Cadmium (Cd) is a toxic heavy metal known to exert severe nephrotoxic effects. Mechanistically, Cd has been reported to disrupt gene expression in renal proximal tubular cells. In addition, alterations in DNA integrity have been reported to be associated with Cd toxicity. Histone proteins play important roles in maintaining DNA integrity, and are responsible for regulating gene transcription. In this study, we examined the involvement of HIST1H4C, a gene encoding the histone H4 protein, in Cd toxicity in HK-2 human proximal tubular cells. It was found that Cd significantly reduced the transcription level of HIST1H4C in HK-2 cells. In addition, HIST1H4C knockdown by siRNA transfection enhanced Cd toxicity in HK-2 cells. Our findings suggest that suppression of gene expression of HIST1H4C may be involved in the elevation of Cd toxicity in proximal tubular cells.

To identify the developmental point of onset of learning and memory function under an operant task, performance of young rats was examined on postnatal days (PDs) 16, 19, 22, 25 and 28, and then compared with that of adult rats (PD60). Each group of Long-Evans male rats with difference days of age was assigned to a series of fixed ratio (FR) operant tasks, in which the number of lever-pressing was required for acquisition of food reward. FR rate started from 1, changed into 2, and finished with 20. Each session conducted once a day, 7 sessions per week, lasted 30 min. Response rate (the number of lever-pressing per minute) and the number of session until animal acquired the learning acquisition criterion were measured. In FR20 learning task, performance of animals at PD32 showed the same shape as those of adult rats, showing an increase of response rate and a decrease of the number of sessions. The results indicated that memory and learning function of rats required for FR20 task might be matured at PD32. Results in the present study implied that the higher brain function used for the operant FR tasks mainly depended on the hippocampal development.

Arsenic is a hazardous element that exists widely in the environment. To elucidate the molecular mechanism involved in the onset of arsenic toxicity, we comprehensively searched for genes affecting the sensitivity of HEK293 cells to arsenite by using a siRNA library targeting approximately 17,000 human genes. As a result, we identified ACOT9, one of the acyl-coenzyme A thioesterases, as the gene conferring arsenite resistance to cells by knockdown.

We examined the effect of methylmercury administration on expression of interleukin genes in brain of mice. We found that gene expression of IL-1β and IL-19 was increased in the brain after the administration of methylmercury.

Sesquiterpene lactones exhibit toxicity in humans and animals by non-selectively interacting with cellular macromolecules. Among the sesquiterpene lactones identified to date, (–)-xanthatin, which was obtained in an extract from Xanthium strumarium (the Cocklebur plant), is reportedly less toxic to animals. Although we have shown that (–)-xanthatin has anti-proliferative effects, coupled with the induction of DNA damage-inducible GADD45γ, on highly aggressive human MDA-MB-231 breast cancer cells, the molecular mechanisms of anti-proliferative activity have not yet been elucidated in detail. Furthermore, evidence for the involvement of DNA damage is currently not sufficient. In the present study, we chemically synthesized pure (–)-xanthatin, and attempted to obtain more concrete evidence for DNA damage caused by (–)-xanthatin, which leads to cell death. The results obtained revealed the marked up-regulation of RhoB, which is up-regulated by DNA damage. We summarized the anti-proliferative effects of (–)-xanthatin in combination with our previous findings.

We identified 3-phenylpropionic acid, citrulline, lactic acid, ornithine, proline, and betaalanine as low-molecular weight substances that are released from cells treated with methylmercury. In this study, we studied their effect on cellular sensitivity to methylmercury. Treating HEK293 and C17.2 cells with each of the six substances minimally affected the proliferation of both cell lines. Among these six substances, however, only citrulline slightly but significantly increased the sensitivity of C17.2 and HEK293 cells to low levels of methylmercury. Citrulline is thought to be a methylmercury toxicity-enhancing factor whose extracellular release is enhanced by methylmercury.

This study attempted to identify substances that are driven out of HEK293 cells by methylmercury. Metabolomic analysis revealed that the levels of 3-phenylpropionic acid, citrulline, lactic acid, ornithine, proline and beta-alanine in the cell culture medium were increased by the treatment of cells with methylmercury. Address to the mechanism underlying the release of these substances will provide useful information to elucidate the toxicity mechanism of methylmercury.

HEK293 cells were cultured in medium containing methylmercury (MeHg), followed by replacement with MeHg-free medium and further culturing. Thus, MeHg preconditioning medium (MeHg-PM) were obtained. Untreated HEK293 cells and C17.2 cells (mouse neural stem cells) were placed in the obtained MeHg-PM for culturing, which resulted in significantly inhibited cell growth. This cell growth inhibition was not affected by heating or proteinase K treatment, suggesting that neither proteins nor peptides caused the growth inhibition.

Of antifoulants that are substitutes for organotin compounds such as tributyltin and triphenyltin, N-(2,4,6-trichlorophenyl)maleimide (IT-354) is listed as a much less toxic agent, although the available information concerning IT-354 toxicity is the results of acute toxicity tests in freshwater fish. In this study, the effects of IT-354 on rat thymic lymphocytes were examined using flow-cytometric techniques with appropriate fluorescent probes in order to estimate the effects of IT-354 on mammalian cells. Treatment of cells with 1-10 μM IT-354 for 1 hr did not increase the population of dead cells (cell lethality). However, 10 μM IT-354 significantly increased the population of living, annexin V-positive cells. Annexin V-positive, living cells are expected to be undergoing apoptosis. IT-354 at 3-10 μM significantly elevated intracellular Ca²⁺ and Zn²⁺ levels mainly by increasing Ca²⁺ influx and intracellular Zn²⁺ release. Furthermore, IT-354 significantly depolarized membranes and decreased cellular non-protein thiol content. Assessments using selected antifouling agents showed that the cellular actions of IT-354 are most likely similar to those of other commonly used antifouling agents. Therefore, the toxic potency of IT-354 on wild mammals is speculated to be similar to those of the other tested antifoulants.

Some household products have high levels of the antimicrobial 2-n-octyl-4-isothiazolin-3-one (OIT). Although the diverse effects of OIT are of concern, information regarding its cellular actions is limited. In a previous study, we found that OIT increased intracellular Zn²⁺ levels in rat thymocytes. However, because Ca²⁺ is considered the essential cation that causes cell injury and death, we examined whether Ca²⁺ and Zn²⁺ were involved in OIT-induced cytotoxicity and proposed the mechanisms underlying these results. The effects of OIT on the membrane and cellular parameters of rat thymocytes were examined with a flow cytometer and appropriate fluorescent probes. OIT (0.3-3 μM) increased intracellular Zn²⁺ levels but not intracellular Ca²⁺ levels. Therefore, the involvement of Zn²⁺ was studied further. The simultaneous application of 0.3 μM OIT and 3 μM ZnCl₂ significantly increased cells with phosphatidylserine-exposed membranes without changing the dead cells. In contrast, applications of 0.3 μM OIT or 3 μM ZnCl₂ alone had no effects. The combination of OIT (0.1-1 μM) and ZnCl₂ (1-3 μM) significantly decreased the cellular non-protein thiol contents. These changes that were induced by their combination were completely suppressed by adding an intracellular Zn²⁺ chelator. These results suggested that submicromolar concentrations of OIT induced Zn²⁺-dependent cytotoxicity in the presence of micromolar concentrations of external Zn²⁺. Because the threshold of OIT levels that affected cellular parameters in the presence of micromolar concentrations of Zn²⁺ are much lower than the OIT contents in some household products, the adverse effects of OIT are of great concern.

Perfluorinated compounds (PFCs) have been used widely, detected worldwide in the environment, and have accumulated highly in animals. As far as we know, there have been no reports which relate the PFC concentration in wild animals to the physicochemical properties. Therefore, we measured the concentrations of 15 currently available PFCs (perfluorocarboxylic acids with x carbons: Cx, perfluorosulfonic acids with x carbons: CxS) in medaka and the environmental water where medaka live. Samples were obtained from 7 points in Japan (Iwate, Ibaraki, Niigata, Hyogo, Yamaguchi, Ehime, and Nagasaki) from July to September in 2013. Twenty to forty medaka were collected from each point, as well as 2 L of water in a clean PET bottle. PFCs were extracted and concentrated using a solid-phase cartridge, and were measured by LC/MS/MS. The medaka samples were treated individually. C5-C9 and C8S were detected mainly in the water, C11-C13 and C8S were detected mainly in medaka. C8S was always detected in high concentrations in the water and medaka. The bioconcentration factors (BCFs) of PFCs were calculated from PFC concentrations of the water and the medaka. The BCFs of C8-C11 were increased exponentially with the length of carbon chain. The BCF of C8S (approx. 5,500) was far greater than C8 (approx. 330) or C9 (approx. 480). However, the BCFs of C8-C11 and C8S tended to increase in proportion with octanol/water partition coefficient (log K_ow).

To obtain information on the possible repeated-dose oral toxicity of β-bromostyrene and its reversibility, Crl: CD (SD) rats were administered β-bromostyrene through gavage at 0, 30, 125, and 500 mg/kg/day once for 28 days, followed by a 14-day recovery period. In the 500 mg/kg group, decrease in spontaneous movement was observed in all males and females on the first dosing day, and one female rat died on Day 3. There were no significant changes in body weight or food consumption. An increase in urine volume and decrease in urine osmolality were observed in males receiving 125 mg/kg and above, and an increase in urine volume was observed in females receiving 500 mg/kg. On blood biochemical examination, increases in total cholesterol, phospholipids, triglycerides, total protein, albumin, inorganic phosphorus, and/or chlorine were observed in the 125 and/or 500 mg/kg groups. Histopathologically, eosinophilic bodies of tubular cells and/or renal tubular degeneration were observed in the kidneys of males in the 125 and 500 mg/kg groups. In the thyroid, hypertrophy of follicular cells was observed in females receiving 125 mg/kg and above and males receiving 500 mg/kg. Furthermore, centrilobular hepatocellular hypertrophy was observed in both sexes receiving 500 mg/kg. These changes observed at the end of the dosing period disappeared or were reduced after the recovery period. Based on these results, the no-observed-adverse-effect-level of β-bromostyrene was judged to be 30 mg/kg/day for both sexes.

Perfluoroalkyl carboxylic acids (PFCAs) are global environmental contaminants that are the cause of concern due to their possible effects on wildlife and human health. Since few studies have investigated the toxicity of long-chain PFCAs, we have performed combined repeated dose toxicity studies with the reproduction/developmental toxicity screening tests. We previously examined perfluoroundecanoic acid (C11), perfluorododecanoic acid (C12), and perfluorooctadecanoic acid (C18). We herein reported our results for perfluorotetradecanoic acid (PFTeDA; C14) and perfluorohexadecanoic acid (PFHxDA: C16). Male and female rats were administered PFTeDA at 1, 3 or 10 mg/kg/day or PFHxDA at 4, 20 or 100 mg/kg/day by gavage, and each female was then mated with a male in the same dose group after 14 days. Males were dosed for a total of 42 days and females were dosed throughout the gestation period until day 5 after parturition. PFTeDA and PFHxDA caused hepatocyte hypertrophy and/or fatty changes in the liver at the middle and high doses. PFTeDA also induced follicular cell hypertrophy in the thyroid at the middle and high doses. The only reproductive/developmental effect observed was an inhibited postnatal body weight gain in pups in the 10 mg/kg/day PFTeDA group. Based on these results, the NOAELs for the repeated dose and reproductive/developmental toxicity were concluded to be 1 and 3 mg/kg/day for PFTeDA and 4 and 100 mg/kg/day for PFHxDA, respectively. Our current and previous results indicate that the toxicity of PFCAs decreases with increases in the carbon chain length from 12 to 18.

2,4,6-Trinitrotoluene (TNT) is a serious occupational and environmental pollutant. We conducted a cross-sectional health survey of workers in a TNT explosives factory in Fuxin, China. For each subject, we determined their blood pressure, hematotoxicity parameters, glutathione concentration, lipid hydroperoxide concentration, superoxide dismutase activity, and nitrite/nitrate (NOx) concentration in serum. Significantly fewer white blood cells were found in samples from male workers exposed to TNT than in samples from control male workers, but hematological parameters (such as the amount of hemoglobin present, the hematocrit value, and the formation of methemoglobin) varied little between the exposed and control workers. Exposure of male workers to TNT was found to cause their blood pressure to decrease significantly, concomitant with a tendency towards increased NOx concentrations in serum. On the other hand, lipid hydroperoxide (an oxidative stress marker) concentrations were significantly higher in female workers exposed to TNT than in control female workers. Our results suggest that TNT has different, deleterious effects in males and females, causing hematotoxic stress in males and oxidative stress in females.

Regression analysis such as linear regression and logistic regression has often been employed to construct toxicogenomic predictive models, which forecast toxicological effects of chemical compounds in human or animals based on gene expression data. While in general these techniques can generate an accurate and sparse model when a regularization term is added to a loss function, they ignore structural relationships behind genes which form vast regulatory networks and interact with each other. Recently, several reports proposed structured sparsity-inducing norms to incorporate prior structural information and make a model reflecting relationships between variables. In this study, assuming that genes regulated by the same transcription factor should be selected together, we applied the latent group Lasso technique on toxicogenomic data with transcription factor networks as prior knowledge. We compared generated classifiers for liver weight gain in rats between the latent group Lasso and Lasso. The latent group Lasso was comparable or superior to the Lasso in terms of predictive performances (balanced accuracy: 74% vs. 72%, sensitivity: 62% vs. 62%, specificity: 86% vs. 83%). Besides, groups selected by the latent group Lasso suggested involvement of Wnt/β-catenin signaling pathway. Such mechanism-related analysis could not have been possible with the Lasso and is one of the advantages of the latent group Lasso.

A selective cylooxygenase-2 (COX-2) inhibitor, rofecoxib, was withdrawn from the worldwide market due to an increased risk of cardiovascular (CV) events. A hypothesis has been proposed that rofecoxib promotes lipid oxidation, which increases the risk of CV events. However, this hypothesis was only predicated on in vitro experiments using isolated human low density lipoprotein and diluted human plasma. In the present study we investigated the effect of rofecoxib on the in vitro and in vivo production of thiorbarbituric acid reacting substance (TBARS) as an indicator of oxidation in plasma and aortas in rats. In vitro experiment, the TBARS production in plasma and aortic homogenate was not changed by the addition of rofecoxib at 2 μM, which concentration is around the maximum plasma concentration at clinical doses, or even at 200 μM. In addition, the production was not increased by rofecoxib in the presence of FeSO₄ as a typical oxidant. Meanwhile the TBARS production in the aorta of rats after 4-weeks administration of 10 mg/kg/day rofecoxib was comparable to that of the control rats. These results in-vitro and in-vivo experiments suggest that rofecoxib would have no or very weak effect on lipid oxidation in clinical usage, and it is thought that the increase of CV events already reported stemmed from causes other than oxidative stress.

Topical drug treatment of the eye exposes ocular tissues to a high drug concentration. Genotoxicity assessment in ocular tissues has not been established to date. Therefore, we investigated the in vitro comet assay by incubating cultured human corneal epithelial (HCE-T) cells with known mutagens. The alkaline comet assay was conducted to measure the DNA strand breakage yield. When the cells were incubated with methyl methanesulfonate (MMS) for 1 hr, hydrogen peroxide (H₂O₂) for 15 min and actinomycin D (AMD) for 1 hr, statistically significant increases of percentage (%) DNA in the tail were noted in MS-, H₂O₂-, and AMD-treated cells at 100, 10, and > 10 μM, respectively. When the cells were treated with mitomycin C (MMC) or 5-bromouracil (5-BrU), %DNA in the tail was unchanged even at the highest concentration. Hedgehog cells were found in MMS- and H₂O₂-treated cells at 1000 and > 100 μM, respectively. The response to each compound was consistent with results previously reported in other cells. In conclusion, the in vitro comet assay using HCE-T cells can detect DNA strand breakage induced by mutagens. This method has a possibility to become a conventional screening tool to assess the genotoxicity of drugs applied to ocular surface.

Cisplatin (CP) is one of important tumour chemotherapeutic agents in humans. Previous reports claim that CP can cause testicular toxicity. The aim of this study was to evaluate the potential effects of CP in the testes of rats. Male Wistar rats were intraperitoneally administered CP at 1.0, 2.5, and 5.0 mg/kg for three consecutive days. After exposure, CP significantly inhibited the testicular activities of succinate dehydrogenase (SDH) and malate dehydrogenase (MDH), but it significantly elevated the activities of acid phosphatase (ACP), alkaline phosphatase (AKP), and lactate dehydrogenase (LDH) in the 5.0 mg/kg group. The decreased levels of superoxide dismutase (SOD), total antioxidant capacity (T-AOC), and metallothionein-1 (MT-1) mRNA as well as the increased levels of malondialdehyde (MDA) and haemoxygenase-1 (HO-1) mRNA showed that CP could increase oxidative stress in rat testes. Western blot analysis showed that the levels of transferrin, vimentin, androgen binding protein (ABP) and inhibinβ-B decreased significantly in the CP 2.5 and 5.0 mg/kg groups compared with the control group. These findings indicated that the inhibited enzymes, oxidative stress, and the down-regulation of Sertoli cell function-related proteins play pivotal roles in CP-induced testicular damage.

Pathophysiological and nutritional conditions often affect the expression of drug-metabolizing enzymes. SHR/NDmcr-cp (cp/cp) rats (SHR/NDcp) are highly suitable as a metabolic syndrome (MS) model. Nevertheless, little is known about the expression profile of cytochrome P450 (CYP) in the liver of SHR/NDcp. We thus attempted to clarify the expression profile of CYP genes and the effect of fish oil (FO) on this profile in the liver of SHR/NDcp. Lower levels of CYP3A2 mRNA and CYP3A activity (testosterone 6β-hydroxylation) were distinctive features in SHR/NDcp compared with their controls (Wistar Kyoto rats (WKY), spontaneously hypertensive rats (SHR), stroke-prone SHR and lean littermates of SHR/NDcp). Differently from CYP3A2, the expression of other CYP isoforms was largely unchanged in SHR/NDcp. The changes in CYP profile observed in SHR/NDcp are similar to those of patients with diabetes and simple hepatic steatosis. Feeding on FO at a high dose (18.8% in the diet) upregulated CYP3A2 gene expression and CYP3A activity in the liver; the extent of these increases was greater in SHR/NDcp than in WKY and lean littermates of SHR/NDcp. This effect was not observed with FO at a normal dose (5% in the diet). These results indicate that, in the context of the CYP profile, SHR/NDcp is an animal model that is suitable for studying MS and imply that FO intake is critical in determining the efficacy or adverse effects of drugs in patients with MS.

Acotiamide hydrochloride hydrate (acotiamide-HH) has been newly developed as an indication for functional dyspepsia, which is characterized by digestive symptoms such as postprandial fullness, abdominal bloating, or early satiation, and is now being prescribed in Japan. As part of a safety assessment, 2-year long-term carcinogenicity studies using rats and mice were conducted. In the mouse carcinogenicity study, no evidence of carcinogenicity was obtained, even in the high-dose-treated group (up to 2000 mg/kg/day). In the rat carcinogenicity study, acotiamide-HH was administered at 200, 600, and 2000 mg/kg/day. Detailed histopathological examination revealed that the incidence of endometrial adenocarcinoma significantly increased in the 600 mg/kg/day treated group. There was no trend of this incidence and no accompanying increase in pre-neoplastic lesions or related histological changes in the genital tissues, suggesting the absence of abnormalities in the sexual endocrine system. Results of genotoxicity and reproductive/developmental studies showed that acotiamide-HH is a non-genotoxic substance and did not affect sexual balance. Acotiamide-HH did not induce an estrogen-dominant hormonal imbalance that could cause the incidence of uterine cancer and did not have initiation activity. Therefore, the proliferation of endometrial adenocarcinoma in this middle dose group in the rat carcinogenesis study was considered an accidental event of naturally occurring tumors. However, the incidence of endometrial adenocarcinoma in this group deviated from the background data collected in the same laboratory during the study period. Therefore, it is considered necessary to conduct another pre-clinical study in order to obtain data that would dispel any concerns of safety.

In photodynamic therapy (PDT) for glioma patients, apoptosis not necrosis is the desirable mode of cell death, as necrotic cell death induces late appearance of obstacles following PDT. We previously demonstrated that increase in both treatment dose of photosensitizer talaporfin sodium (NPe6) and laser fluence (laser energy density) changes the dominant cell death process from apoptosis to necrosis in glioblastoma T98G cells. Here, we investigated the effect of laser irradiance (laser power density), which is another important parameter of PDT, on PDT-induced cell death modalities in cultured T98G cells. When fluence was fixed at 10 J/cm², NPe6 dose-dependently reduced the cell viability, regardless of irradiance (11, 22, 33, and 44 mW/cm²). Morphological observations and biochemical analysis (measurement of caspase-3 activity, staining of cell surface-exposed phosphatidylserine, and staining of propidium iodide) further confirmed that increase in dose of NPe6 changed the dominant cell death process from apoptosis to necrosis, regardless of irradiance. We also noted no influence of irradiance level on the leakage of lactate dehydrogenase from T98G cells following PDT treatment. Taken together, our present and previous findings suggest that dose of NPe6 and laser fluence but not laser irradiance are important parameters to consider in PDT using NPe6 in T98G cells.

Air purifiers, which release positive and negative ions generated by electric discharge, are widely used in a variety of places. In this study, male and female SD rats [Crl:CD(SD)] were exposed by whole-body inhalation (6 hr/day) to ionized air containing positive and negative ions for at least 10 weeks before mating and throughout the mating, gestation, and lactation periods over two generations, and the effects on the reproductive function of parental animals and development of offspring were assessed. The concentrations of the ionized air were set at 0 and 7,000,000 ions/cm3 (280- to 1,000-fold higher than normally used in humans) and each group consisted of 24 F0 rats/sex/group and 20 to 23 F1 rats/sex/group. The ionized air had no general toxicological effects on parental animals in the observation for clinical signs, body weight and food consumption measurement, or pathological examination. As for the effects on the reproductive function, there were no exposure-related changes in mating ability, fertility, pregnancy, parturition, or nursing behavior, nor were there any changes in the estrous cycle or sperm parameters in either generation, nor in the ovarian follicle counts (only F1 females). Moreover, there were no effects on litter size, viability, growth, or development of F1 and F2 offspring, including sexual maturation. Therefore, it was suggested that the ionized air has no reproductive or developmental toxicity in rats.

To understand selenium (Se) circulation in the biosphere, the metabolism of organic Se, in particular, Se metabolites, in animals and plants should be elucidated. In this study, garlic, Allium sativum, a well-known Se accumulator with high Se metabolic ability, was hydroponically cultivated and then exposed to trimethylselenonium ion (TMSe), a urinary metabolite. Thereafter, the Se concentration in several parts of garlic, such as roots, bulbs, and leaves, was determined. To reveal the metabolic pathway of TMSe, the Se species in A. sativum were investigated by speciation using HPLC hyphenated with an inductively coupled plasma mass spectrometer (LC-ICP-MS). Se was mostly accumulated in the roots. TMSe was detected in the extract of each plant part. However, the amount of Se incorporated from the medium was not completely recovered in the garlic, suggesting that a part of TMSe was metabolized into volatile Se. Consequently, we conclude that the majority of TMSe incorporated into garlic is accumulated as is, the rest is partially desmethylated to form a volatile Se compound, such as a dimethylated Se compound.

We previously identified cannabidiolic acid (CBDA), a major component of the fibertype cannabis plant, as an inhibitor of MDA-MB-231 human breast cancer cell migration in vitro (Takeda et al., 2012). Although MDA-MB-231 is a widely used human breast cancer cell line in in vitro and in vivo studies, these cells have to be injected into nude mice (immunodeficient animals) in in vivo trials. Thus, we established the murine breast cancer cell line, 4T1E/M3, which is highly metastatic to bone in BALB/c mice (Takahashi et al., 2008, 2009; Sakai et al., 2012); this murine syngeneic tumor model may be useful for identifying molecular targets for therapeutic interventions. Prior to in vivo experiments using the murine tumor model, we herein performed DNA microarray analyses of 4T1E/M3 cells, treated with CBDA for 48 hr at a sub-toxic concentration (25 μM), in order to comprehensively analyze the effects of CBDA on the genes involved in the bone metastasis of breast cancers. The results obtained revealed that the expression of matrix metalloproteinase-9 (MMP-9), transforming growth factor-β (TGF-β) inducible gene H3 (BIGH3), and parathyroid hormone-related protein (PTHrP) was markedly down-regulated by 0.11-fold, 0.22-fold, and 0.15-fold, respectively; these molecules were mutually involved in the bone metastasis of breast cancer cells.

The purpose of this study was to determine the suitability for measurement of oral bacterial counts (OBC) in dogs using a new device that operates on the principle of dielectrophoretic impedance. Using this device, bacterial counts were successfully measured in swabs collected from the mouths of 5 non-anesthetized beagles. We tried to take samplings from 6 sites in each dog’s mouth and stable counts obtained at an interval of 2 weeks showed no significant difference in any of the 6 sites over time. However, since the counts showed significant differences depending upon the number of times the swab was rubbed on the sampling site, and the time from feeding affects oral bacterial counts, special attention is needed on these 2 issues. The new device allows rapid measurement of oral bacterial counts in dogs under appropriate conditions. The simplicity of this method may make it useful in studies on agents affecting OBC in dogs.

Studies on the low-dose effects of xenoestrogens have yielded conflicting results that may have resulted from differences in estrogen sensitivity between the mouse strains used. We developed a mouse newborn behavioral testing method for evaluating the risk of neurotoxicity of environmental chemicals, by means of determining a newborn’s motor activity through applying the tare function of an analytical balance. Motor activities including crawling, pivoting, and tremors of C57BL/6J and ICR mouse newborns exposed to bisphenol A (BPA) at 200 μg/kg/day on embryonic days 6 through 18 were evaluated for 5 min on postnatal day 1 by the testing method. Motor activities of mature male offspring exposed prenatally to BPA were also evaluated in wheel cage and open field tests. Maternal BPA oral dosing increased the motor activity in newborns of both strains and mature offspring of the C57BL/6J strain. The findings indicate that both mouse strains provide adequate models for the newborn neurobehavioral study of prenatal exposure to environmentally relevant levels of estrogen-mimicking chemicals.

While the primary source of human MeHg exposure is the consumption of fish contaminated with MeHg, it is unknown whether the toxicity of MeHg in fish is equivalent to that of MeHg chloride (MeHgCl) experimentally added to the diet. We investigated developmental and behavioral effects of MeHg derived from fish and MeHgCl added to various diets during the prenatal period in mice from GD 0 to GD 17. From 7 to 9 female C57BL/6NCr mice were assigned to each of the following exposure groups: Control (CL), CL+MeHgCl (CL+MeHg, 1.6 mgHg/kg), low MeHg tuna (LT, 0.2 mgHg/kg), LT+MeHgCl (LT+MeHg, 1.6 mgHg/kg), and high MeHg tuna (HT, 1.6 mgHg/kg). In pups, body weight was depressed and elevated by MeHg exposure in the CL+MeHg and the LT, respectively, compared with other three groups. In neurodevelopmental test, the righting reflex of 4 groups other than CL showed the facilitated developments compared to the CL. The cliff avoidance of the HT developed slower than in the CL+MeHg, LT and LT+MeHg. In water maze test, the swimming speed of the HT decreased in comparison with the CL in males but not females. The latency until falling from a rotating rod of the LT+MeHg was significantly shorter than that of the LT in males but not females. Our results are suggesting the possibility that the toxicological profiles of MeHg derived from fish and reagent MeHg are somewhat different. Our findings also provide evidence that males are more susceptible than females to prenatal MeHg exposure.

The toxicity of organic-inorganic hybrid molecules appears to depend on the toxicity of the organic structure, the metals, and their interaction. However, very little is known about the structureactivity relationship of these molecules. In the present study, we investigated the cytotoxicity of triphenylstibane (Sb25) and its fluorine-substituted derivatives the triarylstibanes, using a culture system of bovine aortic endothelial cells. The results showed that the cytotoxicity of tris(4-fluorophenyl)stibane (Sb33) and tris(3,4,5-trifluorophenyl)stibane (Sb49) was higher than that of Sb25, suggesting that introduction of fluorine atoms into the benzene rings may potentiate the cytotoxicity of Sb25 in vascular endothelial cells. However, interestingly, tris(pentafluorophenyl)stibane (Sb35) was nontoxic. The pnictogen analogues tris(pentafluorophenyl)arsane (As35) and tris(pentafluorophenyl)phosphane (P35) showed a higher cytotoxicity than that of Sb35. In addition, the potentiation was much stronger with P35 than it was with As35. The intracellular accumulation of Sb35 was very low while the accumulation of As35 was higher than that of Sb25. These results collectively suggest that the hydrophobicity and metal of the organometallic compounds do not necessarily predict their cytotoxicity and intracellular accumulation in vascular endothelial cells.

Glucose is important for energy; however, excessive daily intake of sugar may act as a toxin inducing the body to become overweight or obese. High blood glucose level reduces secretion of insulin, and glucose toxicity worsens insulin resistance. We investigated the metabolic fate of excess glucose by changing glucose levels in MRC-5 fibroblasts. Uptake of glucose into fibroblasts, the first stage of glucose metabolism, was measured. Treatment of fibroblasts under diabetic conditions led to rapid glucose incorporation. Glucose was absorbed into the cell almost constantly and reached excessive levels, and its metabolism was assessed by ¹⁴CO₂ output from [U-¹⁴C] D-glucose, the glucose metabolism end product. When fibroblasts were cultured in the presence of high glucose levels, CO₂ production decreased significantly in comparison with normal glucose conditions. Glucose metabolism in the diabetic setting was not accompanied by an increase in glucose uptake. Diabetic patients exercise tight glycemic control to avert disorders from such glucose toxicity. Pyruvate dehydrogenase (PDH) activity is reduced in diabetes; therefore, we investigated the influence of thiamine on PDH activity and intracellular glucose concentration in fibroblast cells exposed to diabetic conditions. Thiamine reversed high glucose-induced PDH inhibition and prevented glucose accumulation. These results, taken together with those of our previous report, suggest that thiamine partially plays a role in modifying the metabolic fate of glucose and reducing glucose toxicity.

Lipophilic toxin pectenotoxin-2 (PTX2) is oxidatively metabolized to pectenotoxin-1 (PTX1), pectenotoxin-3 (PTX3), and pectenotoxin-6 (PTX6) in the Japanese scallop Patinopecten yessoensis. This particular metabolism has been observed only in Japanese scallops, in which PTX6 is the most dominant lipophilic toxin. We investigated the cytotoxicity of PTX2 and its metabolites PTX1,3,6 in a rat cell line (L6) and a human cell line (RD). RD showed an approximately three-fold greater sensitivity than L6 upon exposure to PTXs. The cytotoxicity of PTXs decreased with degree of oxidation in the order PTX2 > PTX1 > PTX3 > PTX6. The calculated half maximal inhibitory concentration (IC₅₀) values of PTX2 obtained for the L6 and RD cell lines were 60 and 23 ng/mL, respectively, while those obtained for PTX6 for both cell lines were over 2,000 ng/mL. These results demonstrate that PTX6 has extremely low cytotoxicity or is non-toxic and that the oxidative metabolism of PTX2 in P. yessoensis is a detoxification process.

Drug-induced hepatotoxicity is a common reason for discontinuing the development of candidate clinical drugs. In the present study, we investigated the utility of three-dimensionally cultured human hepatocytes (spheroids) for prediction of hepatotoxicity, using a panel of model drugs: acetaminophen, benzbromarone, chlorpromazine, cyclosporin A, diclofenac, fialuridine, flutamide, imipramine, isoniazid, ticlopidine and troglitazone. Cultured spheroids showed a significant increase of albumin secretion from 2 to 7 days; the secretion started to decrease at 14 days, but continued from 14 days to 21 days. The morphology of the spheroids was well maintained for 21 days. Long-term exposure of spheroids to hepatotoxic drugs resulted in concentration-dependent depression of albumin secretion and elevation of aspartate aminotransferase (AST) leakage. The estimated 50% effective concentration (IC₅₀) values for decrease of albumin secretion changed from 7 days to 14 days, but similar values were obtained at 14 and 21 days, except for diclofenac. Since the IC₅₀ values and the values of drug concentration inducing 1.2-fold elevation of AST leakage (F1.2) were similar at 14 and 21 days, an incubation period of 14 days was considered sufficient. The coefficient of determination (R²) between IC₅₀ values and F1.2 values of all drugs was 0.335. When cyclosporine A and fialuridine were excluded, the value of R² became 0.887. The results indicate that the proposed human hepatocyte spheroid assay should be helpful in the evaluation of hepatotoxicity during the early development stage of clinical drug candidates.

We developed a newborn mouse behavioral testing method to evaluate the risk of neurotoxicity of environmental toxicants, based on determining a newborn’s motor activity by applying the tare function of an analytical balance. Motor activities of newborn ICR mice exposed prenatally to diethylstilbestrol (DES) at 0.005-0.5 μg/kg/day on days 5 through 18 of gestation were evaluated on postnatal day 1. The activities of male newborns in the 0.05 μg/kg/day group were significantly increased compared to those of the controls, and the increasing tendencies were observed in both sexes of the highest group. The findings indicate that prenatal exposure to low doses of DES causes hyperactivity in newborn mice.

To characterize variability of various musculoskeletal biomarkers by different blood sampling techniques in conscious rats, plasma asparate aminotransferase (AST), creatine kinase (CK) and its isoenzymes, fatty acid binding protein 3 (FABP3), myosin light chain 3 (Myl3) and microRNA (miR-133a) obtained by jugular venipuncture (C-JV) or tail venipuncture (C-TV) were compared with those obtained by jugular venipuncture (A-JV) in isoflurane-anesthetized rats. Plasma CK, FABP3 and Myl3, especially when collected by C-TV, were higher with larger variability than when collected by A-JV, whereas miR-133a displayed large variability in all techniques. Interestingly, higher CK obtained by C-JV or C-TV was largely attributable to higher CK-MM or CK-BB, respectively. Handling and restraint stress were identified as possible factors contributing to larger variability for CK, FABP3 and Myl3. A close correlation between CK and FABP3 was demonstrated both in the C-JV and C-TV techniques. Next, we evaluated the impact of C-JV and C-TV techniques for detecting skeletal myopathy in 2,3,5,6-tetramethyl-p-phenylenediamine-treated rats. In this model, CK and CK-MM obtained by C-TV were significantly increased, but those obtained by C-JV were not modified. In contrast, AST, FABP3, Myl3 and miR-133a obtained by both techniques were drastically elevated to a similar extent. The results suggest that, in conscious rats, the tail venipuncture technique may be more appropriate to detect skeletal myopathy despite the higher variability with this technique than with the jugular venipuncture technique. Furthermore, FABP3, Myl3 and miR-133a may serve as more sensitive biomarkers with large signal-to-noise ratios regardless of the blood sampling technique in conscious rats.

Nanomaterials have been proposed as novel substrates for medical and commercial applications. However, such materials also may have novel toxicities, thus posing environmental and health concerns. We previously reported hepatic injury in mice following the intravenous administration of unmodified silica particles with diameters of 70 nm (SP70); this toxicity was not observed following administration by the same route of micro-size particles with diameters of 300 nm (SP300) or 1,000 nm (SP1000). In the present study, we used electron microscopy to investigate the dynamics of silica nanoparticles administered in mice. SP70 was observed in hepatocytes and in lung epithelial cells. Inclusion within hepatocytes was associated with accumulation of SP70 in the liver sinusoidal endothelial cells and passage through the space of Disse. In contrast, SP300 and SP1000 were not observed within the hepatocytes. To our knowledge, our report represents the first demonstration that silica nanoparticles accumulate in hepatocytes, liver sinusoidal endothelial cells, Kupffer cells, and lung tissue; accumulation of SP70 in liver sinusoidal endothelial cells correlated with the induction of liver injury.

The purpose of this study was to evaluate the effects of sub-chronic exposure to deltamethrin in lower doses on the acquisition of a two-way avoidance task and the levels of amino acid neurotransmitters in hippocampus of mice measured using shuttle-box and LC-MS/MS system. Deltamethrin was given to mice respectively at doses of 0.46, 0.92, 1.80 mg/kg BW daily for 60 days by gavage. Deltamethrin was found to decrease the number of avoidance responses, increase response latency, and increase glutamate levels in the 0.92 and 1.80 mg/kg BW-dose group. As revealed by electron microscopy, in 0.92 and 1.8 mg/kg-dose group mice, morphology of cells were changed and degeneration and necrosis morphological characteristics obviously were appeared. Collectively, results from this study suggest that deltamethrin may have cumulative effects in mice following repeated dosing of deltamethrin using moderately effective doses, beside Na⁺ and Ca²⁺ channels as well as Na⁺ and Ca²⁺-dependent glutamate release, may be involved with neurotoxic action of deltamethrin.

Lepidopteran species fly freely in the environment and their larvae feed on the leaves of host plants which may be exposed to nanomaterials. As an ecological model of nanoparticle exposure, 5th instar larvae of the sweet potato hornworm (Agrius convolvuli) were subcutaneously injected with suspensions of 10 μL (100 μg/mL) titanium dioxide nanoparticles (TiO₂-NPs), 10 μL (100 μg/mL) zinc oxide nanoparticles (ZnO-NPs) or saline (control) and the effects on spermatogenesis were examined in the pupal testis. During the larval wandering stage, larval tissues (except the testis) underwent extremely rapid histolytic changes. Pupation and emergence were not affected by the injection. On pupal day 4, there was a significant decrease in testis weight and the number of sperm bundles in the ZnO-NPs group. Electron microscopic observation revealed that cyst cells surrounding the spermatogenic cells took on small agglomerates of TiO₂-NPs or ZnO-NPs by phagocytosis. As spermatogenesis advanced in the nanoparticle-injected groups, vacuoles of various sizes were found in the nuclei of spermatocytes, the nuclear chromatin of spermatids was uncondensed and some vacuoles were found in the nuclei of sperm bundles. A possible mechanism for this is that abnormal vacuoles disturbed the chromatin condensation, resulting in the decrease of sperm bundles. Toxicity of manufactured TiO₂-NPs and ZnO-NPs were demonstrated detriment to insect spermatogenesis.