embryotoxic mechanisms.

Oral malodor is an unpleasant condition experienced by most individuals around the world. Volatile sulfur compounds (VSCs) may be the main source of oral malodor both in humans and dogs. The purpose of this study was to examine the suitability for measurement of oral malodor levels in dogs using OralChroma^TM, a portable gas chromatograph developed for human use. Oral malodor and periodontal disease in laboratory Beagle dogs (N = 6) were determined by organoleptic assessment and OraStrip® test, respectively. Relatively high concentrations of VSCs in the breath were detected in all six dogs in the order of hydrogen sulfide (H₂S) > methyl mercaptan (CH₃SH) > dimethyl sulfide ((CH₃)₂S). There was no significant influence of meals on VSCs levels. Significant correlations (P < 0.05) were observed among each VSC. These findings for the first time highlight the potential of OralChroma^TM, which allows for noninvasive, rapid and quantitative measurement of oral malodor levels in dogs, and warrants further investigation.

In a previous study, we found that early life exposure to low-dose diethylstilbestrol accelerated onset of abnormal estrous cycles in female C57BL/6J mice. In order to observe the ovarian aging of mice exposed to substances on the candidate list for endocrine-disrupting properties, neonates of C57BL/6J mice were orally administered bisphenol A (BPA) at doses of 5 and 50 μg/kg/day. As a result, normal ovarian aging was observed in females treated with BPA. However, females treated with 5 and 50 μg/kg/day of BPA showed modified T-cell differentiation in the thymus. These results suggested that early life exposure to low-dose BPA did not induce premature ovarian failure but modified T-cell differentiation in female mice.

A gelatin capsule is widely used as a device for oral administration of granular/powdered materials or small amounts of liquid materials in animal experiments. In this study, we investigated the insulin- and glucagon-related parameters following oral administration of gelatin capsules to beagle dogs since a gelatin capsule is made of protein composed of many amino acids as known to stimulate insulin or glucagon secretion. Gelatin capsules (1/2 ounces in size) were administered once orally to three male beagle dogs at the dose level of 0.29 and 1.67 g/kg as gelatin and the plasma levels of insulin, glucagon, glucose, non-esterified fatty acids (NEFA), total ketone bodies (KB) and alanine were determined before dosing and for up to 4 hr after dosing. Plasma insulin, glucagon and alanine levels increased immediately after dosing and subsequently plasma NEFA and total KB levels decreased. Most of these changes disappeared by 4 hr after dosing. No effects on plasma glucose levels were noted. In conclusion, a single oral administration of gelatin capsules increased plasma insulin and glucagon levels transiently and altered plasma levels of some biochemical parameters in dogs. These results might imply that attention should be paid to their own effect on the insulin and glucagon secretion at conducting animal experiments using a lot of gelatin capsules.

WRNIP1 was originally identified as Werner (WRN) helicase interacting protein in a yeast two-hybrid screen. The WRN helicase, which is mutated in the progeroid disorder Werner syndrome, has been implicated in homologous recombination, DNA repair, and other DNA transactions. WRN also interacts with RAD52, which is conserved from yeast to human and may function in homologous recombination under certain conditions. In this study, we addressed the physical and functional relationship between WRNIP1 and RAD52. WRNIP1 and RAD52 formed a complex in cells. WRNIP1 and RAD52 double gene knockout cells grew slightly slower than each of the single gene knockout cell lines and wild-type cells, indicating that WRNIP1 and RAD52 are required to maintain the normal rate of cell growth. Interestingly, the sensitivity to hydrogen peroxide of each single gene knockout cell line was suppressed in double gene knockout cells, suggesting that a new pathway dealing with oxidative stress-induced DNA lesions operates in the absence of both WRNIP1 and RAD52.