Protein disulfide isomerase (PDI) is a multifunctional protein that catalyzes disulfide bond formation and assists protein folding, as well as being a structural subunit of microsomal triglyceride transfer protein (MTP) and prolyl 4-hydroxylase (P4HD), and an estrogen and thyroid hormone-binding protein. Previous reports indicate that some endocrine-disrupting chemicals (EDCs) bind to PDI and disturb its functions, and we executed PDI-knockdown to examine the effects of dysfunction of PDI. In this study, the effects of PDI-knockdown were compared among three cell lines: MCF-7, SH-SY5Y and HeLa. PDI-knockdown induced different levels of cytotoxicity among these cell lines. In MCF-7 cells, PDI-knockdown activated apoptotic signaling, causing cytochrome c release from mitochondria and activation of caspase-9, caspase-6, caspase-7 and poly[ADP-ribose]polymerase-1, and the cytotoxicity induced by PDI-knockdown was suppressed by a pan-caspase inhibitor, z-VAD-fmk. These data suggest that cell death induced by PDI-knockdown is caspase-dependent apoptosis in MCF-7 cells.

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The present study was designed to fully uncover sex and circadian modulatory effects on rat liver. Hepatic transcriptome analyses were performed at 4 hr intervals of a day-night cycle using young adult male and female rats. Sexually dimorphic genes, which were identified by a cross-sex comparison of time series data, included representative sex-predominant genes such as male- or female-predominant cytochrome P450 subfamilies (Cyp2c11, Cyp2c12, Cyp2c13, and Cyp3a2), sulfotransferases, and glutathione S-transferase Yc2. The identified sexually dimorphic genes were over-represented in the metabolism of retinols, xenobiotics, linoleic acids, or androgen and estrogen, or bile acid biosynthesis. Furthermore, transcription factor targets modeling suggested that transcription factors SP1, hepatocyte nuclear factor 4-alpha (HNF4-alpha), and signal transducer and activator of transcription 5b (STAT5b) serve as core nodes in the regulatory networks. On the other hand, Fourier transform analyses extracted universal circadian-regulated genes in both sexes. The circadian-regulated genes included clock or clock-controlled genes such as aryl hydrocarbon receptor nuclear translocator-like (Arntl), period homolog 2 (Per2), and D site albumin promoter binding protein (Dbp). The extracted cyclic genes were over-represented in major tissue activities, e.g. the urea cycle and the metabolism of amino acids, fatty acids, or glucose, indicating that the major liver functions are under circadian control. The transcription factor targets modeling suggested that transcription factors SP1, HNF4-alpha, and c-Myc proto-oncogene protein (c-MYC) serve as major hubs in the circadian-regulatory gene networks. Interestingly, transcription factors SP1 and HNF4-alpha are likely to orchestrate not only sexually dimorphic, but also circadian-regulated genes even though each criterion was rather mutually exclusive. This suggests the cross-talk between those regulations. Sexual dimorphism is likely to interact with circadian rhythmicity via overlapping gene regulatory networks on rat liver.

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Nausea and emesis are often observed as side effects with many medicines and may lead to poor treatment compliance. In the present study, we aimed to establish simple methods for predicting nausea and/or emesis in mice, which do not vomit, using drugs and chemicals known to evoke nausea and/or emesis. The gastrointestinal transit test, the liquid gastric emptying by phenol red solution (Phenol red method) and the solid gastric emptying by resin beads (Beads method) were used and the effects of antispasmogenics (atropine, 0.1-3 mg/kg i.p.; salmon calcitonin, 1-30 units/kg i.m.), nauseants (copper sulfate, 1-30 mg/kg p.o.; apomorphine, 0.01-0.3 mg/kg s.c.) and chemotherapeutics (cisplatin, 0.3-10 mg/kg i.v.; doxorubicin, 0.3-10 mg/kg i.v.) were evaluated. In addition, the effects of ondansetron, a serotonin (5-HT)₃ receptor antagonist, on the inhibition of solid gastric emptying induced by salmon calcitonin, copper sulfate, cisplatin and doxorubicin were also assessed. Only the solid gastric emptying method could detect changes of gastric emptying by all drugs and chemicals. We also found that the inhibition of solid gastric emptying induced by cisplatin and doxorubicin was dose-dependently antagonized by ondansetron. However, ondansetron failed to antagonize the salmon calcitonin-induced delay, but exerted only very weak effects with copper sulfate. Solid gastric emptying may be more suitable than gastrointestinal intestinal transit or liquid gastric emptying in mice to predict nausea and/or emesis. Our results also suggest that chemotherapeutic-induced delay of solid gastric emptying mediated via 5-HT₃ receptors in mice could also be useful for prediction purposes.

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Anhedonia, an affective symptom related to the inability to experience pleasure, is one of the representative symptoms observed in depression. In the present study, considering that repeated nicotine (NC) also causes “depressive” symptoms, the depression-related anhedonic behavioral alterations caused by a typical depression-inducing stressor, immobilization stress (IM), combined with or without NC administration, were examined in mice and compared with the depression-like behavioral alterations caused by NC. In the repeated IM (10 min, 4 days) group, as well as the repeated NC (0.3 mg/kg, s.c., 4 days) group, depression-related behavioral despair was observed in both forced swimming and tail suspension tests. Depression-related anhedonic behavioral alterations, as judged in the sucrose test, were observed only in the IM group. In the group treated with IM plus NC (IM-NC group), NC antagonized the IM-induced anhedonic attenuation of sucrose consumption in the sucrose test. Furthermore, in the IM-NC group, NC attenuated the effects of antidepressants which inhibit the reuptake of monoamines in the forced swimming test. Against the IM-induced anhedonia in the sucrose test, the cannabinoid agonists anandamide and CP 55940, in addition to the antidepressants previously reported, restored the preference for sucrose to control levels, with or without NC co-treatment. The absence of anhedonic behavioral alterations, the antidepressant-like anti-anhedonic effects against IM, and the effects against some antidepressant drugs all seemed to be characteristic of the effects of NC. Neural mechanisms other than those involved in the depression-like effects of NC seemed to contribute to the IM-induced anhedonic component of depression.

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Previously, we investigated endocrine disrupting effects of 17 β-estradiol (E₂) on Japanese quail (Coturnix japonica) in the avian reproduction test according to the testing guidelines, in which new endpoints such as blood vitellogenin (VTG) concentration in parent quails and pathology of F₁ chicks were added, and consequently these additional endpoints suggested to be sensitive markers for detecting any impacts of endocrine disrupting effects (Shibuya et al., 2005b). In the present study, to investigate low dose effects of estrogenic endocrine disrupting chemicals in birds, the avian reproduction study of E₂ at low dose levels was conducted using Japanese quail with additional endpoints such as observations of F₁ chicks until 10 weeks of age, histopathology of F₁ chicks at 14 days and 10 weeks of age and blood VTG concentration in parent quails. Sixteen pairs of 10-week-old quails were fed a low phytoestrogen diet containing E₂ at 0 (control), 0.3, 3, and 30 ppm for 6 weeks, and parent quails, eggs and offspring were examined. F₁ chicks were maintained up to 14 days or 10 weeks of age. Serum E₂ and VTG concentrations in males of the E₂ 3- and 30-ppm groups and in females of the E₂ 30-ppm groups were significantly elevated. In the E₂ 30-ppm group, two parent females died, and toxic changes such as suppression of body weight gain, decrease in food consumption and atrophic and degenerative changes of the reproductive organs were observed in parent quails. In the same group, the number of eggs laid and the fertility rate of eggs were significantly decreased. In addition, the viability of F₁ chicks in the E₂ 30-ppm group were significantly decreased at 10 weeks of age. On the other hand, no abnormalities described above were observed in any parent quails, eggs and F₁ chicks in the E₂ 3- and 0.3-ppm groups, although the fertility rates of eggs in both groups were decreased and the body weight gain of F₁ females in the E₂ 3-ppm group was significantly suppressed. In the histopathological examination of F₁ chicks maintained up to 10 weeks of age, persistent right oviduct and atrophy of the oviduct gland were observed in females of E₂-treatment groups with significantly high incidences. Moreover, cystic dilatation and tubular degeneration of the seminiferous tubules and atrophy of the cloacal gland were also observed in males of the E₂-treatment groups. Thus, the dietary treatment of low dose E₂ (even 0.3 ppm) to parent quails resulted in decreased viability and induction of abnormalities in the oviduct, testis and cloacal gland in F₁ chicks maintained up to 10 weeks of age. These results suggest that additional endpoints such as observations of F₁ chicks until 10 weeks of age, histopathology of F₁ chicks at 14 days and 10 weeks of age and blood VTG concentration in parent quails would be useful and sensitive endpoints for evaluating estrogenic endocrine disrupting effects in the avian reproduction study.

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Perfluorooctane sulfonate (PFOS) is one of the persistent organic pollutants distributed widely in the global environment. We have found that a single oral administration of PFOS induced tonic convulsion in mice and rats when a brief ultrasonic stimulus was applied to the animals. The aim of this study is to examine whether the neurotoxicity is caused by subchronic dietary exposure to PFOS. Rats were treated with dietary PFOS at 0, 2, 8, 32 and 128 ppm for 13 weeks. Animals were carefully observed for pharmacotoxic signs and responses to the ultrasonic stimulus applied biweekly. PFOS increased liver weight and decreased food consumption and body weight. PFOS concentrations in the serum, brain, liver and kidney were increased almost proportional to its total dose, although the ratios of PFOS concentrations in tissues to total doses in the group treated with the highest concentration were a little lower. The ranges of relative concentrations in the brain, liver and kidney to serum concentration were 0.13 to 0.24, 2.7 to 6.3 and 0.82 to 1.6, respectively. PFOS alone did not cause any neurotoxic symptoms; however, 5 rats out of 6 showed tonic convulsion in the 6th week when ultrasonic stimulus was applied to the 128 ppm rats with the total PFOS dose of 338 mg/kg. The ultrasonic stimulus did not cause convulsion in the other groups. Histopathological examination including electron microscopic examination could not detect any abnormality in the brain. Because the acute oral dose of PFOS causing the convulsion was 250 mg/kg (Sato et al., 2009), the convulsion induced by PFOS seemed to depend on its total dose regardless of treatment schedule.

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In order to understand the influence of coefficient of variation (CV) in determining significant difference of quantitative values of 28-day repeated-dose toxicity studies, we examined 59 parameters of 153 studies conducted in accordance with Chemical Substance Control Law in 12 test facilities. Sex difference was observed in 12 parameters and 10 parameters showed large CV in females. The minimum CV was 0.74% for sodium. CV of electrolytes was comparatively small, whereas enzymes had large CV. Large differences in CV were observed for major parameters among 7-8 test facilities. The changes in CV were grossly classified into 11. Our study revealed that a statistical significant difference is usually detected if there is a difference of 7% in mean values between the groups and the groups have a CV of about 7%. A parameter with a CV as high as 30% may be significantly different, if the difference of the mean between the groups is 30%. It would be ideal to use median value to assess the treatment-related effect, rather than mean, when the CV is very high. We recommend using CV of the body weight as a standard to judge the adverse effect level.

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We evaluated the effects of prenatal exposure to low-level mercury (Hg⁰) or methylmercury (MeHg) as well as combined exposure (Hg⁰ + MeHg exposure) on the neurobehavioral function of mice. The Hg⁰ exposure group was exposed to Hg⁰ at a mean concentration of 0.030 mg/m³ for 6 hr/day during gestation period. The MeHg exposure was supplied with food containing 5 ppm of MeHg from gestational day 1 to postnatal day 10. The combined exposure group was exposed to both Hg⁰ vapor and MeHg according to above described procedure. After delivery, when their offspring reached the age of 8 weeks, behavioral analysis was performed. Open field (OPF) tests of the offspring showed an increase and decrease in voluntary activity in male and female mice, respectively, in the MeHg exposure group. In addition, the rate of central entries was significantly higher in this group than in the control group. The results of OPF tests in the Hg⁰ + MeHg exposure group were similar to those in the MeHg exposure group in both males and females. The results in the Hg⁰ exposure group did not significantly differ from those in the control group in males or females. Passive avoidance response (PA) tests revealed no significant differences in avoidance latency in the retention trial between the Hg⁰, MeHg, or Hg⁰ + MeHg exposure group and the control group in males or females. Morris water maze tests showed a delay in the latency to reach the platform in the MeHg and Hg⁰ + MeHg exposure groups compared with the control group in males but no significant differences between the Hg⁰, MeHg, or Hg⁰ + MeHg exposure group and the control group in females. The results of OPF tests revealed only slight effects of prenatal low-level Hg⁰ exposure (0.03 mg/m³), close to the no-observable-effect level (NOEL) stated by the WHO (0.025 mg/m³), on the subsequent neurobehavioral function. However, prenatal exposure to 5 ppm of MeHg affected exploratory activity in the OPF test, and, in particular, male mice were highly sensitive to MeHg. The MeHg and Hg⁰ + MeHg exposure groups showed similar neurobehavioral effects. Concerning the effects of prenatal mercury exposure under the conditions of this study, the effects of MeHg exposure may be more marked than those of Hg⁰ exposure.

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The impact of Morinda citrifolia (noni) juice on fertility and offspring health in three generations of ICR mice was evaluated. The authenticity of the source of noni juice in this study was determined by chemical analysis of known marker compounds. Mice were supplied with 5% noni juice at gestation (day 0) until weaning (21 days postpartum). This procedure was followed through three generations of offspring. Three generations of control mice were also evaluated. There were no intergroup differences in gestation and fertility indices or malformation rates. However, litter sizes of the noni group in the first (F1),  second (F2), and third (F3) generations were, respectively, 29.3% (P < 0.01), 19.8% (P < 0.01) and 19.6% (P < 0.01) larger than corresponding controls. Despite larger litter sizes, there were no decreases in fetal weight in any generation of the noni group. Further, maternal health and offspring viability in the noni groups were equal to or greater than the controls. The results of this study suggest that authentic noni juice has no adverse effect on fertility and fetal development, consistent with previous two-generation studies of noni fruit from French Polynesia, Indonesia, and Hainan , China. On the contrary, noni juice appears to facilitate pregnancy and fetal development.

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This study was performed to investigate the safety of Alchornea cordifolia, Cnestis ferruginea, Lonchocarpus sericeus, Trema orientalis, and Senna alata in respect to genotoxicity. These five medicinal plants are widely distributed in Africa. They are used as a traditional medicine in many African counties for the treatment of microbial, inflammatory, and stress-related diseases. To evaluate the bacterial reverse mutation of these five medicinal plants, the in vitro Ames test using Salmonella typhimurium TA98, TA100, TA1535, and TA1537, and Escherichia coli WP2uvrA, with or without the addition of S9 mixture was performed. Concentrations used for this test were 625, 2,500, and 5,000 μg per plate. A. cordifolia, C. ferruginea, L. sericeus, and T. orientalis showed negative results in the bacterial reverse mutation test, suggesting that it is potentially safe for these plants to be used in medicinal plants supplements at high doses. However, our experiments suggest that S. alata is a potent mutagen. Therefore, further studies are needed to evaluate the carcinogenicity of S. alata in order to adequately assess the risks for human health.

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In order to estimate the effects of the size and surface treatment (coating or non-coating) of titanium dioxide particles on their cytotoxicity and penetration into the cellular membrane, two types of non-treated titanium dioxide (TiO₂)  particles of 20 nm (LU175) and 250 nm (LU205) were exposed to CHO cells, RBL-2H3 cells, A431 cells, B16 melanoma, NHEK(F), and NHSF, and six types of surface-treated or non-treated TiO₂ particles of 35 nm were exposed to RBL-2H3 cells and NHSF. The order of half-maximal inhibitory concentrations (IC50s) of LU175 was NHSF < CHO, RBL-2H3 < A431 < B16 melanoma, NHEK(F). On the other hand, LU205 showed no cytotoxicity against any cells. Surface-treated TiO₂ showed much less cytotoxicity against RBL-2H3 cells than non-treated TiO₂. Then, between 0.5 and 10 mg of LU175 or LU205 was exposed to CHO cells. After 24 hr, the amount of LU175 in cellular cytosol increased dose-dependently. On the other hand, the amount of LU205 in cellular cytosol was much less than that of LU175. The proportion of surface-treated TiO₂ in the cellular cytosol of RBL-2H3 cells differed for each coating material. These results suggested that TiO₂ has different cytotoxicities among cell lines, and that of surface-treated TiO₂ was weaker than that of non-treated TiO₂. TiO₂ located in cytosol might be the main cause of cytotoxicity.

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The purpose of the present study was to elucidate the effects of dietary zinc-deficient feeding and its recovery on liver cytosolic alcohol dehydrogenase (ADH; alcohol: NAD⁺ oxidoreductase, EC1.1.1.1) activities and plasma zinc levels in rats. The weaned male Sprague Dawley rats were randomly divided into the zinc-deficient diet (ZDF: 1.9 mg zinc/kg diet) group and the control diet (53.5 mg zinc/kg diet) group, and were fed for 4 weeks. In the recovery periods, the rats of two groups were fed with the control diet for 3 weeks. Liver cytosolic protein content per body weight in the zinc-deficiency and its recovery period showed no significant changes between both groups. However, zinc-deficiency decreased significantly liver cytosolic ADH specific activity, total liver cytosolic ADH activity and total liver cytosolic ADH activity/body weight by 50%, 76% and 53%, respectively, as compared with the control diet group. Zinc-deficiency also decreased significantly plasma zinc concentration by 84%, as compared with the control diet group. On the contrary, no significant changes in liver cytosolic ADH specific activity, total liver cytosolic ADH activity and total liver cytosolic ADH activity/body weight in the recovery period were observed between both groups. Plasma zinc concentration in the recovery period was almost recovered to the control level. These results suggest that rat liver cytosolic ADH activity was clearly related to dietary zinc intake levels.

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Red peppers are used as a spice for enhancing the palatability of foods. Two major capsaicinoids, dihydrocapsaicin (DHC) and capsaicin (CAP) are responsible for up to 90% of the total pungency of pepper fruits. These capsaicinoids are known to enhance energy metabolism and thermogenesis. However, there is a little information on the effects of capsaicinoids on the lipolysis and carbohydrate metabolism. We studied the effects of DHC and CAP on plasma glucose, free fatty acid (FFA) and glycerol concentrations in rats. Male six-week-old Sprague Dawley rats were divided into the DHC, CAP and control groups. Each capsaicinoid (dose = 3 mg/kg BW/day) was subcutaneously administered to rats for 10 days. DHC increased markedly plasma glucose, FFA and glycerol concentrations on day 1-10 by 14-35%, 61-103% and 108-174%, respectively, as compared with those of the control group. CAP increased relatively plasma glucose concentrations on day 1-3 by 15-17%, as compared with the control group. However, there were no significant differences in plasma glucose concentrations on day 7-10 among three groups. On the contrary, CAP did not change plasma FFA and glycerol concentrations on day 1-3. However, CAP increased markedly plasma FFA and glycerol concentrations on day 7-10 by 54-89% and 92-98%, respectively, as compared with the control group. DHC and CAP did not change the weights of white (perirenal and periepididymal) and brown (interscapular) adipose tissues. In conclusion, the effects of capsaicinoids on plasma glucose, FFA and glycerol concentrations were relatively higher in the DHC than in the CAP, and capsaicinoids did not change the weight of white and brown adipose tissues.

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ATP-binding cassette (ABC) transporter plays an important role for resistance against xenobiotics. There are eleven ABC transporter genes in the genome of fission yeast Schizosaccharomyces pombe. We examined the role of ABC transporter against the toxicity of tributyltin chloride (TBT), a widespread environmental pollutant, in cell growth. Among individual ABC transporter mutants, the growth of a mutant deficient in Bfr1p, a plasma membrane-embedded transporter, was extremely sensitive to TBT. The lethal TBT concentration inducing 50% of cell death (LC₅₀) was 25 µM for the parent strain and 10.2 µM for the bfr1∆ mutant. Thus, Bfr1p was responsible for TBT resistance in S. pombe.

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To investigate the influence of Chlorella (Parachlorella beijerinckii) on the excretion and tissue accumulation of methylmercury (MeHg), we orally administered 5 mg/kg of MeHg chloride (4 mg Hg/kg) to female C57BL/6N mice (aged 10 weeks). The mice were housed in metabolism cages to collect urine and feces for 3 weeks with diets containing 0%, 5%, or 10% P. beijerinckii powder (BP) in a basal diet (CE-2). The lowered blood Hg levels in the 5% and 10% BP groups became significant compared to those of the control group (0% BP) as early as day 7. During the 21 days of testing, significant increases in the cumulative Hg eliminations into urine (5% BP) and feces (5% and 10% BP) were found in the BP groups. Twenty-one days after administration, the organ Hg levels in both BP groups tended to decrease compared to that of the control group. The reduction of Hg levels in the kidney and brain were significant, whereas that in the liver was not. Although tissue Hg levels are known to be closely related to glutathione (GSH) metabolism, no difference was found in GSH levels in the blood or organs between the control group and the 10% BP group. These results suggest that continuous BP intake accelerates the excretion of MeHg and subsequently decreases tissue Hg levels in mice, with no alteration of GSH metabolism. We should conduct further research to elucidate details regarding the mechanism of BP-induced enhancement of MeHg excretion.

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In order to elucidate the transcriptional response of kidney epithelial cells to cadmium, the gene expression pattern was examined in normal rat kidney epithelial cells (NRK-52E cells) exposed to 50 µM cadmium for 4 hr using DNA microarray. Cadmium was found to increase the expression of 73 genes and decrease the expression of 42 genes in NRK-52E cells before the development of cytotoxicity.

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Proteomic analysis was carried out for neuronal vacuolation in rat retrosplenial cortex (RSC) induced by MK-801, a N-methyl-D-aspartate (NMDA) receptor antagonist. Female rats were given a single subcutaneous (sc) injection of either MK-801 (9 mg/kg in saline) or saline. Comparison of changes in proteins in the RSC region between MK-801- and saline-treated groups revealed that MK-801 induced changes in six proteins involved in vesicular transport (vesicle-fusing ATPase) and glycolysis (fructose-bisphosphate aldolase C, triosephosphate isomerase, and glyceraldehyde-3-phosphate dehydrogenase).

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To investigate the effects of chronic exposure to arsenite on the gene expression profiles of mast cells, microarray analysis was performed on rat basophilic leukemia RBL-2H3 cells exposed to arsenite for 28 days. Upregulated genes include calcium-binding S100 proteins such as S100A9, S100A10, S100A6, and S100A13, and granzymes B and C. Among S100 proteins, S100A9 showed the highest expression (8.62-fold of untreated cells) after 4-weeks of exposure to arsenite. As S100A8 and S100A9 comprise a heterodimer called calprotectin, and are implicated in the development of atherosclerosis and cancer, mRNA levels of both S100A8 and S100A9 were analyzed. The results demonstrated that exposure of RBL-2H3 cells to arsenite for a few weeks induces marked increases in mRNA levels of S100A8 and S100A9.

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Cadmium (Cd) is a toxic heavy metal that has been shown to induce vascular diseases, such as atherosclerosis. We used DNA microarray to monitor the transcriptional response of human coronary artery endothelial cells (HCAEC) to a non-lethal dose of Cd (10 µM). Out of 35,035 human genes, Cd enhanced the expression of 3 metallothionein (MT)-I subisoform genes, including MT1E, MT1H and MT1B, and reduced the expression of 12 genes, including ISG20 and TK1, 2-fold or greater.

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