Ube2d ubiquitin-conjugating enzymes promote p53 ubiquitination and proteasomal degradation. We previously showed that cadmium induced p53-dependent apoptosis through the suppression of expression of Ube2d family genes (Ube2d1, Ube2d2, Ube2d3 and Ube2d4) in normal rat proximal tubular cells. Here we examined the effects of inorganic arsenic and inorganic mercury, which induce apoptosis in proximal tubular cells, on cellular protein level of p53 and gene expression of Ube2d family. Inorganic arsenic induced apoptosis with p53 accumulation, and suppressed Ube2d1, Ube2d2 and Ube2d4 expression, but not Ube2d3. On the other hand, although apoptosis was induced in response to inorganic mercury in proximal tubular cells, protein level of p53 was not elevated by inorganic mercury. These results suggest that inorganic arsenic, but not inorganic mercury, may induce p53-dependent apoptotic pathways through downregulation of gene expression of Ube2d family in proximal tubular cells.
HEK293 cells transfected with a double-stranded siRNA to suppress expression of the homeobox gene HOXB13 were highly resistant to oxidative stress-inducing agents, such as hydrogen peroxide, N-ethylmaleimide (NEM), and paraquat. This finding suggests that HOXB13 might enhance cell injury caused by oxidative stress.
Methylmercury (MeHg) is a well-known human neurotoxic agent whose exposure sources are mainly environmental and aquatic-derived food. MeHg is reported to induce central nervous system disability. However, the exact mechanism of MeHg-induced neurotoxicity is still unknown. In this study, to investigate which cell death signaling pathway is related with MeHg-induced cytotoxicity, the effects of MeHg on apoptosis and autophagy were evaluated in HB1.F3 human neural stem cells (NSCs). Human NSCs were treated with 1 μM of MeHg for 48 hr and the effect of MeHg on cell signaling pathway was elucidated. MeHg inhibited Akt1/mTOR signaling that led to induction of caspase-dependent apoptosis and autophagy in the NSCs. Furthermore, retinoic acid (RA)-induced neuronal differentiation was inhibited by MeHg. Taken together, these results suggest that MeHg inhibits the differentiation of human NSCs by induction of caspase-dependent apoptosis and autophagy.
We previously reported that 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) increased the levels of several cytochrome P450 metabolites of the omega-6 polyunsaturated fatty acids (PUFAs), arachidonic acid (ARA) and linoleic acid in the serum, liver, lung and spleen of C57BL/6 mice in an aryl hydrocarbon receptor (AHR)-dependent fashion. These increases correlated with increased levels of CYP1A1, CYP1A2 and/or CYP1B1. In the current study, we measured 77 oxylipins, including 59 that we had not measured previously, and demonstrate that TCDD also markedly increases the levels of many epoxide and diol metabolites of the omega-3 PUFAs, α-linolenic acid, eicosapentaenoic acid (EPA) and docasahexaenoic acid (DHA) in these mice. Since these epoxide metabolites have been reported to have opposite effects on angiogenesis, tumor growth and tumor metastasis compared with the equivalent metabolites of omega-6 PUFA, these observations have important implications with regard to the potential involvement of the cytochrome P450 metabolites of PUFAs in mediating the biological effects of TCDD and other agonists of AHR.
The localization of neuropathological lesions along deep sulci and fissures is one of the characteristics of a cerebrum damaged by methylmercury. Edematous changes in white matter have been proposed as the cause of the localization of lesions; however, the molecular mechanisms underlying methylmercury-induced edema remain unclear. Since the vascular endothelial growth factor (VEGF) system regulates vascular permeability and can be involved in the progression of edematous changes, we examined the effect of methylmercury on the expression of VEGF-related proteins in cultured human brain microvascular endothelial cells and pericytes. After methylmercury exposure, mRNA and protein levels of VEGF-A in pericytes and placenta growth factor (PlGF) and VEGF-receptor-1/-2 in endothelial cells were elevated. The induction of pericyte VEGF-A expression was independent of hypoxia-inducible factor-α and hypoxia-response element signaling. Taken together, these results suggest that methylmercury activates the VEGF system in brain microvessels in a paracrine fashion. When the activation occurs in narrow areas such as along the deep sulci in the cerebrum, hyperpermeability and subsequent edematous changes would cause a circulatory disturbance and result in neural cell damage. We propose this as a reason for the localization of the neuropathological lesions along the deep sulci and fissures in the cerebral cortex, such as the calcarine fissure, in patients with Minamata disease.
The purpose of the present study was to collect the background data on Wistar Hannover [Crl:WI(Han)] (hereafter Wistar Han) rats in embryo-fetal development studies from the 6 safety research facilities of pharmaceutical companies and contract research organizations. In each facility, 20 or 22 female rats were dosed with vehicle solution during the organogenesis period. As a result, no abnormalities in clinical signs and necropsy findings in dams were found. Body weights and food consumption in dams were lower than those in Sprague Dawley (SD) rats. The number of corpora lutea (13.3 vs. 16.0 in SD) and implantations (11.8 vs. 14.7) were fewer, and fetal body weights (3.66 vs. 3.70) and placental weights (0.42 vs. 0.45) tended to be lower than those in SD rats. Regarding the fetal abnormalities, the incidence of several findings such as the persistent left umbilical artery (10.4% vs. 1.1%) and cervical (5.2% vs. 0.4%), full (7.4% vs. 0.9%) or short supernumerary (64.5% vs. 9.9%) and wavy ribs (6.6% vs. 0.3%) was higher than that in SD rats. Our present study showed that they maintained a sufficient number of live fetuses and the difference in the fetal sex ratio was not observed. In conclusion, Wistar Han rats were considered to be a suitable strain for embryo-fetal development toxicity study. Since the incidence of several abnormalities was higher than that in SD rats, it may be said that to accumulate background control data is important to evaluate the embryo-fetal development toxicity study using Wistar Han rats.
The purpose of the present study was to collect background data from repeated dose toxicity studies in Wistar Hannover [Crl:WI(Han)] (hereafter Wistar Han) rats with dosing periods of 4, 13 and 26 weeks from four safety research facilities of pharmaceutical companies and contract research organizations participating in the International Genetic Standardization (IGS) rat forum supported by Charles River Laboratories Japan, Inc. The data from Wistar Han rats were compared with those from Sprague Dawley Crl:CD(SD) rats. In addition, the effects of restricted feeding of SD rats were also investigated by one facility. As a result, body weights and food consumption in Wistar Han rats were lower than those of SD rats. White blood cell (WBC), neutrophil, lymphocyte, monocyte and eosinophil counts were almost half of those noted for SD rats and platelet counts were almost 20% less than those in SD rats. Minimal strain differences were noted in several biochemical parameters including aspartate aminotransferase (AST), alanine aminotransferase, total cholesterol, triglyceride and phospholipids, and in thymus, ovary and testis weights. Ophthalmologic or histopathologic examinations revealed a higher incidence of corneal opacities or corneal mineralization in Wistar Han rats. Restricted feeding of SD rats resulted in intermediate values for body weights and food consumption between the ad libitum fed SD and Wistar Han rats, and WBC and AST were lower than those in the ad libitum fed SD rats. Based on these results, some strain differences might be ascribable to reduced food consumption and associated body weight changes in Wistar Han rats.
Garcinia vilersiana is a traditional medicinal plant in Vietnam. The petroleum ether extract of stem bark of Garcinia vilersiana (GVE) was prepared to evaluate its potential to activate Nrf2, a transcription factor of antioxidant and detoxifying enzymes. Exposure of mouse macrophage RAW264.7 cells to GVE (0.625-2.5 µg/ml) resulted in a significant activation of Nrf2, as evaluated by nuclear accumulation of this transcription factor, and increased antioxidant response element (ARE) binding activity in a time- and concentration-dependent manner. As a result, GVE caused ARE-dependent up-regulation of heme oxygenase-1 (HO-1) in the cells. These results suggest that GVE contains components that have the ability to activate the Nrf2/ARE/HO-1 signaling pathway, leading to cellular protection.
The acute effects of oral administration of diallyl disulfide (DADS), the major organosulfur compound of garlic, on plasma glucose and free fatty acid (FFA) concentrations were examined in rats. Male, 10-week-old Sprague–Dawley rats were divided into DADS-free and DADS-administered (dose = 10, 20, and 40 mg/kg body weight [BW]) groups. Plasma samples were prepared from whole blood drawn from the tail vein 0, 1, 2, 4, and 6 hr after administration. The stomachs were isolated, and the contents were measured 8 hr after administration. In DADS-administered groups, plasma glucose concentrations were increased in a dose-dependent manner 1 hr after the administration. The increase was transient, except in groups administered 40 mg/kg BW of DADS, in which plasma glucose levels remained significantly higher than the DADS-free levels throughout the experimental period. Similar patterns were observed in the plasma FFA concentrations, although the significant differences were lower than those observed in the plasma glucose concentrations. The gastric contents were dose-dependently elevated after DADS administration. The increase was significant when 20 or 40 mg/kg BW of DADS was administered. These results suggest that oral administration of DADS can mobilize energy substrates into the blood, although a higher dose of DADS slows gastric emptying.
Tributyltin-binding proteins (TBT-bps) are members of the fish lipocalins that were isolated from the blood of Japanese flounder (Paralichthys olivaceus) and function in the binding and detoxification of TBT. In this study, we constructed a baculovirus–silkworm expression system and obtained recombinant TBT-bp2 (rTBT-bp2; 31.5 kDa) from the hemolymph of silkworm larvae injected with a recombinant baculovirus containing the TBT-bp2 gene. The binding potential of rTBT-bp2 was investigated and compared to that of the previously available recombinant TBT-bp1 (rTBT-bp1). Both rTBT-bp2 and rTBT-bp1 bound to DAUDA, a typical fluorescent ligand of lipocalins, with dissociation constants of 0.97 and 1.75 µM, respectively. The Hill coefficient value indicated that rTBT-bp2 may have multiple binding sites and strong negative cooperativity. These results suggest that the typical central cavity of lipocalins composed of eight specific β-sheets is conserved in rTBT-bp2, as it is in rTBT-bp1, although rTBT-bp2 has different effects than rTBT-bp1 in TBT binding. In a competition assay, rTBT-bp2 displayed exponential binding affinity to TBT with an inhibition constant of 0.29 µM, demonstrating that TBT binds to the central ligand pocket of rTBT-bp2. However, three fatty acids did not show any affinity to rTBT-bp2. Further studies are required to elucidate the endogenous function of TBT-bps as fish lipocalins and their function in responding to xenobiotics.
This study was investigated the effects of piperonyl butoxide (PBO) on the female reproductive tract. Female Crj:Donryu rats were fed a basal diet containing 5,000, 10,000 or 20,000 ppm PBO for 28 days, and compared with food-restricted rats of comparable body weights to those in the PBO 10,000 or 20,000 ppm groups. Although treatment with 20,000 ppm PBO for 28 days depressed body weight gain, the abnormal estrous cyclicity, mainly prolonged diestrus, was also induced by the PBO treatment which was not correlated with body weight change. 20,000 ppm PBO treatment markedly decreased uterine weights and slightly decreased ovarian weights. 10,000 and 20,000 ppm PBO treatment increased liver weights. These cycle and organ weight changes were linked to atrophic uterus and increased atretic follicles in the ovary. In hormone assays, PBO at both doses reduced serum E2 levels, but did not affect corticosterone levels. An anti-uterotrophic assay showed a slight but significant decrease in absolute uterine weight and a reduction of endometrial epithelium height in the 20,000 ppm group. PBO was positive in an ER α antagonist reporter gene assay, although the activity was much weaker than that of 4-hydroxytamoxifen. These results indicate that high-dose PBO treatment directly induces atrophic changes in the female reproductive tract in rats, and these effects are likely the result of a hypoestrogenic state and the anti-estrogenic activity of PBO.
In the 1950s to 1970s developed countries reported declines in populations of raptorial and fish-eating birds and dichlorodiphenyltrichloroethane (DDT) and its metabolites were considered causative substances because they accumulated significantly in the tissues of wild birds and animals. However, except for the estrogenic effects of o,p’-DDT, a minor component of commercial DDT, there has been no compelling evidence that DDT directly affects avian reproductive systems. To assess the possible impact of DDT on development and reproduction of birds, exposure experiments to the major component of commercial DDT, p,p’-DDT, and its persistent metabolite, p,p’-dichlorodiphenyldichloroethylene (DDE), were performed using Japanese quail (Coturnix japonica) eggs; the test substances (3 to 100 μg/g) were injected into the yolk prior to incubation, and hatched chicks were raised to adulthood. p,p’-DDT had no significant effects on the morphology and function of the reproductive systems, although the hatchability of treated eggs was reduced at the highest dose (100 μg/g). High doses of p,p’-DDE slightly enhanced the eggshell forming ability of female quails; eggshell mass and thickness were increased at 30 μg/g or more although no morphological changes were observed in the oviduct. Transcriptions of the CYP11A1 gene in the ovaries, and of AHR and ARNT in the livers, of adult females were significantly increased at 3 μg/g or more of p,p’-DDT. Except for low hatchability, transovarian exposure to p,p’-DDT or p,p’-DDE did not markedly impair the avian reproductive systems, but the hormonal actions of these compounds are likely to change reproductive and hepatic functions even after maturation.
Ethyl tertiary-butyl ether (ETBE) is an oxygenated gasoline additive synthesized from ethanol and isobutene that is used to reduce CO2 emissions. To support the Kyoto Protocol, the production of ETBE has undergone a marked increase. Previous reports have indicated that exposure to ETBE or methyl tertiary-butyl ether resulted in liver and kidney tumors in rats and/or mice. These reports raise concern about the effects of human exposure being brought about by the increased use of ETBE. The present study was conducted to evaluate the genotoxicity of ETBE using micronucleus induction of polychromatic erythrocytes in the bone marrow of male and female rats treated with ETBE in the drinking-water at concentrations of 0, 1,600, 4,000 or 10,000 ppm or exposed to ETBE vapor at 0, 500, 1,500 or 5,000 ppm for 13 weeks. There were no significant increases in micronucleus induction in either the drinking water-administered or inhalation-administered groups at any concentration of ETBE; although, in both groups red blood cells and hemoglobin concentration were slightly reduced in the peripheral blood in rats administered the highest concentration of ETBE. In addition, two consecutive daily intraperitoneal injections of ETBE at doses of 0, 250, 500 or 1,000 mg/kg did not increase the frequency of micronucleated bone marrow cells in either sex; all rats receiving intraperitoneal injections of ETBE at a dose of 2,000 mg/kg died after treatment day 1. These data suggest that ETBE is not genotoxic in vivo.
We previously reported that methylmercury induces brain-specific upregulation of a chemokine gene, the CCL4 gene. In this study, we investigated the changes in gene expression levels of all chemokines in various tissues (cerebellum, cerebrum, kidney, liver, and spleen) of methylmercury-treated mice. We found that methylmercury induced upregulation of the CCL4 gene and the CCL3 gene, in a brain-specific manner. This finding suggests that upregulation of these two chemokine genes mediates selective damage by methylmercury in the central nervous system.
Methylmercury (MeHg) is an environmental pollutant known to cause neurobehavioral defects, and it is especially toxic to the developing brain. In contrast to the adult, the developing brain consists of a large number of dividing neural progenitor cells (NPCs), which are vulnerable targets for MeHg toxicity. In a previous study, we showed that the embryonic NPCs from the telencephalon are more sensitive to MeHg than other neural cells. Here, we investigated the mechanism of cell death underlying MeHg toxicity. We observed that exposure of NPCs to MeHg caused DNA laddering in a dose- and time-dependent manner. Decreased pro-caspase3 and increased cleaved-caspase3 protein was observed 3-12 hours after incubation of NPCs with MeHg. Moreover, the caspase-inhibitor Z-VAD FMK significantly suppressed MeHg-induced cell death in a dose-dependent manner. These results suggest that environmentally relevant levels of MeHg exposure induce apoptosis in NPCs.
Chloramine T has been widely used as a disinfectant in many areas such as kitchens, laboratories and hospitals. It has been also used as a biocide in air fresheners and deodorants which are consumer products; however, little is known about its toxic effects by inhalation route. This study was performed to identify the subacute inhalation toxicity of chloramine T under whole-body inhalation exposure conditions. Male and female groups of rats were exposed to chloramine T at concentrations of 0.2, 0.9 and 4.0 mg/m3 for 6 hr/day, 5 days/week during 4 weeks. After 28-day repeated inhalation of chloramine T, there were dose-dependently significant DNA damage in the rat tissues evaluated and inflammation was histopathologically noted around the terminal airways of the lung in both genders. As a result of the expression of three types of antioxidant enzymes (SOD-2, GPx-1, PRX-1) in rat’s lung after exposure, there was no significant change of all antioxidant enzymes in the male and female rats. The results showed that no observed adverse effect level (NOAEL) was 0.2 mg/m3 in male rats and 0.9 mg/m3 in female rats under the present experimental condition.
Biological defense factors show diurnal variations in their expression levels or activities. These variations can induce the different sensitivity to external toxicants of a day. We reported earlier that mice showed clear diurnal variation of cadmium (Cd)-induced toxicity, i.e., chronotoxicity. In this report, we investigated additional new evidences for the cadmium (Cd)-induced chronotoxicity, and considered the mechanisms contributed to this chronotoxicity. Male C57BL/6J mice were injected with CdCl2 (6.4 mg/kg, one shot) intraperitoneally at 6 different time points of a day (zeitgeber time (ZT); ZT2, ZT6, ZT10, ZT14, ZT18 or ZT22) followed by monitoring the mortality until 14 days after the injection. We observed extreme difference in survival numbers: surprisingly, all mice died at ZT2 injection while all mice survived at ZT18 injection. Moreover, in non-lethal dose of Cd (4.5 mg/kg), the values of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) used as indexes of hepatotoxicity markedly increased at ZT6 injection while mostly unchanged at ZT18 injection. To consider the mechanisms of this extreme diurnal variation, we examined biochemical studies and concluded that the diurnal variation was not caused by the differences in hepatic Cd level, basal hepatic metallothionein (MT) level, and induction level or induction speed of hepatic MT. We suggested that one of the candidate determination factors was glutathione. We believe that the “chronotoxicology” for metal toxicity may be classic, yet new viewpoint in modern toxicology field.
We examined the alteration of gene expression in HK-2 human proximal tubular cells exposed to cadmium (Cd) using DNA microarray analysis. Cd increased the expression of 30 genes, including 7 genes coding for heat shock proteins, more than 2.0-fold and decreased the expression of 21 genes, including transcription-related genes, such as AP2B1, HOXA7, HOXA9 and TCEB2, less than 0.5-fold prior to the appearance of cytotoxicity in HK-2 cells.
We previously analyzed gene expression in the cerebellum of mice treated with methylmercury using microarrays, identifying 21 different genes that increased expression following the administration of methylmercury. It has already been shown that 5 of these genes encode chemokine molecular species. Among these genes, the expression of CCL4 chemokine was found to be specifically induced in the brain following methylmercury exposure. In this study, we examined the remaining 16 genes showing increased brain-specific induction of expression following methylmercury exposure. As a result, it was shown that the gene expression of Scgb3a1, encoding secretoglobin 3A1, was increased specifically by methylmercury administration.
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