The development of hepatitis is associated with the infiltration and activation of immune cells in liver. N-3 polyunsaturated fatty acids (n-3 PUFAs) rich fish oil (FO) is used to prevent and treat inflammatory diseases. But, the effects of dietary FO on autoimmune hepatitis remain largely unknown. In this study, Concanavalin A (Con A) induced hepatitis was used to evaluate the actions of dietary FO. Unexpectedly, 2-week FO treatment had not shown any protection, on the contrary, exacerbated liver injury in this hepatitis model. The levels of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) statistically increased from 10,501 ± 2,154 and 30,394 ± 2,420 in low fat diet (LFD)/Con A group to 17,579 ± 693 and 49,439 ± 4,628 in FO/Con A group. Simultaneously, FO diet induced more necrotic liver tissues and apoptotic hepatocytes, and up-regulated the hepatic expression of TNF-α and IFN-γ after Con A challenge. Interestingly, FO promoted severe liver injury was accompanied by decreasing the percentage of CD4+ T cell, NK1.1+ cells and CD8+ T cells in CD45+ liver non-parenchymal hepatic cells (NPCs) through inducing apoptosis. Further experiments declared 2-week FO diet intake firstly increased the proportion of CD11b+Gr-1hi neutrophils in liver, but then dramatically expanded CD11b+Gr-1int inflammatory monocytes population after Con A administration. Collectively, our study indicated that high FO intake not only aggravated liver injury, but also altered the population of immune cells in liver. Thus, these results indicated that when dietary FO was used to benefit health in autoimmune diseases, its potential risks of side effect also need paying close attention.
Regulatory T cells (Treg) play a role in suppression of immune response, including anti-tumor immunity. We have recently reported that treatment of naïve CD4 T cells with adenosine A2B receptor antagonist PSB603 under Treg-skewing conditions inhibits expression of Foxp3, a marker of differentiation to Treg, without blocking IL-2 production or CD25 expression, which are activation markers, in CD4 T cells. We hypothesized that PSB603 suppresses cancer growth and metastasis by inhibiting induction of Treg, thereby facilitating anti-tumor immunity. In this study, we first examined the effect of PSB603 on tumor growth in B16 melanoma-bearing C57BL/6 mice. Administration of PSB603 significantly suppressed the increase of tumor volume as well as the increase of Treg population in these mice. The populations of CD4 and CD8 T cells were higher and splenic lymphocyte-mediated cytotoxicity towards B16 melanoma was significantly increased in PSB603-treated mice. We confirmed that PSB603 did not reduce the viability of B16 melanoma cells in vitro. Moreover, we also examined the effect of PSB603 on tumor metastasis in pulmonary metastasis model mice intravenously injected with B16 melanoma cells. The metastasis was also suppressed in PSB603-treated mice, in which the population of Treg was significantly lower. Overall, our results suggest that A2B receptor antagonist PSB603 enhances anti-tumor immunity by inhibiting differentiation to Treg, resulting in a delay of tumor growth and a suppression of metastasis.
Ca2+ overload is one of the mechanisms for H2O2-induced cell death in rat pancreatic β-cell line RIN-5F cells. RIN-5F cells express TRPM2, which is a Ca2+-permeable channel activated by H2O2, and voltage-dependent L-type Ca2+ channels, both of which induce Ca2+ entry by H2O2. This study examined the contribution of these channels to H2O2-induced Ca2+ entry and cell death in RIN-5F cells. Cytosolic Ca2+ concentration was measured using fura-2 as a Ca2+ indicator. Cell death was estimated by trypan blue exclusion. Pre-treatment with poly(ADP-ribose) polymerase (PARP) inhibitors, which inhibit TRPM2 activation, strongly reduced Ca2+ entry by H2O2. The PARP inhibitors used had no effect on the Ca2+ elevation by voltage-dependent L-type Ca2+ channels. On the other hand, pre-treatment with L-type Ca2+ channel blockers, which did not affect TRPM2 activation, partly reduced H2O2-induced Ca2+ entry. Treatment with PARP inhibitors but not L-type Ca2+ channel blockers, around the early phase in H2O2-induced Ca2+ elevation, also reduced the late phase. Moreover, H2O2-induced RIN-5F cell death was strongly attenuated by PARP inhibitors, in compared to L-type Ca2+ channel blockers. Our results suggest that TRPM2 channels rather than L-type Ca2+ channels primarily contribute to H2O2-induced Ca2+ entry and cell death.
Surfactants used in herbicide formulations are generally considered inert with no toxic effects on animals. Polyethoxylated tallow amines (POEAs) are non-ionic surfactants used in many herbicide formulations to promote the penetration of the active matter into plant cuticles. The present study aimed to assess the toxicity of a POEA surfactant system, the Genamin T-200®, on two larval stages of the Pacific oyster, Crassostrea gigas. The embryotoxicity of Genamin T-200® was quantified after 36 hr of exposure, considering both arrested development and abnormalities in D-shaped larvae. The ability of pediveliger larvae to metamorphose was studied after 24 hr exposure to Genamin T-200®. According to the European toxicity classification, the present results suggest that Genamin T-200® could be considered very toxic to embryo larval development, with an EC50 of 262 µg/l, and toxic to metamorphosis processes with an EC50 of 3,027 µg/l. The high toxicity of glyphosate-based formulations compared to the active ingredient and its by-product appears to be due primarily to surfactants.
A widely-used plasticizer di(2-ethylhexyl) phthalate (DEHP) is known to induce apoptosis in neurons, although the mechanisms responsible for DEHP-induced apoptosis is not well explored yet. We recently showed that exposure to DEHP increases the expression of hemeoxygenase (HO)-1, an oxidative stress related enzyme, in the mice brain. In this study, we investigated whether HO-1 is involved in DEHP-induced apoptosis using a mouse neuroblastoma cell line Neuro-2a, which forcibly express SCAT3, a fluorescent indicator of caspase-3 activity. The doses of DEHP at 1, 10 or 100 µM were used in the present study to mimic the level of human exposure to DEHP. Live image analysis of SCAT3-expressing Neuro-2a cells revealed that caspase-3 activity in the cells was significantly increased by DEHP at 100 µM but not 1 or 10 µM. We measured HO-1 mRNA level in Neuro-2a cells exposed to DEHP and found significant increase in HO-1 mRNA level by DEHP at 100 µM but not 1 or 10 µM. Live image analysis of SCAT3-expresisng Neuro-2a cells was further performed to determine the effects of HO-1 siRNA in DEHP-induced apoptosis via caspase-3 activation. We found that knockdown of HO-1 gene nullifies the effects of DEHP to activate caspase-3. These results suggest that HO-1 is involved in DEHP-induced apoptosis. Moreover, this study demonstrates that high-dose DEHP exposure induces caspase-3-dependent apoptosis, which is at least partially mediated by the up-regulation of HO-1 gene, in Neuro-2a cells.
There have been few neurobehavioral toxicology studies on newborn animals. Thus, we developed a mouse newborn behavioral testing method for evaluating the risk of neurotoxicity of environmental toxicants, by means of determining the newborn’s motor activity applying the tare function of an analytical balance. Motor activities including crawling, pivoting, righting or tremors of mouse newborns were evaluated. Tremors of newborns of dams exposed to bisphenol A at 2, 20 or 200 µg/kg/day on days 5 through 18 of gestation were significantly increased when evaluated on postnatal day 1, as well as those of newborns exposed prenatally to diethylstilbestrol at 0.5 µg/kg/day. We suggest that our developed testing method may provide a useful addition to neurobehavioral assessment in very young rodents exposed to environmental hormone mimics.
Anti-angiogenic drugs that target Vascular Endothelial Growth Factor (VEGF) signaling pathways caused hypertension as an adverse effect in clinical studies. Since the hypertension may limit the benefit provided for patients, the demand for non-clinical research that predicts the clinical risk of the hypertension has risen greatly. To clarify whether non-clinical research using rats can appropriately estimate the clinical risk of hypertension caused by VEGF signal inhibitors, we investigated the hemodynamic effects and pharmacokinetics (PK) of the VEGF signal inhibitors cediranib (0.1, 3, and 10 mg/kg), sunitinib (5, 10, and 40 mg/kg), and sorafenib (0.1, 1, and 5 mg/kg) in telemetered rats and examined the correlation between the non-clinical and the clinical hypertensive effect. The VEGF signal inhibitors significantly elevated blood pressure (BP) in rats within a few days of the initiation of dosing, and levels recovered after dosing ended. The trend of the hypertension was similar to that in clinical studies. We found that the AUC at which BP significantly increased by approximately 10 mmHg in rats was comparable to the clinical AUC at which moderate to severe hypertension occurred. These results represent correlations between the non-clinical and the clinical hypertensive effect of VEGF signal inhibitors, suggesting that non-clinical research using telemetered rats would be an effective approach to predict the clinical risk of hypertension caused by VEGF signal inhibitors.
Spontaneous multiple granulomas were present in the animal under the SPF condition and without chemical treatment, in a 19-week-old male Sprague-Dawley control-group rat. Here we describe multiple granulomas and prominent diffuse infiltration by eosinophils in the cecal submucosa, and arteritis in the mesenteric arteries. The multiple granulomas were characterized by central eosinophilic degeneration or necrosis, prominent eosinophils, many multi-nucleated giant cells and abundant fibroblasts. They were restricted to the cecal submucosa. The mesenteric arteritis consisted of fibrinoid necrosis of the intima and media, intense inflammatory cell infiltration and fibrosis in the arterial wall. An affected artery in the cecum was continuous with the mesenteric artery. The foregoing tissue changes in this rat correlate with the high absolute blood eosinophil count found in this animal.
The neuroprotective effects of dexmedetomidine have been reported by many investigators; however its underlying mechanism to reduce neuronal injury during a prolonged anesthesia remains unclear. In this study, we investigated the neurotoxic effects of dexmedetomidine in fetal monkey brains. In the present study, we compare the neurotoxic effects of dexmedetomidine and ketamine, a general anesthetic with a different mechanism of action, in fetal cynomolgus monkeys. Twenty pregnant monkeys at approximate gestation day 120 were divided into 4 groups: non-treatment controls (Group 1); ketamine at 20 mg/kg intramuscularly followed by a 12-hr infusion at 20-50 mg/kg/hr (Group 2); dexmedetomidine at 3 µg/kg intravenously (i.v.) over 10 min followed by a 12-hr infusion at the human equivalent dose (HED) of 3 µg/kg/hr (Group 3); and dexmedetomidine at 30 µg/kg i.v. over 10 min followed by a 12-hr infusion at 30 µg/kg/hr, 10 times HED (Group 4). Blood samples from both dams and fetuses were measured for concentration of dexmedetomidine. Each fetus was perfusion-fixed, serial sections were cut through the frontal cortex, and stained to detect for apoptosis (activated caspase 3 and TUNEL) and neurodegeneration (silver stain). In utero treatment with ketamine resulted in marked apoptosis and degeneration primarily in layers I and II of the frontal cortex. In contrast, fetal brains from animals treated with dexmedetomidine showed none to minimal neuroapoptotic or neurodegenerative lesions at both low- and high-dose treatments. Plasma levels confirmed systemic exposure of dexmedetomidine in both dams and fetuses. In conclusion, these results demonstrate that dexmedetomidine at both low-dose (HED) and high-dose (10 times HED) does not induce apoptosis in the frontal cortex (layers I, II, and III) of developing brain of cynomolgus monkeys.
The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), induces lung tumors in rodents and has been suggested as a causative factor in human lung cancer. NNK is activated by α-hydroxylation at either the methyl or methylene carbon adjacent to the N-nitroso group to yield unstable intermediates that spontaneously decompose to produce alkylating agents. 4-(Hydroxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (HO-methyl NNK) glucuronide, a glucuronide of the reactive intermediate of NNK has been identified. However, there are no available data concerning HO-methyl NNK glucuronide. In the present study, we investigated the tissue distribution of HO-methyl NNK glucuronide in control and phenobarbital (PB)-treated rats after intraperitoneal administration of NNK. In PB-treated rats, HO-methyl NNK glucuronide was detected in plasma, kidney, liver, lung, and pancreas. On the contrary, in the control rats, HO-methyl NNK glucuronide was detected only in plasma, kidney and liver at low concentrations compared with PB-treated rats. The results of cumulative urinary excretion of HO-methyl NNK glucuronide in Wistar and Gunn rats suggested that PB-inducible UDP-glucuronosyltransferase 2B isoforms mainly contribute to the formation of HO-methyl NNK glucuronide.
Wistar Hannover rats have been utilized as one of major strains in regulatory toxicology studies. This study was performed to verify the appropriate age of male sexual maturity in the development and reproductive toxicity (DART) study in Wistar Hannover rats (RccHan:WIST) by comparing reproductive endpoints between 8, 10 and 12 weeks of ages. Although fertility showed a tendency toward decrease in 8-week-old males, copulation index was not different among three ages. Testis weights reached a plateau at 10 weeks of age, whereas weights of other reproductive organs developed until 12 weeks of age. Indices of spermatogenesis (sperm motility, number of sperm in the epididymis and testis and contents of morphologically abnormal sperm) showed age-related progress and did not fully develop except for 12-week-old. For histology, epididymal tubules in 8-week-old animals showed immaturity with tall epithelium. At cesarean section, dams mated with 8-week-old males showed high incidence of preimplantation loss and the number of live fetuses was less than 10. In conclusion, although reproductive performance attained maturity by age of 10 weeks, spermatogenesis was not fully established at 10-week-old, which could result in a low fertility index. Therefore, we recommend that Wistar Hannover male rats at 12-week-old or older are used to conduct DART study properly and evaluate any adverse effects on dams and embryo-fetal development.
In this study, non-thermal multi-gas plasma treatments were performed for Tetrodotoxin (TTX) solution, and TTX decomposition was analyzed by liquid chromatography coupled with electrospray time-of-flight mass spectrometry. The TTX mass spectrum signal was reduced by plasma irradiations to different levels by using various gas species. Nitrogen plasma exhibited the optimal capability for TTX decomposition, followed by oxygen, argon, and carbon dioxide plasmas. The TTX concentration decreased 100-fold by nitrogen plasma treatment for 10 min.
Protein expression changes were examined in day 10.5 rat embryos cultured for 24 hr in the presence of ethanol by using two-dimensional electrophoresis and mass spectrometry. Exposure to ethanol resulted in quantitative changes in many embryonic protein spots (16 decreased and 28 increased) at in vitro embryotoxic concentrations (130 and 195 mM); most changes occurred in a concentration-dependent manner. For these protein spots, 17 proteins were identified, including protein disulfide isomerase A3, alpha-fetoprotein, phosphorylated cofilin-1, and serum albumin. From the gene ontology classification and pathway mapping of the identified proteins, it was found that ethanol affected several biological processes involving oxidative stress and retinoid metabolism.
Biological risk assessment studies of chemical substances that induce DNA lesions have been primarily based on the action of DNA polymerases during replication. However, DNA lesions interfere not only with replication, but also with transcription. There is no simple method for the detection of the DNA lesion-induced inhibition of transcription. Here, we report an assay for estimating the toxicity of chemical substances by visualizing transcription in mammalian cellsusing nucleotide analog 5-ethynyluridine (EU) and its click chemistry reaction. Ultraviolet light and representative chemical substances (camptothecin, 4-nitroquinoline-1-oxide, mitomycin C, and cisplatin, but not etoposide) of DNA- damaging agents show toxicity, as indicated by RNA synthesis inhibition in response to DNA damage in HeLa cells. Using titanium dioxide, we observed RNA synthesis inhibition in response to the rutile form, but not the anatase form, indicating that rutile titanium dioxide is a toxic substance. Because this method is based on the transcriptional response to DNA lesions, we can use terminally differentiated neuron-like PC12 cells, the differentiation of which can be induced by nerve growth factors, for evaluating chemical substances. Ultraviolet light and some chemicals (camptothecin, 4-nitroquinoline-1-oxide, mitomycin C, and cisplatin, but not etoposide) inhibited RNA synthesis in non-differentiated PC12 cells. Conversely, camptothecin and cisplatin did not inhibit RNA synthesis in differentiated PC12 cells, but 4-nitroquinoline-1-oxide, mitomycin C, and etoposide did. And using titanium dioxide, we did not observed any RNA synthesis inhibition. These data suggest that this method might be used to estimate the potential risk of chemical substances in differentiated mammalian cells, which are the most common cell type found in the human body.
Arsine (AsH3) is used in many industries, but there is insufficient knowledge about the potential for percutaneous absorption. In order to examine possible percutaneous absorption of arsine, we conducted inhalation studies. Arsine was generated by reducing arsenic trioxide with NaBH4. Male 5-week-old Hos:HR-1 hairless mice were subjected to a single percutaneous exposure or whole-body inhalation exposure of ca. 300 ppm arsine for 5 min. The examination was performed 0-6 hr after the exposure. Total arsenic in whole blood and hematocrit (Ht) values were measured. Generation of an arsenic–hemoglobin (As-Hb) adduct in the blood was detected using high-performance liquid chromatography with an inductively coupled plasma mass spectrometer (HPLC-ICP-MS). Ht values in the inhalation group significantly decreased after 3 hr, but those in the percutaneous exposure group did not. Total arsenic in the inhalation group was 9.0-14.2 mg/l, which was significantly higher than that in the percutaneous group. The As-Hb adduct was detected only in mice in the inhalation group. Histopathological changes were noted only in the inhalation group, with marked deposition of eosinophilic globules in the proximal convoluted tubules of the kidneys, the Kupffer cells of the liver, and the red pulp in the spleen, but not in the lungs. Immunohistochemically, these eosinophilic globules were stained positively by hemoglobin (Hb) antibody. In the present study, arsine-induced hemolysis and deposition of Hb occurred in the kidney via the inhalation route but not via percutaneous exposure. The presence of As-Hb adduct may be a useful indicator for confirming arsine poisoning.
Tributyltin (TBT) has long been recognized as a major environmental pollutant that can cause significant damage to the cellular functions as well as disruption of endocrine homeostasis. TBT induces apoptosis accompanied by production of reactive oxygen species (ROS) in mammalian and yeast cells. We observed that the budding yeast cells exposed to this compound at low concentrations exhibited cell growth arrest, but not cell death. Flow cytometric analysis of yeast cells without synchronization and morphological assessment of cells synchronized at M phase by nocodazole treatment indicated that TBT-exposed Saccharomyces cerevisiae cells were arrested at G1 phase of the cell cycle. This arrest was recovered by the addition of N-acetylcysteine, suggesting the involvement of ROS production by TBT. This is the first study to evaluate the action of TBT on cell cycle events.
Epithelial-mesenchymal transition (EMT) plays a pivotal event in the development of pulmonary fibrosis. We have previously reported that methotrexate (MTX)-induced alveolar epithelial cell injury followed by pulmonary fibrosis as a result of the recruitment and proliferation of myofibroblasts. However, there is no data concerning whether EMT occurs in MTX-induced pulmonary fibrosis. In the present study, therefore, we investigated the expression of EMT markers such as E-cadherin, α-SMA, and vimentin by immunofluorescence analysis in mouse lung tissues after administration of MTX. We found that vimentin and α-SMA-positive cells of the MTX-induced pulmonary fibrosis were increased; on the other hand, E-cadherin was decreased, indicating that epithelial cells act as the main source of mesenchymal expansion. These results exhibited the down-regulation of E-cadherin expression and the up-regulation of α-smooth muscle actin (α-SMA) in primary mouse alveolar epithelial cells (MAECs) and A549 cell lines. Additionally, MTX-induced A549 cells exhibited an EMT-like phenotype accompanied by the elevation of the expression of interleukin-6 (IL-6) and transforming growth factor (TGF)-β1, as well as an enhancement of migration. All of these findings suggest that MTX-induced pulmonary fibrosis occurs via EMT.
Cigarette smoke induces skeletal muscle wasting by a mechanism not yet fully elucidated. Branched-chain amino acids (BCAA) in the skeletal muscles are useful energy sources during exercise or systemic stresses. We investigated the relationship between skeletal muscle wasting caused by cigarette smoke and changes in BCAA levels in the plasma and skeletal muscles of rats. Furthermore, the effects of BCAA-rich diet on muscle wasting caused by cigarette smoke were also investigated. Wistar Kyoto (WKY) rats that were fed with a control or a BCAA-rich diet were exposed to cigarette smoke for four weeks. After the exposure, the skeletal muscle weight and BCAA levels in plasma and the skeletal muscles were measured. Cigarette smoke significantly decreased the skeletal muscle weight and BCAA levels in both plasma and skeletal muscles, while a BCAA-rich diet increased the skeletal muscle weight and BCAA levels in both plasma and skeletal muscles that had decreased by cigarette smoke exposure. In conclusion, skeletal muscle wasting caused by cigarette smoke was related to the decrease of BCAA levels in the skeletal muscles, while a BCAA-rich diet may improve cases of cigarette smoke-induced skeletal muscle wasting.
Although some studies have described the function of ADAM8 (a disintegrin and metalloprotease 8) related with rheumatoid arthritis, cancer and asthma, etc., the concrete role of ADAM8 in acute liver injury is still unknown. So mice respectively received anti-ADAM8 monoclonal antibody (mAb) of 100 μg/100 μl, 200 μg/100 μl or 300 μg/100 μl in PBS or PBS pre-injection. Then acute liver injury was induced in the mice by intraperitoneal (i.p.) injection of carbon tetrachloride (CCl4). Serum AST and ALT level, Haematoxylin-eosin (H&E) staining, the expression level of vascular endothelial growth factor (VEGF), cytochrome P450 1A2 (CYP1A2) and proliferating cell nuclear antigen (PCNA) were detected in the mice after CCl4 administration. Our results showed that anti-ADAM8 mAb pre-injection could effectively lower AST and ALT levels (P < 0.05 or P < 0.01) and reduce liver injury (P < 0.05 or P <0.01), induce the expression of VEGF, CYP1A2 and PCNA (P <0.05 or P < 0.01) in dose-dependent manner compared with the control mice which received PBS pre-injection. In summary, our study suggested that ADAM8 might promote liver injury by inhibiting the proliferation of hepatocytes, angiogenesis and affecting the metabolism function of liver during acute liver injury induced by CCl4. Anti-ADAM8 mAb injection might be suitable as a potential method for acute liver injury therapy.
Pau d’arco is a plant-derived traditional medicine that acts by poorly understood molecular mechanisms. Here, we studied the effect of pau d’arco on the cytoprotective transcription factor Nrf2. An aqueous extract of pau d’arco stimulated Nrf2-dependent gene expression and led to nuclear localization of Nrf2 in vitro. Chromatographic separation and mass spectrometry of the extract identified benzene trioles or benzene tetraoles within the active fractions. The extract stimulated the mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase (MEK)/extracellular-signal-regulated kinase (ERK1/2) pathway. The pharmacological inhibition of MEK, but not of p38 mitogen-activated protein kinase, glycogen synthase kinase-3 or phosphoinositide 3-kinase was required for the activation of Nrf2-dependent gene expression by pau d’arco, but not for the nuclear translocation of Nrf2. In vivo pau d’arco increased the expression of Nrf2-target genes in the intestine. The results suggest that the activation of Nrf2 could mediate beneficial effects of pau d’arco, in particular in the intestine.
Possible teratogenicity of 3 different asbestos (crocidolite, chrysotile and amosite) was assessed in CD1(ICR) mice. Dams on day 9 of gestation were given a single intraperitoneal administration at dose of 40 mg/kg body weight of asbestos suspended in 2% sodium carboxymethyl cellulose solution in phosphate buffered saline, while dams in the control group were given vehicle (10 ml/kg body weight). Dams and fetuses were examined on day 18 of gestation. To compare with the control group, the mean percentage of live fetuses in implantations in the group given crocidolite and the incidence of dams with early dead fetuses in the groups given chrysotile or amosite were increased. While no external or skeletal malformation was observed in the control group, the incidence of external malformation (mainly reduction deformity of limb) in the group given amosite, and the incidences of skeletal malformation (mainly fusion of vertebrae) in the all dosed groups were significantly increased. The result indicated that asbestos (crocidolite, chrysotile and amosite) have fetotoxicity and teratogenicity in mice.
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