Dietary Fructooligosaccharides Reduce Mercury Levels in the Brain of Mice Exposed to Methylmercury.

Methylmercury (MeHg) exposure during pregnancy is a concern because of its potential health risks to fetuses. Intestinal microbiota has important roles in the decomposition and fecal excretion of MeHg. We investigated the effect of nondigestible saccharides on the accumulation and excretion of Hg after MeHg exposure. Female BALB/cByJ mice were fed a basal diet or the same diet supplemented with 5% fructooligosaccharides (FOS) or 2.5% glucomannan. Six weeks after feeding, mice were administered MeHg chloride (4 mg Hg/kg, per os (p.o.)), and urine and feces were collected for 28 d. FOS-fed mice had lower total Hg levels in all tissues (including the brain) compared with that of controls. The glucomannan diet had no effect on tissue Hg levels. No differences in tissue concentrations of inorganic Hg among groups were found. Fecal Hg excretion was markedly higher in FOS-fed mice than that in controls, but urinary Hg excretion was similar. FOS-fed mice had a higher proportion of inorganic Hg in feces than that of controls, with a significant increase in fecal Hg excretion. Analysis of fecal bacterial population showed the relative abundance of Bacteroides in FOS-fed mice to be higher than that in controls. The results suggest that FOS enhanced fecal Hg excretion and decreased tissue Hg levels after MeHg administration, possibly by accelerating MeHg demethylation by intestinal bacteria (the candidate genus Bacteroides). This demethylation also reduces MeHg absorption in the large intestine. In conclusion, daily FOS intake may decrease tissue Hg levels in animals and humans exposed to MeHg.

Cadmium induces iron deficiency anemia through the suppression of iron transport in the duodenum.

Cadmium (Cd) is an environmental contaminant that triggers toxic effects in various tissues such as the kidney, liver, and lung. Cd can also cause abnormal iron metabolism, leading to anemia. Iron homeostasis is regulated by intestinal absorption. However, whether Cd affects the iron absorption pathway is unclear. We aimed to elucidate the relationship between the intestinal iron transporter system and Cd-induced iron deficiency anemia. C57BL/6J female and male mice, 129/Sv female mice, and DBA/2 female mice were given a single oral dose of CdCl2 by gavage. After 3 or 24 h, Cd decreased serum iron concentrations and inhibited the expression of iron transport-related genes in the duodenum. In particular, Cd decreased the levels of divalent metal transporter 1 and ferroportin 1 in the duodenum. In addition, human colon carcinoma Caco-2 cells were treated with CdCl2. After 72 h, Cd decreased the expression of iron transport-related factors in Caco-2 cells with a pattern similar to that seen in the murine duodenum. These findings suggest that Cd inhibits iron absorption through direct suppression of iron transport in duodenal enterocytes and contributes to abnormal iron metabolism.

Effect of gestational cadmium exposure on fetal growth, polyubiquitinated protein and monoubiqutin levels in the fetal liver of mice.

Cadmium (Cd) is an environmental pollutant present in contaminated water, food and soil. Cd adversely affects fetal development. We exposed pregnant mice to daily oral doses of 5 and 10 mg/kg Cd and examined fetal growth. It was demonstrated that the exposure to Cd (10 mg/kg) during gestation caused fetal growth retardation (FGR). Investigation of the ubiquitin-proteasome system in fetal livers of mice exposed to gestational Cd revealed increased polyubiquitinated protein accumulation, contrasting with decreased levels of monoubiquitin protein. Moreover, the expression level of Ubc (encoding polyubiquitin C protein) was significantly decreased in 5 and 10 mg/kg Cd-treated groups in comparison with the control group. Therefore, we propose that decrease of monoubiquitin level and accumulation of polyubiquitinated protein in the fetal liver may be important factors in Cd-induced FGR.

Accumulation of p53 via down-regulation of UBE2D family genes is a critical pathway for cadmium-induced renal toxicity.

Chronic cadmium (Cd) exposure can induce renal toxicity. In Cd renal toxicity, p53 is thought to be involved. Our previous studies showed that Cd down-regulated gene expression of the UBE2D (ubiquitin-conjugating enzyme E2D) family members. Here, we aimed to define the association between UBE2D family members and p53-dependent apoptosis in human proximal tubular cells (HK-2 cells) treated with Cd. Cd increased intracellular p53 protein levels and decreased UBE2D2 and UBE2D4 gene expression via inhibition of YY1 and FOXF1 transcription factor activities. Double knockdown of UBE2D2 and UBE2D4 caused an increase in p53 protein levels, and knockdown of p53 attenuated not only Cd-induced apoptosis, but also Cd-induced apoptosis-related gene expression (BAX and PUMA). Additionally, the mice exposed to Cd for 6 months resulted in increased levels of p53 and induction of apoptosis in proximal tubular cells. These findings suggest that down-regulation of UBE2D family genes followed by accumulation of p53 in proximal tubular cells is an important mechanism for Cd-induced renal toxicity.

Gene expression differences in the duodenum of 129/Sv and DBA/2 mice compared with that of C57BL/6J mice.

We compared the cadmium (Cd) concentration in the liver and kidney of different strains of mice after exposure to 50 ppm Cd for 30 days via drinking water. Cd concentration in the liver and kidney of C57BL/6J mice were higher than those of 129/Sv and DBA/2 mice. Since orally ingested heavy metals are absorbed in the small intestine and then widely distributed to target tissues, microarray analyses were performed to compare the expression levels of transport-related genes in the duodenum between C57BL/6J mice and 129/Sv or DBA/2 mice. The expression levels of 9 and 11 genes were elevated more than 2.0-fold and 13 and 12 genes were reduced less than 0.5-fold in 129/Sv mice and DBA/2 mice, respectively. Among these low expressed genes, 10 genes (Slc2a2, Slc5a1, Slc16a2, Slc22a13, Slc22a18, Slc25a11, Slc36a1, Slco6c1, Abca3 and Abcd1) were common between the two types of strains. These results suggest that some of those genes might be involved in Cd absorption and its toxicity.

Ultrastructural changes in the air-blood barrier in mice after intratracheal instillations of Asian sand dust and gold nanoparticles.

The purpose of this study was to investigate a possible translocation pathway of intratracheally instilled gold nanoparticles after the induction of acute pulmonary injury by Asian sand dust. ICR mice were intratracheally instilled with 800µg Asian sand particles (CJ-2 particles) 24h before instillation of 50-nm gold nanoparticles. Lungs from mice treated with Asian sand particles and gold nanoparticles showed an acute focal inflammation with an increased expression of proinflammatory cytokines (IL-6 and TNF-α) and oxidative stress markers (Cu/Zn SOD and iNOS) in alveolar macrophages, type I alveolar epithelial cells, and endothelial cells at the alveolar walls. Electron microscopy revealed a destruction of the alveolar walls with an increased number of endocytic vesicles in the cytoplasm of both type I epithelial cells and endothelial cells; gold nanoparticles were demonstrated in these endocytic vesicles. These findings suggest that translocation of the exposed nanoparticles may be enhanced in the lung tissues with acute inflammatory changes.

Vanadium toxicity in mice: possible impairment of lipid metabolism and mucosal epithelial cell necrosis in the small intestine.

Because precise information as to the toxicity of vanadium is required for practical use of vanadium compounds as antidiabetic drugs, we examined vanadium toxicity in mice fed normal diet or high-fat diet (C57BL/6N, male, 7 weeks) by oral administration of ammonium metavanadate (AMV) with a maximum dose of 20 mgV/kg/day. Marked lipid accumulation in hepatocytes, renal epithelial cells, and mucosal epithelial cells of the small and large intestines and severe degeneration, necrosis, and loss of mucosal epithelial cells in the small intestine were observed. These pathological changes were more severe in mice fed high-fat diet than mice fed normal diet, and the intensity of the changes increased with increase in the administered dose of AMV. By electron microscopy, the number and size of lipid droplets in hepatocytes were increased. In the small intestine, a TUNEL assay showed a decreased number of positive cells, and positive cells for acrolein immunohistochemistry were observed specifically in the mucosal epithelial cells indicating degeneration and necrosis in the AMV-treated group, suggesting that a possible factor responsible for cell necrosis in the small intestine could be oxidative stress. In conclusion, AMV may impair cellular lipid metabolism, resulting in lipid accumulation, and induce mucosal epithelial cell necrosis in the small intestine.

Cadmium toxicity is caused by accumulation of p53 through the down-regulation of Ube2d family genes in vitro and in vivo.

Cadmium (Cd) causes renal dysfunction with damage to kidney proximal tubule cells; however, the precise mechanisms of the toxicity remain unclear. Previously, we found that the expression of Ube2d4 gene, which is a member of the ubiquitin-conjugating enzyme Ube2d family, is suppressed by Cd in NRK-52E rat renal tubular epithelial cells. To investigate the mechanisms of Cd-induced renal toxicity, we examined the effects of Cd on the ubiquitin-proteasome system, particularly the expression and function of Ube2d family members in the NRK-52E cells and mice. Cd markedly decreased the expression of Ube2d1, Ube2d2, Ube2d3 and Ube2d4 prior to the appearance of cytotoxicity in the NRK-52E cells. Cd also dramatically increased p53 protein levels in the cells, without stimulation of p53 gene expression or inhibition of proteasome activity. In addition, Cd induced phosphorylation of p53 and caused apoptosis in the NRK-52E cells. In vivo, we examined the effect of orally administrated Cd for 12 months on the expression of Ube2d genes and accumulation of p53 in the mouse kidney. Chronic Cd exposure also caused suppression of Ube2d genes expression and accumulation of p53. Cd did not induce severe kidney injury, but caused apoptosis in the renal tubules. These results suggest that the Cd-induced accumulation of p53 may be due to inhibition of p53 degradation through the down-regulation of Ube2d family genes, and that Cd induces p53-dependent apoptosis in renal tubular cells. Moreover, Ube2d family members may be one of the critical targets of renal toxicity caused by Cd.

Attenuation of cadmium-induced testicular injury in metallothionein-III null mice.

AIMS: In order to evaluate the role of metallothionein (MT)-III in cadmium (Cd)-induced testicular toxicity, we examined the sensitivity of MT-III null mice to severe testicular injury caused by Cd. MAIN METHODS: Male MT-III null mice, MT-I/II null mice and wild-type mice were given a subcutaneous injection of CdCl(2) (15µmol/kg). The testis was collected from each mouse at 6, 12 and 24h after Cd administration. KEY FINDINGS: Testicular hemorrhages by evaluating morphology, hemoglobin content and histological parameters in the 3 types of mice were elevated by Cd injection in a time-dependent manner. The degree of hemorrhage in Cd-injected MT-I/II null mice was similar to that in the wild-type mice. In contrast, hemorrhage in the MT-III null mice was attenuated compared with that in wild-type mice and MT-I/II null mice. Cd levels, MT-I and MT-II mRNA levels and Cd-binding molecules in the testis were similar between MT-III null mice and wild-type mice. In microarray analysis, high expression of purine-nucleoside phosphorylase 2 (Pnp2), retinal degeneration 3 (Rd3), and cadherin-like 24 (Cdh24) was revealed in the testis of MT-III null mice under normal or Cd-treated conditions. SIGNIFICANCE: MT-III null mice were found to show attenuation of Cd-induced severe testicular toxicity. These results suggest the lack of MT-III contributes to protection of testis from Cd. In addition, regulation of Pnp2, Rd3, and Cdh24 mRNA levels may involve the sensitivity of MT-III null mice to Cd.

Resistance of metallothionein-III null mice to cadmium-induced acute hepatotoxicity.

We examined the sensitivity of metallothionein (MT)-III null mice to cadmium (Cd)-induced acute hepatotoxicity. MT-I/II null mice were also used to compare Cd toxicities between MT-III null mice and MT-I/II null mice. Male MT-I/II null mice, MT-III null mice and wild-type mice were given s.c. injection of Cd (5-20 micromol/kg) and then the blood and liver were collected from each mouse under ether anesthesia at 2 days after the administration. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities elevated by injection of Cd were significantly higher in the MT-I/II null mice than in the wild-type mice. In the MT-III null mice, ALT and AST activities were not elevated following the injection of Cd. Further, marked morphological changes such as necrosis of hepatocytes, severe hemorrhage and congestion were observed by injection of Cd in both MT-I/II null mice and wild-type mice, whereas the degree of injury was found to be more extensive in MT-I/II null mice. In contrast, only occasional damage was observed in the liver of MT-III null mice treated with the same dose of Cd. These morphological observations were consistent with the results of ALT and AST activities. In the present study, it was clearly found that MT-III null mice were resistant to Cd hepatotoxicity, although MT-I/II null mice were sensitive to its toxicity. MT-III may be an accelerative factor in Cd-induced acute hepatotoxicity.

Dichotomous effects of lead acetate on the expression of metallothionein in the liver and kidney of mice.

Metallothionein (MT) is a low-molecular-weight cysteine-rich protein which has a high affinity for metals and plays important roles in the protection against metal toxicity. As little information is available concerning the mechanism of MT induction by lead (Pb) compounds, we investigated the induction of MT by Pb acetate both at mRNA and protein levels in mice. Administration of Pb increased the levels of MT-I mRNA in the liver and kidney in six strains of mice. However, MT protein was detected only in the liver, and little or no increases in MT protein were detected in the kidney of any strains of mice. Speciation of metals in the liver cytosol showed that the major metal bound to MT was zinc but not Pb. The increases in plasma concentrations of interleukin-6 suggest that the production of interleukin-6 by Pb administration is involved in the induction of MT in the liver. Treatment of renal cells with Pb in vitro also resulted in the increase in MT mRNA but little increase in MT protein. These data suggest that Pb exerts a dual effect on MT expression; enhancement of MT gene transcription both in the liver and kidney and suppression of MT mRNA translation in the kidney.

Induction of metallothionein by manganese is completely dependent on interleukin-6 production.

Metallothionein (MT) is a cysteine-rich protein that binds to and is inducible by heavy metals such as cadmium and zinc. However, the precise mechanism of MT induction by other metals remains unclear. In the present study, we investigated the mechanism of MT induction by manganese, focusing on the involvement of cytokine production. Administration of MnCl(2) to mice resulted in the induction of MT dose-dependently in the liver with little accumulation of manganese. Speciation analysis of metals in the liver cytosol showed that the major metal bound to the induced MT was zinc. Administration of MnCl(2) caused an increase in mRNA levels of interleukin-6 (IL-6) in the liver as well as an increase in serum levels of IL-6 but not those of other inflammatory cytokines. Subsequently, serum levels of serum amyloid A (SAA), an acute-phase protein induced by IL-6, increased with a peak at 24 h. However, no increase in serum alanine aminotransferase activity was observed, suggesting that manganese enhanced the production of IL-6 and SAA without causing liver injury. In response to IL-6, the expression of a zinc transporter, ZIP14, was enhanced in the liver, possibly contributing to the synthesis of hepatic zinc-MT. In IL-6-null mice, the induction of hepatic MT by treatment with MnCl(2) was completely suppressed to the control level. These results suggest that manganese is a unique metal that induces the synthesis of hepatic MT completely depending on the production of IL-6 without accompanying liver injury.

Characterization of an immortalized hepatic stellate cell line established from metallothionein-null mice.

Hepatic stellate (HS) cells were isolated from the livers of metallothionein (MT)-null and control mice and used to establish IMS/MT(-) and IMS/N cell lines, respectively, using SV40 virus transformation. Cellular morphology, incorporation of vitamin A and expression of alpha-SMA, desmin and SV40 T-antigen were used to confirm that both cell lines were immortal HS cells. The growth rates of both cell lines were similar and there was little difference between cell line sensitivity to zinc. MT-null IMS/MT(-) cells were more sensitive to cadmium and mercury, although both cell lines accumulated almost equal amounts of cadmium during a 24-hr culture period. As HS cells play an important role in hepatic fibrosis and are activated by heavy metals such as cadmium or reactive oxygen, the MT-null HS cell line derived in this study should be a useful experimental model for examination of the role of MT in HS cell activation.

Pentavalent vanadium induces hepatic metallothionein through interleukin-6-dependent and -independent mechanisms.

Metallothionein (MT) is a low-molecular-weight cysteine-rich protein which has a high affinity for metals. The synthesis of MT is induced by heavy metals such as cadmium and zinc. However, little is known about the induction of MT by tetravalent or pentavalent metals. We investigated the induction of MT synthesis by a pentavalent vanadium compound in mice. Hepatic MT concentrations were increased by subcutaneous injection of ammonium metavanadate (AMV) dose-dependently, and to the similar levels as those induced by zinc chloride. However, accumulation of vanadium in the liver was very low, while high concentrations of vanadium were detected in the kidney. High performance liquid chromatography/inductively coupled argon plasma-mass spectrometry (HPLC/ICP-MS) chromatogram of the liver cytosol of AMV-treated mice revealed that the major metal bound to MT was not vanadium, but zinc. The chromatogram of the liver cytosol of MT null mice demonstrated the existence of a low-molecular-weight vanadium-binding protein that is different from MT. A time-course study showed that concentrations of serum interleukin-6 (IL-6) and serum amyloid A (SAA), an acute-phase protein, increased after the AMV injection. To confirm the involvement of IL-6 in MT induction by AMV administration, IL-6 null and wild-type mice were injected with AMV. In IL-6 null mice, hepatic MT induction by AMV administration decreased significantly to about a half of wild-type mice. These data suggest that both IL-6-dependent and -independent mechanisms are involved in MT induction by vanadium compounds in mice.

Induction of hepatic metallothionein by trivalent cerium: role of interleukin 6.

Metallothionein (MT) is a small sulfydryl-rich protein that binds to and is inducible by heavy metals such as mercury, cadmium, zinc, and copper. However, little is known about the induction of MT by trivalent metals except for bismuth. In this study, we examined the induction of MT synthesis by cerium, a trivalent lanthanoid metal. Administration of cerium chloride (CeCl3) to mice resulted in accumulation of cerium and induction of MT in the liver in a dose-dependent manner. Distribution profiles of metals in the soluble fraction of the liver of CeCl3-treated mice analyzed by high performance liquid chromatography/inductively coupled argon plasma-mass spectrometry (HPLC/ICP-MS) demonstrated that the metal bound to MT-I and MT-II was zinc, but not cerium. Administration of CeCl3 caused increases in the activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and the levels of serum amyloid A (SAA), an acute phase protein. Among inflammatory cytokines examined, interleukin 6 (IL-6) exhibited a marked increase in the serum at 3 h after the CeCl3 administration. In order to evaluate the involvement of IL-6 in the induction of MT by cerium, we examined MT induction by CeCl3 in IL-6 null mice. Both the induction of hepatic MT and the increases in SAA levels were markedly suppressed in IL-6 null mice. These results suggest that IL-6 plays an important role in the induction of hepatic MT by cerium.

Copper(II) protects yeast against the toxicity of cisplatin independently of the induction of metallothionein and the inhibition of platinum uptake.

We have made the unexpected discovery that copper sulfate protects Saccharomyces cerevisiae from the toxic effects of cisplatin. Addition of copper to the culture medium of yeast cells at concentrations above 0.1 microM significantly reduced the toxicity of cisplatin. Since a high-affinity copper transporter, Ctr1, has been reported to play a major role in the uptake of cisplatin, we examined the effects of copper on the cellular uptake of cisplatin. We found that the cellular concentration of platinum was not significantly affected by treatment of cells with 1 microM copper. It is known that mammalian metallothionein is induced by copper and is involved in acquired resistance to cisplatin. Copper significantly increased the level of mRNA for yeast metallothionein at a concentration that has effectively reduced the toxicity of cisplatin. However, the toxicity of cisplatin in cells with a disrupted gene for ACE1, a factor that regulates transcription of the yeast gene for metallothionein, was also significantly reduced by treatment with copper. These results suggest that copper protects yeast cells from cisplatin toxicity independently of induction of the synthesis of metallothionein and of the inhibition of platinum uptake. Since copper is one of the trace elements that are essential for cell function and since a relatively low concentration of copper (0.1 microM) significantly reduced cisplatin toxicity, it is possible that copper might play an important role in the expression of cisplatin toxicity.

Functional characterization of two variant human GSTO 1-1s (Ala140Asp and Thr217Asn).

Glutathione-S-transferase class Omega (GSTO 1-1) belongs to a new subfamily of GSTs, which is identical with human monomethylarsonic acid (MMA(V)) reductase, the rate limiting enzyme for biotransformation of inorganic arsenic, environmental carcinogen. Recombinant GSTO 1-1 variants (Ala140Asp and Thr217Asn) were functionally characterized using representative substrates. No significant difference was observed in GST activity towards 1-chloro-2,4-dinitrobenzene, whereas thioltransferase activity was decreased to 75% (Ala140Asp) and 40% (Thr217Asn) of the wild-type GSTO 1-1. For MMA(V) reductase activity, the Ala140Asp variant exhibited similar kinetics to wild type, while the Thr217Asn variant had lower V(max) (56%) and K(m) (64%) values than the wild-type enzyme. The different activities of the enzyme variants may influence both the intracellular thiol status and arsenic biotransformation. This can help explain the variation between individuals in their susceptibility to oxidative stress and inorganic arsenic.

Preliminary investigation on the cytotoxicity of tellurite to cultured HeLa cells.

Cytotoxicity of tellurite to cultured HeLa cells was examined by cell viability, lactate dehydrogenase (LDH) assay, and tellurite uptake. The experimental results show that the toxicity of tellurite depends on its concentrations and exposure time. HeLa cells exposed to tellurite for 2 h at 9.1 x 10(-4) to 4.5 x 10(-3) mmol/L did not exhibit cytotoxic effects as measured by cell viability. Exposure to tellurite for 24 h at the same concentrations markedly reduced the cell viability to 57% of the control during the first 5 minutes. Additionally, HeLa cells incubated at 2.7 x 10(-2) to 0.27 mmol/L of tellurite for 2 h retained 53% to 67% of cell viability. Even after 24 h exposure, the HeLa cells incubated at 9.1 x 10(-4) to 4.5 x 10(-2) mmol/L of tellurite still retained 57% to 66% of cell viability. Furthermore, tellurite toxicity was also demonstrated in supernatant of the culture at 37 degrees C by LDH assay. It was found that exposure to tellurite for 90 minutes did not stimulate LDH activity. However, tellurite uptake seems to be more sensitive than the cell viability and LDH activity release tests, as it significantly increases with the increasing of exposure time.