Orexin-A has been suggested to control hypertension, feeding behavior, and obesity. We recently established that long-term consumption of thiamine water by obese diabetic rats leads to reduced obesity and metabolic disorders. In addition, we found that drinking thiamine water daily may modulate oxidative stress-related diseases, such as diabetes and its complications. In the present study, we focused on obesity-related hypertension and plasma orexin-A levels in Otsuka Long–Evans Tokushima Fatty (OLETF) rats under oxidative stress conditions and assessed their cerebral ADP-ribosylated protein expression after drinking thiamine water. The thiamine water-drinking group was administered 2 g thiamine/L in drinking water. Plasma orexin-A content was measured by ELISA testing. ADP-ribosylated protein expression was analyzed in the brain of OLETF rats using Western blotting. Primary experimental characteristics, body weight, and caudal blood pressure were similar among the groups. However, at 28 weeks of thiamine water-drinking, significant decreases in body weight and systolic blood pressure were observed in the diabetic-thiamine group compared to those in the diabetic-control group. Moreover, obese diabetic rats exhibited increased plasma orexin-A levels and poly-ADP-ribosylated protein levels in the brain. Notably, the enhanced plasma orexin-A level and cerebral oxidative stress conditions of the obese diabetic rats were attenuated by drinking thiamine water. The relationship between consumption of thiamine in drinking water and obesity-related hypertension and cerebral oxidative stress status via modulation of plasma orexin-A levels requires further investigation. It is noteworthy that the upregulation of orexin signaling may not only cause hypertension, but also maintain obesity in polyphagia-induced OLETF rats.

To evaluate the safety of chicken breast extract containing plasmalogen, the following three tests were conducted: i) chromosome aberration study with mammalian cells; ii) bacterial reverse mutation test; and iii) 90-day repeated oral dose study in rats. The chromosome aberration study showed that there was no statistically significant increase in the structural aberration rate and the polyploidy aberration rate between the group that was treated with chicken breast extract and the negative-control group. Under the present test conditions, chicken breast extract did not induce chromosomal aberrations. Similarly, in the reverse mutation test, there was no increase in the number of revertant colonies in all of the bacterial strains that more than doubled the negative-control value related to the dose, regardless of the presence or absence of metabolic activation. In other words, chicken breast extract did not exhibit a gene mutation–inducing effect under the present test conditions. No effects were observed in the 90-day repeated oral dose study with the administration of chicken breast extract. Under the present test conditions, the no-observed-adverse-effect level for both male and female rats was 1000 mg/kg/day.

SM (Sulfur mustard) is an oily, hydrophobic, and lipophilic chemical agent that damages cells with intricate patterns. CEES (2-chloroethyl ethyl sulfide soluble) is a standard SM analog commonly employed in the toxicity mechanism study. To obtain ideal results in vitro, researchers should disperse CEES well in the medium, avoiding the presence of undissolved droplets. However, such a purpose is not easy to reach under the conventional solution preparation, and the information about droplet formation and function is little available. Here, we showed that phospholipid and triglyceride, two essential components of serum lipids, could prevent CEES from dissolving in water after vortex, which kept partial CEES as small droplets. By detecting CEES level, we proved that residual droplets slowed CEES hydrolysis and conversion. Under the microscope, CEES droplets were observed to degrade and diffuse with time to induce the necrosis and mitochondrial membrane potential decline from nearby cells. In conclusion, the damage pattern of CEES droplets is quite different from that of dissolved CEES, and a low level of phospholipid and triglyceride is beneficial for preventing droplets formation in preparing CEES solution.

Isomaltodextrin-producing enzymes (PP enzyme) include two extracellular enzymes (α-glucosyltransferase and α-amylase) produced by Paenibacillus alginolyticus PP710. These enzymes are essential for producing isomaltodextrin (IMD), a highly branched α-glucan, from starch. In this study, we evaluated the safety of this PP enzyme. No genotoxicity was observed when the PP enzyme was assayed in standardized bacterial reverse mutation and chromosome aberration tests. An acute toxicity study in rats showed no toxic effects of PP enzyme at 2,000 mg/kg. No animals died, and no effects of enzyme administration were observed in a 14-day repeated oral-dose toxicity study in rats at the maximum dose of 1,000 mg/kg/day. The no observed adverse effect level was determined to be 1,000 mg/kg/day in a 90-day subchronic gavage toxicity study in rats. No animals died, and no abnormal findings due to consumption of the PP enzyme were observed in this study. These safety evaluation results demonstrated that the PP enzyme was safe for application as an ingredient in the manufacturing of the food ingredient IMD.

In this study, we evaluated the toxicity of 16 veterinary drugs approved in Japan (antimicrobial substances, antiparasitic agents, and disinfectants) using a fish acute toxicity test, Daphnia swimming inhibition test, algae growth inhibition test, and earthworm acute toxicity test. Test organisms included Oryzias latipes, Daphnia magna, Pseudokirchneriella subcapitata, and Eisenia fetida. The tests were performed in accordance with the Organisation for Economic Co-operation and Development guidelines for testing chemicals. In the Globally Harmonized System of Classification and Labelling of Chemicals, when the EC/LC₅₀ value of a substance is < 1 mg/mL, then the substance is classified as Category 1, or highly toxic to aquatic organisms. The results of the present study indicate that the toxicity of tylosin phosphate, lincomycin, oxytetracycline chloride, and chlortetracycline to algae; that of ivermectin to crustaceans and fish; and that of didecyldimethylammonium chloride to all organisms examined should be classified as Category 1. In terrestrial organism, weak toxicity was observed with ivermectin, fenbendazole, and didecyldimethylammonium chloride. These data are considered to be valuable data for actually knowing the toxicity of veterinary drugs, in particular, since there is hardly any data following the guidelines using Eisenia fetida. Our results also suggest that the environmental risk of the tested veterinary drugs might be low at present researched environmental levels in river. In addition, our data indicate that the possibility of many veterinary drugs adversely affecting the environment is low if they are properly used.

In this study, we developed screening-level hepatotoxicity prediction models using test data on in vitro assays, which measure key events at molecular levels that are possibly linked to hepatotoxicity. Hepatotoxic chemicals were retrieved from repeated-dose toxicity databases of the Hazard Evaluation Support System Integrated Platform and the Toxicogenomics Project. In vitro assay data with specified protein targets likely leading to hepatotoxicity were selected using the hepatotoxic chemicals. In total, 47 in vitro assays were selected for constructing the hepatotoxicity prediction models. Then, two predictive models were constructed. Model A returns “Hepatotoxic” if the query chemical is tested, and the test result is “Active” in any of the selected in vitro assays. Model B returns “Hepatotoxic” if an analog of the query chemical is tested, and the test result is “Active” in any of the selected in vitro assays. External validation of the two models was performed using repeated-dose toxicity test data from the Toxicity Reference Database. Model A and Model B had sensitivity values of 0.67 and 0.72 and specificity values of 0.74 and 0.72, respectively. Our models could predict the hepatotoxic chemicals underlying the toxic mechanisms that are not established by the existing knowledge base model. On the other hand, false negatives were found to involve mechanisms requiring metabolic activation. Because our hepatotoxicity prediction model is based on the biological activity of key molecular events leading to the toxicity endpoint, scientific justification would be more acceptable as adverse outcome pathway information becomes more available.

Sulforaphane [1-isothiocyanato-4-(methyl-sulfinyl)butane] is an isothiocyanate derivative from cruciferous vegetables, with anti-proliferative actions on various cancer and tumor cells. In this paper, we envisaged the effects of sulforaphane on various functions (growth inhibition, cytotoxicity and enhancement of O₂^--generating activity) of human monoblastic leukemia U937 cells. Sulforaphane showed strong cytotoxicity, resulting in inhibition of proliferation in a dose-dependent manner. In addition, cell differentiation induced by 1 μM all-trans retionic acid (RA) remarkably caused the enhanced resistance against cytotoxicity of sulforaphane. Moreover, the RA-induced O₂^--generating activity was also enhanced by sulforaphane in a dose dependent manner. When U937 cells were cultured in the presence of 1 μM RA and 2 μM sulforaphane, the O₂^--generating activity increased more than 2.5-fold compared with that in the absence of the latter. Semiquantitative RT-PCR showed that co-treatment with RA and sulforaphane slightly enhanced transcription of only p47-phox gene among five essential components (p22-phox, gp91-phox, p40-phox, p47-phox and p67-phox) for the O₂^--generating system in phagocytes. On the other hand, immunoblot analysis revealed that co-treatment with RA and sulforaphane caused accumulation of protein levels of p47-phox (upto ~130%) and p67-phox (upto ~240%) compared with those of the RA-treatment alone. These results indicated that sulforaphane may enhance the RA-induced O₂^--generating activity in U937 cells via accumulation of p47-phox and p67-phox proteins. These data suggested that sulforaphane may serve as an effective drug for leukemia treatment.

Hesperidin is a flavonoid with many nutritional benefits including antioxidant activity in food formulations; however, hesperidin is practically insoluble in water. A commercial enzymatic process has been developed in which a glucose molecule is attached to hesperidin increasing the solubility by approximately 100,000 times. The substance is called glucosyl hesperidin (GH) with the main component being monoglucosyl hesperidin (MGH; 75 to 85%). This paper presents results of OECD-compliant toxicity studies with GH, including 4-week and 13-week sub-chronic toxicity, and teratogenicity studies in rats, and chromosomal aberration and mouse micronucleus formation tests. There were no deaths and no treatment-related adverse effects in the 4-week (highest dose 15,000 ppm) or the 13-week sub-chronic (highest dose 50,000 ppm) studies. There were no statistically significant treatment-related adverse effects on any parameter evaluated. The NOEL in the 4-week study was calculated as 1,280 mg/kg/day in females and 1,206 mg/kg/day for males, and in the 13-week study, the NOEL was 3,428 and 3,084 mg/kg/day, for females and males, respectively. In the teratogenicity study, the NOAEL was 1,000 mg/kg/day of treatment for both dams and fetuses. No genotoxicity was observed in the chromosomal study at 5,000 μg/mL and no micronuclei at 2,000 mg/kg, respectively. The results of these OECD-compliant studies support the safe use of GH as a food and beverage ingredient.

The diabetic state is considered to be one of the risk factors of drug-induced liver injury (DILI) because of the lower levels of glutathione for detoxification by conjugation with drugs. Carbamazepine (CBZ) -induced hepatotoxicity in humans is rare and unpredictable with the present state of knowledge, but it is somehow related to disturbance of glutathione metabolism, although data in this regard are limited. In order to estimate the potential risk of DILI in patients with type 2 diabetes mellitus (T2DM), we investigated the liver injury from CBZ, which is often used in the treatment of painful diabetic neuropathy in diabetic patients, using SD rats and Spontaneously Diabetic Torii-Lepr^fa (SDT fatty) rats as a model for human T2DM. The SDT fatty rats appropriately mimic the diabetic state in humans and have similar profiles of glucose metabolism, hepatic function tests and glutathione synthesis to those in patients with T2DM. Short-term oral dosing with CBZ to the SDT fatty rats revealed that liver injury was detected in the SDT fatty rats but not in the SD rats and the difference was considered to be due to lower hepatic detoxification of the metabolites of CBZ by depleted hepatic glutathione synthesis. In conclusion, the potential for CBZ to induce liver injury is considered to be higher in diabetic patients than in non-diabetic humans.

Drug-induced mitochondrial dysfunction can lead to severe adverse effects. Accordingly, new in vitro assay systems for assessing mitochondrial-related toxicity are required. Current systems evaluate drug-induced mitochondrial dysfunction based on cell death. However, if mitochondria are damaged without cell death, these methods run the risk of overlooking toxic or dangerous compounds. To solve this problem, we attempted to measure morphological changes semi-quantitatively by transmission electron microscopy and to detect subtle changes in mitochondrial function. To this end, we exposed HepG2 cells cultured in galactose-containing medium to benzbromarone (BBR), which impairs mitochondrial function. After 24 hr of BBR exposure, we compared the rate of cell death between galactose and glucose cultures. Before the onset of cell death, BBR increased the ratio of damaged mitochondria to a greater extent in galactose-cultured than glucose-cultured HepG2 cells. Our results suggested that this new in vitro assay system could detect mitochondrial-related toxicity before the onset of cell death.

Many environmental chemicals caused rat hyperactivity, a key feature of attention deficit hyperactivity disorder (ADHD). These were confirmed by many epidemiological studies, showing the correlation in their exposure levels in patients with ADHD. The nanomaterials have been difficult to be evaluated health risk because of its physicochemical nature. In this study, we examined the possibility that silver nanoparticle might be ADHD related chemical. Oral exposure of rat pups to silver nanoparticles (3mg/kg) with 1% bovine serum albumin as a carrier protein at 5 days old increased 1.3~1.4 fold spontaneous motor activity while without carrier proteins, it failed to do so. Thus, dispersion of silver nanoparticle solution by carrier proteins was necessary to examine the toxicological action of silver nanoparticles in the animal experiment.

TTA-121 is a novel oxytocin nasal spray with high bioavailability and is expected to increase oxytocin delivery to the brain by adjusting osmolality and viscosity of the formulation. As nonclinical safety studies to support the conduct of the Phase 1 and Phase 2 studies of TTA-121, a 6-week repeated intranasal dose toxicity study of TTA-121 in monkeys was conducted. In the present study, TTA-121 was administered intranasally to male and female cynomolgus monkeys once daily for 6 weeks at 0, 26.88, 134.4, and 672.0 U/body/day followed by a 4-week recovery period to investigate the toxicity of oxytocin and systemic exposure to oxytocin. No animal died or was euthanized due to moribundity. There were no test article-related changes in clinical signs, body weight, food consumption, ophthalmology, electrocardiography, urinalysis, hematology, blood chemistry, necropsy, organ weights, or histopathology at any dose level during the dosing or recovery periods. The toxicokinetic analysis indicated that systemic exposures of oxytocin increased with the dose ranging from 26.88 to 672.0 U/body/day on the first and final day of dosing. Based on these results, the no-observed adverse effect level (NOAEL) of TTA-121 was 672.0 U/body/day.

Autism spectrum disorder (ASD) is a highly prevalent neurodevelopmental disorder. Although there is no established treatment for the core symptoms of ASD, recent research has indicated a potentially therapeutic effect of intranasally administered oxytocin. TTA-121 is a novel oxytocin nasal spray with high bioavailability and is expected to increase oxytocin delivery to the brain by adjusting osmolality and viscosity of the formulation. As nonclinical safety studies to support the conduct of the Phase 1 and Phase 2 studies of TTA-121, a 6-week repeated intranasal dose toxicity study of TTA-121 in rats was conducted. In the present study, TTA-121 was administered intranasally to male and female rats at 0 (placebo), 1.2, 6, and 30 U/body/day once daily for 6 weeks followed by a 4-week recovery period to evaluate potential toxicity and systemic exposure to oxytocin. The toxicokinetic analysis indicated that systemic exposure of oxytocin increased with a dose ranging from 1.2 to 30 U/body/day at the first and last dosing. No deaths or moribundity were observed. There were no toxicologically significant changes in clinical signs, functional observational battery, body weight, food consumption, water consumption, ophthalmology, urinalysis, hematology, blood chemistry, organ weights, necropsy or histopathology at any dose during the dosing or recovery period. Based on these results, the no-observed-adverse-effect level of TTA-121 was 30 U/body/day for male and female rats, suggesting that there is a sufficient safety margin. We believe that TTA-121 is expected to be sufficiently safe to treat males and females with ASD.

Ovariectomized rats were used as an animal model of osteoporosis in this study. They were given ad libitum access to a diet with high or low calcium to phosphorus ratio, or a normal diet, for a period of 10 weeks and effects on osteoporosis were assessed. Our results showed that the experimental diets affect urine levels of calcium and phosphorus, but do not affect the levels of biochemical, histopathological, or bone-related biomarkers. These results indicate that the administration of feed with different ratios of calcium to phosphorus (the phosphorus content was kept constant and the calcium content varied from 0.2× to 5× relative to the phosphorus content) over a period of 10 weeks does not accelerate the development of osteoporosis in ovariectomized rats with normal renal function to maintain homeostasis.

S-(1, 2-Dichlorovinyl)-L-cysteine (DCVC) is derived from trichloroethylene (TCE) and known to cause renal cell injury after metabolic activation by cysteine conjugate β-lyase. We examined the in vivo disposition of [³⁵S] DCVC in mice after intraperitoneal administration at a dose of 30 mg/kg (2.42 MBq/6 mg/mL). DCVC and its related-substances were absorbed rapidly and distributed highly in the kidneys. Whole-body autoradiography analyses revealed high accumulation of DCVC and its relatedsubstances in hyaline cartilage of growth plates of the femoral epiphysis, vertebral endplates of the spinal column, costal cartilage, and tracheal cartilage, in addition to the kidneys. These findings suggest that TCE and DCVC are likely to be a cause of damaged cartilage.

In the therapy of ocular diseases, different active pharmaceutical ingredients are often instilled concomitantly as different ophthalmic solutions at an appropriate interval (unfixed-combination therapy) or simultaneously as one formulation (fixed-combination therapy). In this study, we aimed to compare the in vitro cytotoxicity of fixed- and unfixed-combination ophthalmic solutions using the glaucoma therapeutic agents, timolol and brimonidine. Cultured human corneal epithelial cell line was used as a test system. Exposure period was set at 5 min. Compared with the fixed-combination treatment, the unfixed-combination treatment (timolol followed by brimonidine, and vice versa) led to a significant reduction in the percentage of cell viability. In conclusion, it was suggested that a fixed-combination ophthalmic solution was expected to cause less cellular damage to the ocular surface, compared to an unfixed-combination ophthalmic solution.

β-Carotene, a natural additive that is often used for food coloring, is converted into vitamin A in the body and it removes reactive oxygen species. β-Carotene accumulates in the liver and adipose tissue, where it can induce adverse effects if present in excess. In this study, we determined whether β-carotene supplementation of a high-fat/high-sucrose diet enhances liver inflammation and fibrosis in Mongolian gerbils, which accumulate β-carotene in the liver. Ten-week-old male Mongolian gerbils were divided into four groups and fed either a normal diet, a high-fat/high-sucrose diet, a high-fat/high-sucrose diet supplemented with a low dose of β-carotene (0.001%), or a high-fat/high-sucrose diet supplemented with a high dose of β-carotene (0.004%). An oral glucose tolerance testing was performed after 12 weeks, and non-fasted serum and tissue samples were collected after 13 weeks of diet feeding. The high dose of β-carotene increased triglycerides and total cholesterol serum concentrations. Intake of a high-fat/high-sucrose diet with a high dose of β-carotene increased the β-carotene content, volume occupied by fat droplets, degree of fibrosis, and protein levels of matrix metalloprotease-9 (a marker of inflammation in steatohepatitis) in the liver. These data suggest that the intake of excessive amounts of β-carotene for 13 weeks exacerbates high-fat/high-sucrose diet-inducible inflammation and fibrosis in the liver in Mongolian gerbils. This model is suitable for studying the mechanism of β-carotene-induced liver injury, including liver fibrosis.

D-allulose is a rare sugar with an almost zero calorie and is known to suppress postprandial hyperglycemia and fat mass accumulation. Although D-allulose has been reported to be safe in healthy subjects and overweight/obese adults, its safety in borderline diabetes and diabetes patients has not been evaluated. Therefore, we conducted an open trial aimed to investigate the long-term safety of D-allulose in borderline diabetes and type 2 diabetes. Subjects took 5 g of D-allulose with meals three times daily for 12 continuous weeks. The general blood biochemical parameters, hematological parameters, urinary parameters, and anthropometric indicators were measured at 0, 2, 4, 8, and 12 weeks of the consumption periods and 4 weeks after completing consumption. Adverse events were assessed by the principal physician on each examination day. A total of 12 and 6 subjects with borderline and type 2 diabetes, respectively, were analyzed. No serious clinical problems were found in this study, although significant cholesterol variations and the improvements of some indicators of hepatic function were observed. In conclusion, the long-term ingestion of D-allulose is safe in borderline diabetes and type 2 diabetes. D-allulose can potentially suppress postprandial hyperglycemia and fat mass accumulation, and thus might be useful in diabetes.

Rare sugar D-allulose prevents obesity; however, an excessive and continuous intake of D-allulose may induce weight increases in the liver and kidney without apparent pathological and functional abnormalities. Conversely, there has not been reported about how these parameters will change after cessation of D-allulose intake. In this study, effects of a 10-week D-allulose cessation on liver and kidneys weights and biomarkers were investigated in rats previously fed a D-allulose containing diet for 4 weeks. Wistar rats were fed a control diet (C, n=16) or a 3% D-allulose diet (DA, n=16) for 4 weeks, and then the half of rats in the C and DA subgroups were dissected, while the other half of rats were fed the control diet for 10 weeks (C-C and DA-C, n=8, respectively). At the end of the first 4 weeks period, halves of rats in each diet group were euthanized, and the serum, urine, liver, and kidneys were used for pathological and biochemical analyses. The remaining rats were also similarly treated at the end of latter 10 weeks treatment. At week 4, the relative weights of the liver and kidneys were higher in the DA group than in
the C group, but these differences were disappeared by cessation of D-allulose. No abnormal parameters related to liver and kidneys functions were observed in the serum and urine. These findings suggested that D-allulose-induced increases in the liver and kidneys weights could be recovered to the normal levels by D-allulose cessation without accompanying functional and pathological abnormalities.

Cisplatin (CP) is used as a chemotherapeutic drug for the treatment of various kinds of cancer. However, it is becoming increasingly difficult to ignore its side effects, especially nephrotoxicity which has to do with oxidative stress and inflammation. Procyanidin (PRO) has been proved to be a powerful antioxidant. Therefore, we investigated whether PRO could prevent Cisplatin-induced nephrotoxicity and explored the underlying mechanism. In cellular experiment, reactive oxygen species (ROS), the malondialdehyde (MDA) levels, the activities of total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-PX) were measured for the assessment of cisplatin-induced oxidative cell damage. CCK-8 reagent and flow cytometry were used to detect the cell viability and apoptosis. Furthermore, the level of oxidative-related protein Nrf2 and HO-1 were carried out by western blot analysis. We found that cisplatin gave rise to the elevated levels of ROS and MDA and the decrease of the activities of T-SOD and GSH-PX with a related lower viability and higher apoptosis in HK-2 cells. Inversely, the pretreatment of PRO mitigated the oxidative damage, promoted the cell viability and lowered the apoptosis, activated nuclear related factor 2 (Nrf2) and elevated the expression of heme oxygenase-1 (HO-1), the above cytoprotection of PRO was blocked by siNrf2 or siHO-1. These results demonstrated that PRO has the potential to prevent cisplatin-induced nephrotoxicity through activation of Nrf2/HO-1 signaling pathway.

The Sprague-Dawley (SD) rat has been widely used for general toxicity and toxicokinetic (TK) studies, and is also useful for phototoxicity assessments. We previously showed that phototoxicity assessments could be incorporated into general toxicity study. However, this research was performed at only one facility. Thus, the effects of repeated administration and TK blood collection were investigated in three facilities to explore the possibility of incorporating phototoxicity assessments into general toxicity study. Lomefloxacin and pirfenidone were tested as the phototoxic compounds. Six-week-old male and female SD rats were allocated to two groups for each compound: single-dose and repeated-dose. The single-dose group was irradiated after a single administration of the drug without blood collection for TK. The repeated-dose group was irradiated after 8 days of repeated administration of the drug with TK blood collection (total 0.72-0.84 mL) after the 1st and 7th administration. Phototoxic reactions on the ventral skin, dorsal skin, and auricle skin were observed macroscopically at 2, 24, 48, and 72 hr after irradiation, and skin reaction scores were evaluated. The phototoxic compounds produced skin reactions in rats at all facilities regardless of the presence or absence of repeated administration and TK blood collection. However, there were differences in the degree of skin reaction between the two groups and among the facilities. Although further studies are needed to standardize this new evaluation system, we expect that the incorporation of phototoxicity assessments will contribute to shortening the research and development period and support the 3R principle for animal experiments.

N,N’-[(Cyclohexylmethylene)di-4,1-phenylene]bis[2-(1-pyrrolidinyl)acetamide] (1) is a novel antiprion compound, termed a designer molecular chaperone, that we recently developed. The administration of compound 1 prolonged the survival time of prion-infected mice and slowed the development of neurological and psychological symptoms in prion-infected macaques. The aim of this study was to investigate the oral toxicity of compound 1 to rats and cynomolgus monkeys. Compound 1 was administered orally to rats at doses of 31.3, 125, and 500 mg/kg. Although two of ten rats died at a dose of 500 mg/kg, no serious safety problems were identified at doses of 31.3 and 125 mg/kg. Repeated oral administration of compound 1 to rats at a dosage of 31.3 mg/kg/day for a week led to no significant toxic effects in the rats. An acute toxicity test in cynomolgus monkeys revealed that the administration of compound 1 at a dose of 60 mg/kg induced vomiting and fecal abnormalities. The monkeys did not die even at a dose of 250 mg/kg. A dose-dependent increase in the plasma concentrations of compound 1 in the cynomolgus monkeys as measured by LC/MS/MS analysis indicated that compound 1 migrated into the bloodstream. These results suggest that compound 1 might have potential as a therapeutic agent for prion diseases.

Antimony (Sb) is a useful metalloid in many industries and a therapeutic agent for leishmaniasis in developing countries. Thus, it is expected that humans and wild animals face the risk of exposure to Sb. Although Sb is generally recognized as a toxic element, the mechanisms underlying its toxicity have not been fully elucidated yet. The objective of this study was to evaluate species differences in Sb distribution in blood and urine between rat and hamster. Antimony was more preferably accumulated in rat red blood cells (RBCs) than hamster RBCs. On the other hand, it has been reported that arsenic is bound to a specific cysteine residue in rat hemoglobin, which results in the substantial accumulation of arsenic in rat RBCs. These have led us to formulate the hypothesis that Sb, which belongs to the same group in the periodic table as arsenic, is also accumulated in the same manner as arsenic. However, because Sb was less accumulated than arsenic even in rat RBCs, Sb seemed to have less affinity for the cysteine residue than arsenic. Trivalent Sb showed greater accumulation than pentavalent Sb in rat RBCs. Consequently, species differences in Sb distribution between rat and hamster could be attributed to the affinity for the specific cysteine residue in hemoglobin.

A standard animal model for phototoxicity evaluation does not appear in any guideline. Sprague-Dawley (SD) rats have been widely used in general toxicity and toxicokinetic studies and can be used in phototoxicity evaluation to reduce animal usage. To standardize phototoxicity procedures of SD rat, we investigated skin site- and sex-related differences in sensitivity to drug-induced phototoxicity at 3 facilities. Six-week-old male and female SD rats were orally administered 30 or 100 mg/kg lomefloxacin and light irradiation 1 hr after dosing; an ultraviolet (UV) irradiation device (10 J/cm2, UVA) or solar simulator (18 J/cm2, UVA) was used as light sources. Phototoxic reactions on ventral skin, dorsal skin, and auricle were observed macroscopically at 2, 24, 48, and 72 hr after irradiation. Plasma concentrations of lomefloxacin were also measured in non-irradiated, conscious rats. Skin reaction scores for ventral skin were highest and those of dorsal skin were lowest among the skin sites examined at all dose levels and facilities. Although drug concentrations in plasma were almost similar between sexes or higher in males than females, skin reaction scores appeared higher in females than males for ventral or dorsal skin. A difference in skin reaction scores among facilities was also observed; however, the order of skin sites based on sensitivity was approximately the same. We therefore suggest that appropriate conditions be drafted at each facility as differences in sensitivity to phototoxicity are dependent on skin site or sex in SD rats. Furthermore, we encourage multi-site validation studies to standardize experimental conditions in in-vivo phototoxicity studies.

Methylmercury is an environmental pollutant that causes central nervous system injury. We reported that the expression of the inflammatory cytokines TNF-α and IL-1β was specifically induced in the brains of methylmercury-treated mice. In addition, we recently found that cytotoxic microglia (M1-microglia) may be involved in the induction of inflammatory cytokine expression by methylmercury in mouse cerebral slice cultures. In the current study, we investigated the involvement of M1-microglia in the neuronal cell death caused by methylmercury using mouse cerebral slice cultures. The results revealed that methylmercury activated steady state microglia (M0-microglia) to M1-microglia, but this activation was suppressed by pretreatment with minocycline, a microglial activation inhibitor. In addition, under the same conditions, minocycline suppressed neuronal cell death by methylmercury. These results suggest that methylmercury may induce neuronal cell death via activation to M1-microglia.

Cyclopentyl methyl ether (CPME, CAS No. 5614-37-9) is used as an alternative to other ethereal solvents in pharmaceutical chemical process development. Although brief summaries on the repeated dose toxicity or genotoxicity of CPME are available, no detailed toxicity information has been reported. This study performed a detailed hazard characterization of CPME using new reproductive/developmental toxicity data and existing data for a 28-day repeated dose toxicity and genotoxicity study of CPME. Sprague-Dawley (SD) rats were treated with CPME by gavage at doses of 50, 150, or 450 mg/kg/day in the reproductive/developmental toxicity study or at doses of 15, 150, and 700 mg/kg/day in the 28-day study. In the reproductive/developmental toxicity study, lower body weight in males and longer gestational lengths were observed in the F0 animals receiving the 450 mg/kg/day dose. In the F1 animals, lower body weight gain during days 1-7 of life was detected in the 450 mg/kg/day groups in both sexes. In the 28-day study, 6 of 10 males showed poor clinical conditions, resulting in unscheduled deaths in the 700 mg/kg/day group. Based on these findings, the NOAELs for both the reproductive/developmental and 28-day repeated dose toxicity studies were estimated at 150 mg/kg/day. The results of all genotoxicity studies including the bacterial reverse mutation test, in vitro mammalian cell chromosome aberration test, and in vivo mouse micronucleus test were negative. A permitted daily exposure (PDE) of 15 mg/day was suggested based on the present findings to help determine the PDE for the ICH guidelines on impurities in pharmaceuticals.

Discarded scallop internal organs, especially the hepatopancreas, are rich in n-3 polyunsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid. However, they have not been utilized because of their contamination with toxic substances, such as cadmium (Cd), and the occurrence of diarrheic shellfish toxins (DST). We have successfully prepared a high-quality scallop oil (SCO) from the internal organs of the Japanese giant scallop (Patinopecten yessoensis), including the hepatopancreas without Cd and DST. These pollutants were removed by liquid-liquid partitioning followed by adsorption to active carbon of fine particle size with high pore surface volume. In this study, we prepared SCO from scallop internal organs obtained from two different processing areas (Mutsu and Uchiura bays, Japan), and referred to them as SCO-M and SCO-U, respectively. Genotoxicity of SCO-M and SCO-U was evaluated by the in vitro bacterial reverse mutation test (Ames test) and in vivo micronucleus test in accordance with the Organisation for Economic Co-operation and Development guidelines. SCO-M and SCO-U showed negative results in the Ames test in the presence or absence of metabolic activation with S9 mix. In addition, no genotoxic effects of SCO-M and SCO-U were observed at all tested doses in the micronucleus test. Based on the results of the present study, it can be concluded that SCO-M and SCO-U are safe products in terms of genotoxicity under these experimental conditions.

The benchmark dose (BMD) approach is one of the important techniques in dose-response assessment for the risk assessment of chemicals and adapted by various international organizations. We investigated the appropriateness of the statistical parameters and model selection criteria for BMD lower bound (BMDL) estimation by BMD software (BMDS) (developed by the US Environmental Protection Agency) and PROAST (developed by the National Institute for Public Health and the Environment of the Netherlands). Publicly available repeated-dose toxicity study data (226 dichotomous datasets and 151 continuous datasets) were used for the investigation. Our findings were applied to establish BMD technical guidance for BMDS for the evaluation of various endpoints in repeated-dose toxicity studies. Under the Japan Chemical Substance Control Law (CSCL), the DRA-BMDS guidance (i.e., Division of Risk Assessment-BMDS guidance) is used for the evaluation of a “Priority Assessment Chemical Substance.” Namely, selecting of an extra risk of 10% (dichotomous data) or a level change of 1SD (continuous data) as a default benchmark response. Running all the models without or with parameter constraints. Selecting the model that calculated the lowest BMDL but excluding the one that estimated a BMD/BMDL ratio ≥ 10 or lowest dose/BMDL ratio ≥ 10. We believe that the DRA-BMDS guidance can assist risk assessors in the selection of the BMD model.

Kidney proximal tubules are composed of S1, S2, and S3 segments having different properties of excretion and substance reabsorption. Since renal toxicants such as cadmium and cisplatin cause segment-specific toxicity, it is important to examine the segment-specific transport and detoxification systems of renal toxicants. Here, we investigated the gene expression profiles of immortalized S1, S2, and S3 cells derived from each segment of mouse kidney proximal tubules. Microarray analyses showed distinct expression of various genes in each cell line. We compared the expression levels of selected genes related to the transport and detoxification of renal toxicants. Some genes showed segment-specific expression patterns similar to those observed in in vivo studies. The gene expression profiles of each cell line shown in this study will provide a foundation for the future utilization of immortalized S1, S2, and S3 cells for toxicity screenings as well as for the elucidation of renal toxicity mechanisms.

Titanium dioxide nanoparticles (TiNP) are widely used commercially and exist in a broad range of applications and consumer products such as exterior wall paints, antibacterial agents, white pigments, and sunscreens. We previously reported that the testis is a fragile organ against titanium toxicity as compared to the liver; TiNP has been shown to decrease both the sperm motility and the sperm numbers, that is, TiNP quantitatively and qualitatively change the sperm functions. There are, however, few reports regarding to the influence of TiNP on fertility ability. In this paper, we evaluated the influence of TiNP on fertilization rate using in vitro fertility (IVF) assay. Male C57BL/6J mice were administered orally with TiNPs (10 mg/kg or 100 mg/kg). Mice were sacrificed 24 hr after the administration. As a result, TiNP (10 mg/kg group) significantly decreased the fertilization rate. In the higher dose group (100 mg/kg), the degree was weaker than in the lower dose group. Our results indicate that TiNP reduces not only the sperm motility but also the fertility, and it will be useful information in considering the influence of TiNP on next generation.

Alzheimer’s and Parkinson’s disease are neurodegenerative disorders of unknown cause for which there is no cure or way of preventing or slowing its progression. Various genetic and environmental factors are thought to be involved in the onset of neurodegenerative diseases. Oxidative stress, such as the generation of reactive oxygen species and lipid peroxidation, is a major factor in initiating the disease process. However, oxidative stress in cells is known to be suppressed by drug-metabolizing enzymes, such as NAD(P)H:quinone oxidoreductase 1 (NQO1), and antioxidant enzymes, such as catalase. Here, we used PC12 cells, which are a recognized model for neuronal cell death and neurite extension, to investigate whether Magnoliae Cortex derived from the Magnoliaceae plant family can induce these enzyme activities. Our results reveal that Magnoliae Cortex extract induces the activity of both NQO1 and catalase. In addition, the cytotoxic effect of hydrogen peroxide and 6-hydroxydopamine was significantly suppressed by pretreatment of the cells with Magnoliae Cortex extract. Based on our findings, we conclude that induction of these enzyme activities by Magnoliae Cortex extract leads to an enhancement of its cytoprotective effect.

Taiwanofungus camphoratus is an edible and medicinal mushroom originating in Taiwan. Several researches have revealed T. camphoratus possessed various biological activities, including anti-cancer, immunomodulation, liver protection and anti-inflammation. Recently, it has been widely used in food supplements and drug development for its health benefits and medicinal properties. Therefore, the safety issue is the primary concern for consumers. The aim of this study was to evaluate the toxicological effects of T. camphoratus extract that was composed of extracts from cut-log cultivated fruiting body and solid-state culture of T. camphoratus. The genotoxicity tests, rodent and non-rodent repeated dose toxicity studies were performed. The results of the genetic toxicology tests including in vitro bacterial reverse mutation assay, in vitro chromosomal aberration test, and in vivo mouse bone marrow micronucleus assay were all negative that indicated neither mutagenicity nor clastogenicity was caused by T. camphoratus extract. Moreover, 13-week and 26-week repeated dose oral toxicity studies in rats showed that no significant adverse effects of T. camphoratus extract were found up to dosages of 3400 mg/kg and 1700 mg/kg for male and female rats, respectively. The results of 28-day repeated dose oral toxicity study in beagle dogs showed no-observed-adverse-effect-level (NOAEL) of T. camphoratus extract up to dosage of 1500 mg/kg for male and female dogs. Accordingly, these results provided the safety information of T. camphoratus extract that supported for using in food supplements or medicinal usage.

Halitosis or oral malodor is correlated with the concentration of volatile sulfur compounds (VSCs) produced in the oral cavity by metabolic activity of periodontal pathogenic germs. Our previous study demonstrated that VSCs in canine breath air can be measured using a portable VSC monitor. The aim of this study was to assess the association between oral malodor and periodontal disease in dogs. Forty-three laboratory Beagle dogs (1-16 years of age, 24 males, 19 females) were included in this study. Oral halitosis was evaluated by the organoleptic test score (OS) and by measuring the oral levels of VSCs: hydrogen sulfide (H₂S; HS), methyl mercaptan (CH₃SH; MM), and dimethyl sulfide (CH₃SCH₃; DMS) using OralChroma™. The calculus index (CI) and the gingival index (GI) were measured as periodontal parameters. Oral levels of halitosis parameters (OS, HS, MM, CI, and GI) in Group 2 dogs (7-16 years of age) were significantly higher than those in Group 1 dogs (1-6 years of age). In addition, significant positive relationships were found between oral malodor and periodontal disease, both of which are age-dependent in dogs. The present study suggested that aging is an important factor for oral malodor and periodontal disease in dogs.

Repeated-dose oral toxicity of L-hydroxyproline (Hyp) was assessed in male and female SD rats by gavage for 28 days at dose level of 40, 200, 1,000 or 4,000 mg/kg/day. The reversibility of treatment-related changes was also examined by providing 14-day recovery period in the control group and 4,000 mg/kg group. In the results, reduced body weight gain with decreased food consumption was noted in males at 4,000 mg/kg/day. The histopathological evaluation revealed increased incidences of focal dilatation of the renal tubules with narrowing of the tubular cell liner and focal interstitial fibroplasia in the kidney of males at 1,000 and 4,000 mg/kg and females at 4,000 mg/kg. The lack of kidney specific serum chemical or urinalysis findings supported that the renal changes were very mild. Based on the above results, it was concluded that the no observed adverse effect level (NOAEL) of Hyp was 200 mg/kg/day.

Benzotriazole ultraviolet stabilizers (BUVSs) are widely used as ultraviolet filters in various consumer and industrial products. For this purpose, we examined the effects of 10 BUVSs and benzotriazole itself on transcriptional activation mediated by nuclear receptors: pregnane X receptor (PXR), constitutive androstane receptor (CAR) and peroxisome proliferator-activated receptor alpha (PPARα). UV-090 and UV-9 showed rat PXR-agonistic activity in the concentration range of 1-30 μM in reporter gene assay using simian kidney COS-1 cells. UV-090 showed the highest activity (REC₂₀ value: 3.85 × 10^-6 M). UV-090 was also positive in rat CAR activation assay, while UV-P showed inverse agonistic activity towards CAR. In the presence of the CAR agonist artemisinin (10 μM), UV-P also showed dose-dependent CAR-antagonistic activity in the concentration range of 10-30 μM. UV-090 and UV-9 activated rat PPARα. Overall, these results suggest that UV-090, UV-9 and UV-P modulate PXR, CAR and/or PPARα activation.

Organophosphorus compounds (OPCs) are widely used as pesticides, but often show high toxicity in mammalian cells. To assess the toxic potential of OPCs, we examined the cytotoxicity of paraoxon, methyl-paraoxon, parathion, and methyl-parathion exposures on NB41A3 neuroblastoma mouse cell lines. The LC50s (median lethal concentrations) at 24 hr of exposure were determined from the acute toxicity test including time course experiments. The LC50 values suggest higher toxicity of paraoxon (0.42 mM) compared to parathion (0.66 mM). In addition, the methylated derivatives of both OPCs indicated similar but slightly lower levels of toxicity compared to paraoxon and parathion (0.46 mM for methyl-paraoxon and 0.77 mM for methyl-parathion). However, the results from time course experiments indicated obvious reduction of cell viability for parathion and methyl-parathion as early as 1 hr of exposure, whereas the effects of paraoxon and methyl-paraoxon were not significant before 6 hr of exposure. We also report the most affected biological processes in NB41A3 cells in response to parathion exposure using microarray experiment. Among the statistically overrepresented biological processes are the ones related to neuronal development, apoptosis, cell stress, and cell signaling.

Nanomaterials with nanoscale microstructures have new properties in which reactions to stimuli such as heat, light, and voltage differ from those of macroscale materials. For that reason, the development of nanotechnology using nanomaterials has been remarkable, and these technologies have been put to practical use in various fields such as medicine and electronics. Nanomaterials have been researched as new materials with superior properties that have not been seen in the past, but concerns remain about the influence of nanomaterials on living bodies. Silver nanoparticles are materials with excellent optical, electrical, and antibacterial properties. However, few reports have described the influence of silver nanoparticles on the living body and interactions between chemicals such as pharmaceuticals. We therefore investigated the effect of silver nanoparticles on the living body and drug interactions. We administered silver nanoparticles with particle diameters of 10, 50, and 200 nm (SnP10, SnP50, and SnP200, respectively) to mice through the tail vein. As a result, acute liver injury was induced only in the SnP10 group. Furthermore, liver injury was induced by co-administering SnP10 with carbon tetrachloride, streptomycin, or cisplatin. SnP10 appears to induce liver injury through acute and drug interactions.

In this study, we assessed embryonic developmental toxicity and teratogenicity of lithium (Li) on medaka (Oryzias latipes) and predict the molecular mechanisms of their effects using a nanosecond pulsed electric field (nsPEF) technique and bioinformatics analysis. The microscopic observation revealed that the 1 mg/L LiCl treatment causes the most severe deformation effects, such as thrombus, heart hypertrophy, deformation of eyes, and growth retardation to embryos. The RNA-seq analysis identified 2,483 up- and down-regulated genes, such as histogenesis and organ growth related genes, in 2 day post-fertilization embryos after treatment with nsPEF and 1 mg/L LiCl. In addition, bioinformatic analyses showed that LiCl affects several aspects of gene ontology, such as molecular functions and cellular components, and some pathways, such as spliceosome, cell cycle, selenocompound metabolism, TGF-β signaling, and RNA degradation. The upregulation of GSK3B (signal transduction and cell growth), BAX (apoptosis), and MAP3K8 (cell death, arrest of cell cycle, and inflammation) genes were also observed in embryos exposed to LiCl. Our results suggest that the incorporation of Li compounds into medaka eggs using nsPEF shows adverse effects to the development and teratogenicity, and that these toxic effects may be affected by the alterations of certain gene expression in medaka embryos.

The aim of this study was to investigate whether the Japanese herbal medicine Juzentaiho-to (JTX) showed attenuating effects on isoniazid- and rifampicin-induced liver injury. Seven-weekold male Institute of Cancer Research mice were orally administered JTX or saline once a day at 9:00 for 3 days. Additionally, the mice received a mixture of 80 mg/kg isoniazid and 160 mg/kg rifampicin (10 mL/kg) via intraperitoneal injection three times (at 19:30) per 24 hr period. Twenty-four hours after the last administration of isoniazid/rifampicin, the mice in each group were sacrificed and blood was removed to obtain the plasma and livers. Mice that had received isoniazid/rifampicin showed high plasma levels of alanine aminotransferase, aspartate aminotransferase, and interleukin-6. In addition, the mice injected with isoniazid/rifampicin displayed increased hepatic lipid peroxidation and receptor-interacting protein-1 and -3 levels. Treatment with JTX prevented an isoniazid/rifampicin-induced increase in levels of alanine aminotransferase and aspartate aminotransferase, lipid peroxidation, and receptor-interacting protein changes. Our results suggest that JTX protects against isoniazid/rifampicin-induced hepatic injury by modulating oxidative stress and inflammatory responses.

(-)-Epigallocatechin-3-gallate (EGCg), a major catechin in green tea, eliminates reactive oxygen species and development of lifestyle-related diseases. However, excessive EGCg intake could induce adverse effects, particularly liver injury. We examined whether optimal dietary doses of EGCg reduces the risk of liver injuries in non-obese type 2 diabetic Goto-Kakizaki (GK) rats by examining gene expression of the proinflammatory cytokines interleukin (IL)-1β, IL-18 and tumor necrosis factor-α (TNF-α) and fibrosis-related matrix metalloproteinases (MMPs) in the liver. GK rats at 9 weeks of age were fed a control high-fat diet or a high-fat diet containing 0.1%, 0.2% or 0.5% EGCg (w/w) for 25 weeks. Expression of mRNA and proteins related to inflammation were determined by qRT-PCR and western blot analysis, respectively. IL-1β and IL-18 mRNA in the liver were reduced by EGCg supplementation at concentrations of 0.1% and 0.1%-0.2%, respectively, but not at concentrations of 0.2% and 0.5% (IL-1β) or 0.5% (IL-18) EGCg. TNF-α mRNA in the liver was reduced by supplementation with EGCg at concentrations of 0.1%-0.5%. Expression of MMP2 in the liver was reduced by EGCg supplementation at a concentration of 0.2%, but not 0.1% or 0.5%. Importantly, IL-18 protein levels in the liver and serum were reduced by 0.1% EGCg, but not by 0.5%. EGCg supplementation at concentrations from 0.1% to 0.5% did not induce increases in the expression of liver injury marker genes, while low doses (0.1%–0.2%) of EGCg in GK rats reduced expression of injury-associated genes in the liver.

Glycidol fatty acid esters (GEs) are food process contaminants detected in edible oils. It has been thought that glycidol is released from GEs by lipase in vivo, and shows genotoxicity. While DNA damage from glycidol has been reported, there is very little information on the DNA damaging potency of GEs in vivo. Therefore, we estimated DNA damage of glycidol and glycidyl oleate, which is one type of GEs, using the standard comet assay and a formamidopyrimidine glycosylase(Fpg)-modified comet assay. ICR male mice were orally administrated glycidol and glycidyl oleate (1.0 and 2.0 mmol/kg body weight) at 24 and 3 hr prior to dissection. In the standard comet assay, DNA damage (tail length and % tail DNA) in liver, kidney and blood samples of glycidol-treated groups were increased in a concentration-dependent manner. In Fpg-modified comet assay, glycidol showed DNA damage with higher sensitivity compared with the standard comet assay. DNA damage was not observed in the administration group of glycidyloleate in the standard comet assay. However, in Fpg-modified comet assay, glycidyl oleate showed significant DNA damage in the liver, kidney and blood samples compared with the standard comet assay. In this study, it was revealed that glycidol and glycidyl oleate induce DNA damage, such as oxidative and alkylation damage, recognized by Fpg protein.

Diabetic hyperglycemia is typically accompanied by various protein modifications, indicating hyperglycemic glucotoxicity. Overactivation of poly [adenosine diphosphate (ADP)-ribose] polymerase 1 (PARP-1) has been implicated in the pathogenesis of oxidative stress-related diseases including diabetes and its complications. Furthermore, obesity and diabetes are known to be associated with a substantial risk of chronic liver disease. We have previously reported that thiamine supplementation prevented obesity and diabetes-related liver disease. As a step forward, in the present study, we focus on hepatic ADP-ribosylation that reflects PARP-1 activation and an increased oxidative stress condition. Otsuka Long-Evans Tokushima Fatty (OLETF) rats were randomly divided into the following groups: thiamine-supplemented and unsupplemented control groups. The thiamine-supplemented group received 2 g of thiamine/L of drinking water for 33 weeks. ADP-ribosylated protein expression was analyzed in the livers of OLETF rats using Western blotting. Moreover, the fasting blood glucose level was measured in these rats. The obese diabetic OLETF rats exhibited high ADP-ribosylated protein expression in the liver. Interestingly, hepatic ADP-ribosylated protein expression and fasting blood glucose levels were lower in the thiamine-supplemented OLETF group than in the control OLETF group. These results suggest that thiamine supplementation attenuates oxidative stress by inhibiting hepatic ADP-ribosylation in OLETF rats. The beneficial effect of high-dose thiamine on oxidative stress-related diseases could be attributed to its inhibitory effect on PARP-1 activation, in addition to its role as a coenzyme. Furthermore, we found that thiamine supplementation prevented fasting hyperglycemia, suggesting that high-dose thiamine modifies the hepatic glucose metabolism and obesity-induced hepatic insulin resistance.