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Abstract: Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme activated by binding to DNA breaks, which causes PARP-1 automodification. PARP-1 activation is required for regulating various cellular processes, including DNA repair and cell death induction. PARP-1 involved in these regulations is localized in the nucleoplasm, but approximately 40% of PARP-1 can be found in the nucleolus. Previously, we have reported that nucleolar PARP-1 is delocalized to the nucleoplasm in cells exposed to DNA-damaging agents. However, the functional roles of this delocalization in cellular response to DNA damage is not well understood, since this approach simultaneously induces the delocalization of PARP-1 and its automodification. We therefore devised an approach for separating these processes. Unmodified PARP-1 was first delocalized from the nucleolus using camptothecin. Then, PARP-1 was activated by exposure of cells to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). In contrast to treatment with MNNG alone, delocalization of PARP-1 by CPT, prior to its activation by MNNG, induced extensive automodification of PARP-1. DNA repair activity and consumption of intracellular NAD+ were not affected by this activation. On the other hand, activation led to an increased formation of apoptotic cells, and this effect was suppressed by inhibition of PARP-1 activity. These results suggest that delocalization of PARP-1 from the nucleolus to the nucleoplasm sensitizes cells to DNA damage-induced apoptosis. As it has been suggested that the nucleolus has a role in stress sensing, nucleolar PARP-1 could participate in a process involved in nucleolus-mediated stress sensing. [Copyright &y& Elsevier]

Neuronal morphological changes in the epidermis are considered to be one of causes of abnormal skin sensations in dry skin-based skin diseases. The present study aimed to develop an in vitro model optimised for human skin to test the external factors that lead to its exacerbation. Human-induced pluripotent stem cell-derived sensory neurons (hiPSC-SNs) were used as a model of human sensory neurons. The effects of chemical substances on these neurons were evaluated by observing the elongation of nerve fibers, incidence of blebs (bead-like swellings), and the expression of nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2). The nerve fiber length increased upon exposure to two common cosmetic preservatives—methylparaben and phenoxyethanol—but not to benzo[a]pyrene, an air pollutant at the estimated concentrations in the epidermis. Furthermore, the incidence of blebs increased upon exposure to benzo[a]pyrene. However, there was a decrease in the expression of NMNAT2 in nerve fibers, suggesting degenerative changes. No such degeneration was found after methylparaben or phenoxyethanol at the estimated concentrations in the epidermis. These findings suggest that methylparaben and phenoxyethanol promote nerve elongation in hiPSC-SNs, whereas benzo[a]pyrene induces nerve degeneration. Such alterations may be at least partly involved in the onset and progression of sensitive skin. [ABSTRACT FROM AUTHOR]

Infection with the gastric pathogen Helicobacrer pylori (H. pylori) causes chronic gastritis, peptic ulcer, and gastric adenocarcinoma. These diseases are associated with production of reactive oxygen species (ROS) from infiltrated macrophages and neutrophiles in inflammatory sites. Metallothionein (MT) is a low-molecular-weight, cysteine-rich protein that can act not only as a metal-binding protein, but also as a ROS scavenger. In the present study. we examined the role of MT in the protection against H. pylori-induced gastric injury using MT-null mice. Female MT-null and wild-type mice were challenged with H. pylori SS1 strain, and then histological changes were evaluated with the updated Sydney grading system at 17 and 21 wk after challenge. Although the colonization efficiency of H. pylori was essentially the same for MT-null and .wild-type mice, the scores of activity of inflammatory cells were significantly higher in MT-null mice than in wild-type mice at 17 wk after challenge. Histopathological examination revealed erosive lesions accompanied by infiltration of inflammatory cells in the infected MT-null mice but not in wild-type mice. Furthermore, activation of NF-κB and expression of NF-κB-mediated chemokines such as macrophage inflammatory protein-1α and monocytes chemoattractant protein-1 in gastric cells were markedly higher in MT-null mice than in wild-type mice. These result, suggest that MT in the gastric mucosa might play an important role in the protection against H. pylori-induced gastric ulceration. [ABSTRACT FROM AUTHOR]

This study examined the role of placenta metallothionein (MT) in maternal-to-fetal mercury transfer in MT-null and wild-type mice after exposure to elemental mercury (Hg0) vapor. Both strains were exposed to Hg0 vapor at 5.5–6.7 mg/m3 for 3 h during late gestation. Twenty-four hours after exposure to Hg0 vapor, accumulation of mercury in the major organs, except the brain, of MT-null maternal mice was significantly lower than that in organs of wild-type mice. In contrast to mercury levels in maternal organs, fetal mercury levels were significantly higher in MT-null mice than in wild-type mice. In placenta, mercury concentrations were not significantly different between the two strains.. Although MT levels in major organs, except the brain, of wild type mice were markedly elevated after the exposure to Hg0 vapor, the placental MT levels were not elevated. However, endogenous MT level in the placenta is significantly higher than that in other organs, except the liver. Gel filtration profile of the placental cytosol in the wild-type mice revealed that a large amount of placental mercury was associated with MT. In MT-null mice, mercury in placental cytosol appeared mainly in the high-molecular-weight protein fractions. Mercury in the placenta was localized mainly in the yolk sac and decidual cells in the deep layer of the decidua in both mouse strains. The similar localization of MT was found in the placenta of wild type mice. These results suggest that MT in the placenta has a defensive role in preventing maternal-to-fetal mercury transfer. [Copyright &y& Elsevier]

Abstract We previously reported that abnormal copper release from mutated Cu, Zn-superoxide dismutase (SOD1) proteins might be a common toxic gain-of-function in the pathogenesis of familial amyotrophic lateral sclerosis (FALS) [Ogawa et al. (1997) Biochem. Biophys. Res. Commun., 241, 251–257.]. In the present study, we first examined metallothioneins (MTs), known to bind copper ions and decrease oxidative toxicity, and found a twofold increase in MTs in the spinal cord of the SOD1 transgenic mice with a FALS-linked mutation (G93A), but not in the spinal cord of wild-type SOD1 transgenic mice. We then investigated whether the clinical course of FALS mice could be modified by the reduced expression of MTs, by crossing the FALS mice with MT-I- and MT-II-deficient mice. FALS mice clearly reached the onset of clinical signs and death significantly earlier in response to the reduction of protein expression. These results indicated that the copper-mediated free radical generation derived from mutant SOD1 might be related to the degeneration of motor neurons in FALS and that MTs might play a protective role against the expression of the disease. [ABSTRACT FROM AUTHOR]

The tumor suppressor p53 regulates various stress responses via increasing its cellular levels. The lowest p53 levels occur in unstressed cells; however, the functions of these low levels remain unclear. To investigate the functions, we used empirical single-cell tracking of p53-expressing (Control) cells and cells in which p53 expression was silenced by RNA interference (p53 RNAi). Here, we show that p53 RNAi cells underwent more frequent cell death and cell fusion, which further induced multipolar cell division to generate aneuploid progeny. Those results suggest that the low levels of p53 in unstressed cells indeed have a role in suppressing the induction of cell death and the formation of aneuploid cells. We further investigated the impact of p53 silencing by developing an algorithm to simulate the fates of individual cells. Simulation of the fate of aneuploid cells revealed that these cells could propagate to create an aneuploid cell population. In addition, the simulation also revealed that more frequent induction of cell death in p53 RNAi cells under unstressed conditions conferred a disadvantage in terms of population expansion compared with Control cells, resulting in faster expansion of Control cells compared with p53 RNAi cells, leading to Control cells predominating in mixed cell populations. In contrast, the expansion of Control cells, but not p53 RNAi cells, was suppressed when the damage response was induced, allowing p53 RNAi cells to expand their population compared with the Control cells. These results suggest that, although p53 could suppress the formation of aneuploid cells, which could have a role in tumorigenesis, it could also allow the expansion of cells lacking p53 expression when the damage response is induced. p53 may thus play a role in both the suppression and the promotion of malignant cell formation during tumorigenesis. [ABSTRACT FROM AUTHOR]

Compares the kinetics of poly(ADP-ribose) synthesis by the polymerase and subsequent polymer degradation by poly(ADP-ribose) glycohydrolase with the rate of DNA repair. Effect of the presence of short poly(ADP-ribose) chains on poly(ADP-ribose)polymerase; Causes of negative charge repulsion during the synthesis.

Cadmium (Cd) is an environmental contaminant that causes renal toxicity. We have previously demonstrated that Cd induces renal toxicity by altering transcriptional activities. In this study, we show that Cd markedly inhibited the activity of transcription factor MEF2A in HK‐2 human proximal tubule cells, which generated significant cytotoxicity in the cells. This reduction in the nuclear levels of MEF2A protein may be involved in the Cd‐induced inhibition of MEF2A activity. We also demonstrate that one of the glucose transporters, GLUT4, was downregulated not only by Cd treatment but also by MEF2A knockdown. Knockdown of SLC2A4, encoding GLUT4, eliminated both cell viability and Cd toxicity. Cd treatment or SLC2A4 deficiency reduced the cellular concentration of glucose. Therefore, the suppression of SLC2A4 expression, which mediates the reduction in cellular glucose, is involved in Cd toxicity. The Cd toxicity induced by the reduction in GLUT4 may be associated with a reduction of cellular ATP levels in HK‐2 cells. The levels of Slc2a4 mRNA in the kidney of mice exposed to Cd for 6 or 12 months were significantly lower than those in the control group. These results demonstrate that Cd exerts its cytotoxicity through the suppression in SLC2A4 expression and the subsequent inhibition of MEF2A transcriptional activity. Cd‐induced suppression of SLC2A4 expression also reduces cellular ATP levels, partly by reducing glucose levels. This study suggests that the glucose transporter plays an important role in the renal toxicity of Cd, and provides a crucial breakthrough in our understanding of the mechanism of Cd toxicity. [ABSTRACT FROM AUTHOR]

Cultured cell populations are composed of heterogeneous cells, and previous single-cell lineage tracking analysis of individual HeLa cells provided empirical evidence for significant heterogeneity of the rate of cell proliferation and induction of cell death. Nevertheless, such cell lines have been used for investigations of cellular responses to various substances, resulting in incomplete characterizations. This problem caused by heterogeneity within cell lines could be overcome by investigating the spatiotemporal responses of individual cells to a substance. However, no approach to investigate the responses by analyzing spatiotemporal data is currently available. Thus, this study aimed to analyze the spatiotemporal responses of individual HeLa cells to cytotoxic, sub-cytotoxic, and non-cytotoxic doses of the well-characterized carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Although cytotoxic doses of MNNG are known to induce cell death, the single-cell tracking approach revealed that cell death occurred following at least four different cellular events, suggesting that cell death is induced via multiple processes. We also found that HeLa cells exposed to a sub-cytotoxic dose of MNNG were in a state of equilibrium between cell proliferation and cell death, with cell death again induced through different processes. However, exposure of cells to a non-cytotoxic dose of MNNG promoted growth by reducing the cell doubling time, thus promoting the growth of a sub-population of cells. A single-cell lineage tracking approach could dissect processes leading to cell death in a spatiotemporal manner and the results suggest that spatiotemporal data obtained by tracking individual cells can be used as a new type of bioinformatics data resource that enables the examination of cellular responses to various external substances. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) is a toxic heavy metal that is widely present in the environment. Renal proximal tubule disorder is the main symptom of Cd chronic poisoning. Our previous study demonstrated that Cd inhibits the total activities of peroxisome proliferator-activated receptor (PPAR) transcription factors in human and rat proximal tubular cells. In this study, we investigated the involvement of PPAR in Cd renal toxicity using the HK-2 human proximal tubular cell line. Among PPAR isoform genes, only PPARD knockdown significantly showed resistance to Cd toxicity in HK-2 cells. The transcriptional activity of PPARδ was decreased not only by PPARD knockdown but also by Cd treatment. DNA microarray analysis showed that PPARD knockdown changed the expression of apoptosis-related genes in HK-2 cells. PPARD knockdown decreased apoptosis signals and caspase-3 activity induced by Cd treatment. PPARD knockdown did not affect the intracellular Cd level after Cd treatment. These results suggest that PPARδ plays a critical role in the modification of susceptibility to Cd renal toxicity and that the apoptosis pathway may be involved in PPARδ-related Cd toxicity. [ABSTRACT FROM AUTHOR]

To investigate the in vivo effects of cobalt chloride on gene expression at early time points, DNA microarray analysis was performed on the liver of mice injected subcutaneously with cobalt chloride. The liver tissue samples were taken 0.5, 1, and 3 hr after injection. Of the 14 genes up-regulated at 0.5 hr after injection, 7 are related to immunological responses, and 4 of the 7 were found to be involved in the activation of interferon. [ABSTRACT FROM AUTHOR]

DNA microarray was used to monitor the transcriptional response of human brain microvascular endothelial cells (HBMEC) and human coronary artery endothelial cells (HCAEC) to sodium arsenite (iAsIII). iAsIII enhanced the expression of 10 and 6 genes, and reduced the expression of 45 and 20 genes in HBMEC and HCAEC, respectively. Among the 64 genes whose expression was changed in some way, HBMEC and HCAEC had 5 up-regulated and 12 down-regulated genes in common. [ABSTRACT FROM AUTHOR]

DNA repair is known as a defense mechanism against genotoxic insults. However, the most lethal type of DNA damages, double-strand DNA breaks (DSBs), can be produced by DNA repair. We have previously demonstrated that when long patch base excision repair attempts to repair a synthetic substrate containing two uracils, the repair produces DSBs (Vispe, S. and Satoh, M. S. (2000)1. Biol. Chem. 275, 27386-27392 and Vispe, S., Ho, E. L., Yung, T. M., and Satoh, M. S. (2003)1. Biol. Chem. 278, 35279 -35285). In this synthetic substrate, the two uracils are located on the opposite DNA strands (separated by an intervening sequence stable at 37 °C) and represent a high risk site for DSB formation. It is not clear, however, whether similar high risk sites are also induced in genomic DNA by exposure to DNA damaging agents. Thus, to investigate the mechanisms of DSB formation, we have modified the DSB formation assay developed previously and demonstrated that high risk sites for DSB formation are indeed generated in genomic DNA by exposure of cells to alkylating agents. In fact, genomic DNA containing alkylated base damages, which could represent high risk sites, are converted into DSBs by enzymes present in extracts prepared from cells derived from clinically normal individuals. Furthermore, DSBs are also produced by extracts from cells derived from ataxia-telangiectasia patients who show cancer proneness due to an impaired response to DSBs. These results suggest the presence of a novel link between base damage formation and DSBs and between long patch base excision repair and human diseases that occur due to an impaired response to DSB. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Poly(ADP-ribosyl)ation is a post-translational modification that occurs immediately after exposure of cells to DNA damaging agents. In vivo, 90% of ADP-ribose polymers are attached to the automodification domain of poly(ADP-ribose) polymerase-1 (PARP-1), the main enzyme catalyzing this modification reaction. This enzyme forms complexes with transcription initiation, DNA replication, and DNA repair factors. In most known cases, the interactions occur through the automodification domain. However, functional implications of the automodification reaction on these interactions have not yet been elucidated. In the present study, we created fluorescent protein-tagged PARP-1 to study this enzyme in live cells and focused on the interaction between PARP-1 and topoisomerase I (Topo I), one of the enzymes that interacts with PARP-1 in vitro. Here, we demonstrate that PARP-1 co-localizes with Topo I throughout the cell cycle. Results from bioluminescence resonance energy transfer assays suggest that the co-localization is because of a direct protein-protein interaction. In response to DNA damage, PARP-1 de-localization and a reduction in bioluminescence resonance energy transfer signal because of the automodification reaction are observed, suggesting that the automodification reaction results in the disruption of the interaction between PARP-1 and Topo I. Because Topo I activity has been reported to be promoted by PARP-1, we then investigated the effect of the disruption of this interaction on Topo I activity, and we found that this disruption results in the reduction of Topo I activity. These results suggest that a function for the automodification reaction is to regulate the interaction between PARP-1 and Topo I, and consequently, the Topo I activity, in response to DNA damage. [ABSTRACT FROM AUTHOR]

Attenuation of the renal toxicity of cis-diamminedichloroplatinum (CDDP) is important in the use of this effective but cytotoxic anticancer agent. We have previously shown that the renal toxicity of CDDP can be efficiently reduced by the induction of metallothionein (MT) by preadministration of bismuth compounds in mice. Bismuth subnitrate (BSN) is used as an antigastric ulcer agent and as an antidiarrheic agent, and is suitable for inducing MT in the kidney in cancer patients. However, due to the low absorption rate of Bi from the gastrointestinal tract, the efficacy of BSN in inducing renal MT is low. In the present study, we examined the effects of citrate as a vehicle for oral administration of BSN on the tissue distribution of Bi and induction of MT in the kidneys and tumors in mice inoculated with Meth-A fibrosarcoma. Renal levels of MT and Bi were markedly increased in the mice given BSN dissolved in citrate solution compared with those given BSN suspended in saline. On the other hand, the use of citrate increased Bi accumulation in the tumor only slightly and did not increase tumor MT levels. Administration of BSN with citrate efficiently depressed the renal toxicity of CDDP, but did not affect its antitumor activity. Since both BSN and citrate are used clinically as pharmaceuticals, the combination regimen of BSN and citrate may be readily applicable as a countermeasure against the adverse side effects of CDDP without affecting its antitumor activity. [ABSTRACT FROM AUTHOR]

Abstract Transgenic mice carrying familial amyotrophic lateral sclerosis (FALS)-linked mutant Cu/Zn superoxide dismutase (SOD1) genes such as G93A (G93A-mice) and G85R (G85R-mice) genes develop limb paresis. Introduction of human wild type SOD1 (hWT-SOD1) gene, which does not cause motor impairment by itself, into different FALS mice resulted in different effects on their clinical courses, from no effect in G85R-mice to acceleration of disease progression in G93A-mice. However, the molecular mechanism which causes the observed difference, has not been clarified. We hypothesized that the difference might be caused by the stability of mutant SOD1 proteins. Using a combination of mass spectrometry and enzyme-linked immunosorbent assay, we found that the concentration of G93A-SOD1 protein was markedly elevated in tissues of transgenic mice carrying both G93A- and hWT-SOD1 genes (G93A/hWT-mice) compared to that in G93A-mice, and also found that the concentration of G93A-SOD1 protein had a close relation to the disease duration. The concentration of metallothionein-I/II in the spinal cord, reflecting the degree of copper-mediated oxidative stress, was highest in G93A/hWT-mice, second in G93A-mice, and normal in the mice carrying hWT-SOD1 gene. These results indicated that the increase of G93A-SOD1 protein was responsible for the increase of oxidative stress and disease acceleration in G93A/hWT-mice. We speculate that coexpression of hWT-SOD1 protein is deleterious to transgenic mice carrying a stable mutant such as G93A-SOD1, because this mutant protein is stabilized by hWT-SOD1 protein, but not to transgenic mice carrying an unstable mutant such as G85R-SOD1, because this mutant protein is not stabilized by hWT-SOD1. [ABSTRACT FROM AUTHOR]

Hypothesized model of the inhibitory effect of Cd on Fe transport. • Cadmium decreases serum iron concentrations in mice. • Cadmium inhibits the expression of iron transport-related genes in the mouse duodenum. • Cadmium decreases duodenum levels of divalent metal transporter 1 and ferroportin 1. • Cadmium decreases the expression of iron transport-related factors in Caco-2 cells. 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 CdCl 2 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 CdCl 2. 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. [ABSTRACT FROM AUTHOR]

Aspergillus fumigatus is an opportunistic mold that infects patients who are immunocompromised or have chronic lung disease, causing significant morbidity and mortality in these populations. While the factors governing the host response to A. fumigatus remain poorly defined, neutrophil recruitment to the site of infection is critical to clear the fungus. Galectin-3 is a mammalian β-galactose-binding lectin with both antimicrobial and immunomodulatory activities, however the role of galectin-3 in the defense against molds has not been studied. Here we show that galectin-3 expression is markedly up-regulated in mice and humans with pulmonary aspergillosis. Galectin-3 deficient mice displayed increased fungal burden and higher mortality during pulmonary infection. In contrast to previous reports with pathogenic yeast, galectin-3 exhibited no antifungal activity against A. fumigatus in vitro. Galectin-3 deficient mice exhibited fewer neutrophils in their airways during infection, despite normal numbers of total lung neutrophils. Intravital imaging studies confirmed that galectin-3 was required for normal neutrophil migration to the airspaces during fungal infection. Adoptive transfer experiments demonstrated that stromal rather than neutrophil-intrinsic galectin-3 was necessary for normal neutrophil entry into the airspaces. Live cell imaging studies revealed that extracellular galectin-3 directly increases neutrophil motility. Taken together, these data demonstrate that extracellular galectin-3 facilitates recruitment of neutrophils to the site of A. fumigatus infection, and reveals a novel role for galectin-3 in host defense against fungal infections. Author summary: The environmental mold Aspergillus fumigatus commonly causes lung infections in people with impaired immunity or those suffering from a chronic lung disease. While neutrophils are a key cell type necessary for the eradication of this infection, the precise mechanism of their recruitment to the site of infection remains incompletely understood. Here we show that the secreted mammalian protein galectin-3 plays an important role in helping neutrophils reaching the fungus within the airways. We found that both mice and humans produce galectin-3 when infected with A. fumigatus, and mice lacking galectin-3 were more susceptible to infection than normal mice. Galectin-3-deficient mice had impaired neutrophil recruitment to the site of infection. In the absence of galectin-3, neutrophils exhibited reduced motility in mouse lungs and in tissue culture. Our study offers insights into the mechanisms underlying the recruitment of neutrophils to the airways during A. fumigatus infection and reveals a new role for galectin-3 in increasing neutrophil motility. [ABSTRACT FROM AUTHOR]

The muscle membrane, sarcolemma, must be firmly attached to the basal lamina. The failure of proper attachment results in muscle injury, which is the underlying cause of Duchenne muscular dystrophy (DMD), in which mutations in the dystrophin gene disrupts the firm adhesion. In patients with DMD, even moderate contraction causes damage, leading to progressive muscle degeneration. The damaged muscles are repaired through myogenesis. Consequently, myogenesis is highly active in patients with DMD, and the repeated activation of myogenesis leads to the exhaustion of the myogenic stem cells. Therefore, approaches to reducing the risk of the exhaustion are to develop a treatment that strengthens the interaction between the sarcolemma and the basal lamina and increases the efficiency of the myogenesis. Galectin-3 is an oligosaccharide-binding protein and is known to be involved in cell-cell interactions and cell-matrix interactions. Galectin-3 is expressed in myoblasts and skeletal muscle, although its function in muscle remains elusive. In this study, we found evidence that galectin-3 and the monosaccharide N-acetylglucosamine, which increases the synthesis of binding partners (oligosaccharides) of galectin-3, promote myogenesis in vitro. Moreover, in the mdx mouse model of DMD, treatment with N-acetylglucosamine increased muscle-force production. The results suggest that treatment with N-acetylglucosamine might mitigate the burden of DMD. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This study examined the emergence of delayed behavioral effects in offspring mice exposed to low levels of mercury vapor (Hg0) during the lactation period. Female offspring of mice were repeatedly exposed to Hg0 at 0.057 mg/m³, similar to the current threshold value (TLV), for 24 hr until the 20th day postpartum. The behavioral effects were evaluated with locomotor activity in the open field (OPF), learning activity in the passive avoidance response (PA) and spatial learning ability in the Morris water maze (MM) at the ages of 3 and 15 months. Hg0-exposed mice did not differ from controls in the three behavioral measurements at 3 months of age, and no neurobehavioral effects were observed. On the other hand, the mice exhibited significantly more central locomotion in the OPF task when tested at 15 months of age, but no abnormality in other behavioral performance. Immediately after postnatal exposure, the brain mercury concentration of offspring was about 150 times that of the control, in which the concentrations were approximately 0.4 µg/g. The results indicate that mice exposed to Hg0 at concentrations around TLV during the developing period resulted in the emergence of delayed behavioral effects at a later stage in life. [ABSTRACT FROM AUTHOR]

Abstract: Zinc is an essential micronutrient, deficiency of which results in growth retardation, immunodeficiency, and neurological diseases such as dysgeusia. Several zinc coordination compounds are used for zinc supplementation; however, supplemented zinc ions have no specificity and interact with various groups of molecules. Here, we found that, from a library of 30 zinc coordination compounds, bis(L-cysteinato)zincate(II), designated Z01, functioned as a metallothionein (MT) inducer. Z01 induced MT expression mediated by the transcription factor MTF-1, without inducing cell-stress-related heme oxygenase-1 gene expression at specific concentration. The zinc ion was necessary for the MT induction. 65Zn incorporation following treatment with 65Zn-labeled Z01 suggested that Z01 did not act as zinc ionophore despite its hydrophilicity. Electrophoretic mobility shift assays revealed that Z01 facilitates MTF-1–MRE complex formation, and, by inference, transfer of zinc from Z01 to MTF-1. Phosphorylated ERK levels were increased by ZnSO4 treatment but not by Z01. Although our data do not definitely prove that Z01 is an MTF-1-specific activator, our observations suggest that zinc coordination compounds can regulate zinc distribution and act as zinc donors for specific molecules. [Copyright &y& Elsevier]

Dental amalgam is a source of exposure to elemental mercury vapor in the general population. The aim of this study was to elucidate the effect of elemental mercury vapor exposure from dental amalgam restorations on gene expression profiles. Out of 26,962 rat genes, mercury vapor was found to increase the expression of 1 gene (Atp1b3) and decrease the expression of 1 gene (Tap1) in the cerebrum, increase the expression of 1 gene (Dnaja2) in the cerebellum, increase the expression of 2 genes (Actb and Timm23) and decrease the expression of 1 gene (Spink3) in the liver, increase the expression of 2 genes (RT1-Bb and Mgat5) and decrease the expression of 6 genes (Tnfaip8, Rara, Slc2a4, Wdr12, Pias4 and Timm13) in the kidney. [ABSTRACT FROM AUTHOR]

Metallothionein (MT) is known to be involved in various physiological roles and diseases. However, a standard method for MT measurement has not been established until recently. Therefore, we have developed an easy and specific enzyme-linked immunosorbent assays (ELISA) to determine MT-1 and MT-2. In order to evaluate the method we developed, MT-1/2 in liver, kidney and brain was determined in wild type (WT), MT-1/2 knockout (KO) and MT-3 KO mice, with and without Cd treatment. MT 1/2 in urine was determined in genetically disordered LEC rats (an animal model of Wilson disease). MT-1/2 concentrations in the liver, kidney and brain in MT-1/2 KO mice were significantly lower compared to those of WT and MT-3 KO mice. MT-1/2 concentrations in the livers of WT mice significantly increased with Cd administration, but not in MT-1/2 KO mice. Similar results were observed by immunohistochemical staining. To confirm the molecular weight (MW) of MT detected in organs by the ELISA, analysis with a Sephadex G-75 was performed. Two peaks of MT-1/2 (MW small and large) were detected in WT and MT-3 KO mice. The small MT peak was mostly depleted in MT 1/2-KO mice, while a large MT peak remained. A significant increase in MT-1/2 concentration was detected in the urine of LEC rats with age and especially at the hepatitis stage. In conclusion, MT-1/2 ELISA and immunohistochemical staining was highly correlated with MT-1/2 determination in experimental animal specimens and could be a robust analytical tool for physiological and toxicological studies. [ABSTRACT FROM AUTHOR]

This study examined the neurobehavioral changes and alteration in gene expression in the brains of metallothionein (MT)-I/II null mice exposed to low-levels of mercury vapor (Hg0) during postnatal development. MT-I/II null and wild-type mice were repeatedly exposed to Hg0 at 0.030 mg/m (range: 0.023-0.043 mg/³), which was similar to the current threshold value (TLV), for 6 hr per day until the 20th day postpartum. The behavioral effects were evaluated with locomotor activity in the open field (OPF), learning ability in the passive avoidance response (PA) and spatial learning ability in the Morris water maze (MM) at 12 weeks of age. Hg0-exposed MT-I/II null mice showed a significant decrease in total locomotor activity in females, though learning ability and spatial learning ability were not affected. Immediately after Hg0 exposure, mercury concentrations in the brain did not exceed 0.5 µg/g in any animals. Hg0 exposure resulted in significant alterations in gene expression in the brains of both strains using DNA microarray analysis. The number of altered genes in MT-I/II null mice was higher than that in wild-type mice and calcium-calmodulin kinase II (Camk2a) involved in learning and memory in down-regulated genes was detected. These results provide useful information to elucidate the development of behavioral toxicity following low-level exposure to Hg0. [ABSTRACT FROM AUTHOR]

Abstract: We measured the levels of some biological metals: copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), and zinc (Zn) in the cerebrospinal fluid (CSF) in patients with neurodegenerative diseases (52 patients with amyotrophic lateral sclerosis (ALS)), 21 patients with Alzheimer''s disease (AD), and 20 patients with Parkinson''s disease (PD) by inductively coupled plasma mass spectrometry (ICP-MS). The diagnoses were additionally supported by neuroimaging techniques for AD and PD. In ALS, the levels of Mg (p <0.01 significant difference), Fe, Cu (p <0.05), and Zn (p <0.10) in CSF were higher than those in controls. Some patients showed very high levels of Cu and Zn before the critical deterioration of the disease. In AD, the levels of Cu and Zn in CSF were significantly higher in patients with late–onset AD (p <0.01). In PD, we found significantly increased levels of especially Cu and Zn in particular (p <0.01) and Mn (p <0.05) in CSF. A multiple comparison test suggested that the increased level of Mg in ALS and that of Mn in PD were the pathognomonic features. These findings suggest that Cu and Zn in particular play important roles in the onset and/or progression of ALS, AD, and PD. Therefore, Cu-chelating agents and modulators of Cu and Zn such as metallothionein (MT) can be new candidates for the treatment of ALS, AD, and PD. [Copyright &y& Elsevier]

Poly(ADP-ribose) polymerase-1 (PARP-1) modifies various proteins, including itself, with ADP-ribose polymers (automodification). Polymer synthesis is triggered by binding of its zinc finger 1 (Zn1) and 2 (Zn2) to DNA breaks and is followed by inactivation through automodification. The multiple functional domains of PARP-1 appear to regulate activation and automodification-mediated inactivation of PARP-1. However, the roles of these domains in activation-inactivation processes are not well understood. Our results suggest that Zn1, Zn2, and a domain identified in this study, the double-stranded DNA binding (DsDB) domain, are involved in DNA break-dependent activation of PARP-1. We found that binding of the DsDB domain to double-stranded DNA and DNA break recognition by Zn1 and Zn2, whose actual binding targets are likely to be single-stranded DNA, lead to the activation of PARP-1. In turn, the displacement of single- and double-stranded DNA from Zn2 and the DsDB domain caused by ADP-ribose polymer synthesis results in the dissociation of PARP-1 from DNA breaks and thus its inactivation. We also found that the WGR domain is one of the domains involved in the RNA-dependent activation of PARP-1. Furthermore, because zinc finger 3 (Zn3) has the ability to bind to single-stranded RNA, it may have an indirect role in RNA-dependent activation. PARP-1 functional domains, which are involved in oligonucleic acid binding, therefore coordinately regulate PARP-1 activity depending on the status of the neighboring oligonucleic acids. Based on these results, we proposed a model for the regulation of PARP-1 activity. [ABSTRACT FROM AUTHOR]

The purpose of the present study was to investigate whether cilostazol, a phosphodiesterase-III inhibitor and antiplatelet drug, would prevent tPA-associated hemorrhagic transformation. Mice subjected to 6-h middle cerebral artery occlusion were treated with delayed tPA alone at 6 h, with combined tPA plus cilostazol at 6 h, or with vehicle at 6 h. We used multiple imaging (electron microscopy, spectroscopy), histological and neurobehavioral measures to assess the effects of the treatment at 18 h and 7 days after the reperfusion. To further investigate the mechanism of cilostazol to beneficial effect, we also performed an in vitro study with tPA and a phosphodiesterase-III inhibitor in human brain microvascular endothelial cells, pericytes, and astrocytes. Combination therapy with tPA plus cilostazol prevented development of hemorrhagic transformation, reduced brain edema, prevented endothelial injury via reduction MMP-9 activity, and prevented the blood-brain barrier opening by inhibiting decreased claudin-5 expression. These changes significantly reduced the morbidity and mortality at 18 h and 7 days after the reperfusion. Also, the administration of both drugs prevented injury to brain human endothelial cells and human brain pericytes. The present study indicates that a phosphodiesterase-III inhibitor prevents the hemorrhagic transformation induced by focal cerebral ischemia in mice treated with tPA. [ABSTRACT FROM AUTHOR]

Abstract: 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 CdCl2 (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. [ABSTRACT FROM AUTHOR]

Interleukin-6 (IL-6) is known to be involved in the pathogenesis of various inflammatory diseases, but its role in the development of pulmonary emphysema remains unclear. Wild-type (WT) and IL-6-deficient mice received either phosphate-buffered saline (PBS) or porcine pancreatic elastase (PPE) intratracheally. The development of emphysema was determined by measuring the mean linear intercept (Lm). The lung specimens were also subjected to immunohistochemistry for single-stranded DNA to detect apoptotic cells. Lung mechanics and airway responsiveness to inhaled methacholine were analyzed. Bronchoalveolar lavage (BAL) fluid was subjected to evaluation of inflammatory cell accumulation and cytokine measurement. PPE treatment caused significant increases in Lm and lung compliance, which was attenuated by IL-6 deficiency. The increases in apoptotic cells in the lung were attenuated in IL-6 null mice. Airway responsiveness was not affected by PPE challenge or IL-6 deficiency. Intratracheal PPE increased the cell counts in BAL fluid throughout the observation, which was suppressed in IL-6 null mice. In BAL fluid, PPE-induced increases in the levels of macrophage inflammatory protein (MIP)-1α and eotaxin were mitigated by IL-6 deficiency. PPE-induced up-regulation of matrix metalloproteinase (MMP)-12 in the lung was attenuated by IL-6 deficiency. These results indicate that IL-6 may play an important role in the development of elastase-induced lung inflammatory changes. [ABSTRACT FROM AUTHOR]

The purpose of this study was to examine whether intracellular metallothionein (MT) protects against benzo[a]pyrene (B[a]P)-induced forestomach and lung carcinogenesis. Ten-week-old male MT-I/II null mice and wild-type mice were orally administered B[a]P at a dose of 100 or 250 mg/kg twice a week for 4 weeks. B[a]P-induced mortality was higher in the MT-I/II null mice than in the wild-type mice. The incidence of tumors in the forestomach and lung was 78.6% and 7.1% in the wild-type mice treated with 100 mg/kg B[a]P, respectively. In the MT-I/II null mice treated with B[a]P, tumor incidence in the forestomach and lung was 100% and 33.3%, respectively. The tumor area in the forestomach and lung in the MT-I/II null mice treated with B[a]P was greater than that of wild-type mice. These results suggest that MT acts as a biological protective factor against carcinogenesis induced by B[a]P. [ABSTRACT FROM AUTHOR]

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 μmol/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. [ABSTRACT FROM AUTHOR]

Abstract: The knowledge of vascular toxicity is important for understanding the neurotoxicity of methylmercury. In the present study, we investigated the cell-density-dependent susceptibility of human brain microvascular pericytes to methylmercury-induced toxicity by using a cell-culture system. The susceptibility of sparse pericyte cultures to methylmercury was greater than that of the dense cultures. In addition, the sparse cultures were more susceptible to methylmercury than to inorganic mercury and cadmium. The intracellular accumulation of methylmercury in the sparse cells was significantly higher than that in the dense cells. Methylmercury is transported through the L-type large neutral amino acid transporter (LAT 1) in the form of a complex with cysteine. The mRNA- and protein-level expressions of LAT 1 in the sparse cells were markedly higher than those in the dense cells; in addition, the LAT 1 expression was increased by methylmercury. However, there was no reduction in the levels of glutathione and metallothionein, which are involved in the defense mechanisms against methylmercury, in the sparse cells. The present data revealed that pericytes are markedly susceptible to methylmercury-induced cytotoxicity at low cell densities. The susceptibility of the sparse pericytes is postulated to be due to the not only constitutively higher but also methylmercury-induced expression of LAT 1, which increased the intracellular accumulation of methylmercury. [Copyright &y& Elsevier]

Abstract: Metallothioneins (MTs) are metal binding proteins and have four isoforms. MT-3, known as growth inhibitory factor (GIF), exists mainly in the central nervous system. It regulates zinc levels and exhibits a neuroprotective effect in the various types of brain diseases. However, the reports demonstrate that the relation between MT-3 and psychiatric disorder is still unknown. In the present study, the authors carried out behavioral tests on MT-3 knock-out (KO) mice. The duration of the MT-3 KO mice''s social interactions were significantly shorter than that of the wild-type (WT) mice. The acoustic startle response of the MT-3 KO mice showed diminished prepulse inhibition (PPI) at all prepulse intensities. However, the locomotor activity tests of the MT-3 KO mice displayed normal circadian rhythm, activity, and habituation to a novel environment. In the novel object recognition test, the MT-3 KO mice exhibited normal memory. These findings indicate that abnormalities of psychological behavior were observed in the MT-3 KO mice. Further experiments will be needed to clarify the involvement of MT-3 in higher brain function. [Copyright &y& Elsevier]

Metallothionein (MT) is known to reduce chemical carcinogenesis. Carcinogenesis induced by benzo[a]pyrene (B[a]P) which is an environmental chemical carcinogen is related to DNA adduct formation and oxidative damage through metabolic activation. Ten-week-old male MT-I/II null mice and wild-type mice were given a single injection of B[a]P (250 mg/kg, p.o.), and B[a]P-induced DNA damage was evaluated at 6-48 hr later. The frequencies of micronucleated reticulocytes (MNRET) in MT-I/II null mice were significantly increased compared with that of wild-type mice at 48 hr after B[a]P administration. At 48 hr after B[a]P administration, comet scores were significantly increased in MT-I/II null mice but not in wild-type mice. 8-Hydroxy-2'-deoxyguanosine (8-OHdG), a biomarker of oxidative DNA damage, was significantly increased in liver of MT-I/II null mice at 6 and 12 hr after B[a]P administration, although that of wild-type mice was only slightly changed. Because cytochrome P450 (CYP) plays a major role in the process of B[a]P metabolic activation, we attempted to reveal the effect of MT on metabolic activation of B[a]P. Although CYP1A activities were elevated in the livers of MT-I/II null mice and wild-type mice treated with B[a]P, it was not different between both strains of mice. In addition, MT levels in the livers of wild-type mice were significantly increased by the B[a]P treatment, whereas MT was not detected in livers of MT-I/II null mice with or without B[a]P treatment. These results demonstrate that MT acts as an endogenous defensive factor against B[a]P-induced DNA damage. [ABSTRACT FROM AUTHOR]

Abstract: Metallothioneins (MTs) are metal-binding proteins and have four isoforms. MT-III was, at first, found in the brains of patients with Alzheimer''s disease. MT-III exists mainly in the central nervous system, and the main effects are thought to be anti-oxidative and regulate zinc levels. In some previous reports, MT-III exhibited neuroprotective effects in various pathological situations, but its detailed effects are still unclear. In the present study, we examined neuronal damage after a middle cerebral artery occlusion (MCAO) in MT-III knockout (KO) mice to elucidate the relationship between MT-III and cerebral infarction. There was no significant difference in cerebral infarction after 24-h permanent MCAO between the wild-type and MT-III KO mice. On the other hand, after 2-h MCAO and 22-h reperfusion, cerebral infarction in the MT-III KO mice was aggravated compared with the wild-type mice. Furthermore, fatal rate of MT-III KO mice increased from 3 days after MCAO, and neurological deficits at 5 and 7 days after MCAO of MT-III KO mice were worse than those of wild-type. We examined terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining and the immunostaining of an oxidative stress marker, 8-hydroxy-2′-deoxyguanosine (8-OHdG), at 24 h after transient MCAO. In the penumbra lesion, the positive cell numbers in both staining assays were higher in the MT-III KO mice than those of the wild-type mice. These findings indicate that neuronal damage was aggravated by reperfusion injury in the MT-III KO mice compared with the wild-type mice, suggesting that MT-III plays anti-oxidative and neuroprotective roles in transient cerebral ischemia. [Copyright &y& Elsevier]

We present herein transcriptional changes in mouse spinal cords in response to physical exercise on a treadmill using a DNA microarray. By 30-min exercise, the expression of 3 genes was enhanced and expression of 29 genes reduced. By continuous 2-week (30-min exercise per day), the expression of 1 gene was enhanced and expression of 13 genes reduced. [ABSTRACT FROM AUTHOR]

Vascular toxicity is an important feature of the neuropathy induced by methylmercury. Methylmercury does not cause nonspecific cell damage, but rather retards the repair of wounded monolayers of cultured human brain microvascular endothelial cells by inhibiting their proliferation. Since vascular endothelial cell proliferation during the repair process strongly depends on the fibroblast growth factor-2 (FGF-2) system, we investigated the effects of methylmercury on the expression of FGF-2 and related proteins (i.e., FGF receptor 1 and perlecan) in cultured human brain microvascular endothelial cells. Of the mRNAs examined, FGF-2 mRNA expression was significantly lowered by methylmercury in not only wounded monolayers but also dense and sparse cultures of endothelial cells; a lower expression of FGF-2 protein in the cells was confirmed. In addition, exogenous FGF-2 partially abrogated the proliferation-inhibitory effect of methylmercury. The results of this study suggest that suppression of FGF-2 expression is one of the mechanisms underlying the inhibitory effect of methylmercury in damaged endothelial cell monolayers. The FGF-2 system may be one of the important biological systems behind the vascular toxicity of methylmercury. [ABSTRACT FROM AUTHOR]

The integration of knowledge concerning the regulation of MT, a highly conserved, low molecular weight, cystein-rich metalloprotein, on its proposed functions is necessary to clarify how MT affects cellular processes. MT expression is induced/enhanced in various tissues by a number of physiological mediators. The cellular accumulation of MT depends on the availability of cellular zinc derived from the diet. MT modulates the binding and exchange/transport of heavy metals such as zinc, cadmium, or copper under physiological conditions and cytoprotection from their toxicities, and the release of gaseous mediators such as hydroxyl radicals or nitric oxide. In addition, MT reportedly affects a number of cellular processes, such as gene expression, apoptosis, proliferation, and differentiation. Given the genetic approach, the apparently healthy status of MT-deficient mice argues against an essential biological role for MT; however, this molecule may be critical in cells/tissues/organs in times of stress, since MT expression is also evoked/enhanced by various stresses. In particular, because metallothionein (MT) is induced by inflammatory stress, its roles in inflammation are implied. Also, MT expression in various organs/tissues can be enhanced by inflammatory stimuli, implicating in inflammatory diseases. In this paper, we review the role of MT of various inflammatory conditions. [ABSTRACT FROM AUTHOR]

Abstract: Metallothionein (MT) is a free radical scavenger induced by inflammatory stimuli; however, its roles in inflammation have not been fully investigated. In the present study, we genetically determined the role of MT in ozone (O3)-induced lung inflammation using MT-I/II null (–/–) mice. Subacute (65 h) exposure to O3 (0.3 ppm) induced lung inflammation and enhanced vascular permeability, which was significantly greater in MT(–/–) than in corresponding wild-type mice. Electron microscopically, O3 exposure induced vacuolar degeneration of pulmonary endothelial and epithelial cells, and interstitial edema with focal loss of the basement membrane, which was more prominent in MT(–/–) than in wild-type mice. O3 -induced lung expression of interleukin-6 was significantly greater in MT(–/–) than in wild-type mice; however, lung expression of the chemokines examined was comparable in both genotypes of mice in the presence of O3. Following O3 exposure, the formation of oxidative stress-related molecules/adducts, such as heme oxidase-1, inducible nitric oxide synthase, 8-hydroxy-2′-deoxyguanosine, and nitrotyrosine, in the lung was significantly greater in MT(–/–) than in wild-type mice. Collectively, MT protects against O3-induced lung inflammation, at least partly, via the regulation of pulmonary endothelial and epithelial integrity and its antioxidative property. [Copyright &y& Elsevier]

In order to elucidate the involvement of metallothionein (MT) in radiation carcinogenesis, we examined the susceptibility of MT-I/II null mice to carcinogenesis and oxidative DNA damage resulting from X-irradiation. Eight-week-old female MT-I/II null mice and wild-type mice were exposed to whole-body X-irradiation at doses of 1.0, 1.5 or 2.0 Gy once a week for 6 weeks. Incidence of thymic lymphoma was determined at 24 weeks after the first exposure to X-irradiation. The frequency of thymic lymphomas induced by X-irradiation (at 1.5 and 2.0 Gy) was significantly higher in MT-I/II null mice than in wild-type mice. In addition, although the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) were increased in the serum and urine of both strains of mice 24 hr after exposure to a single bout of whole body X-irradiation, these increases were significantly greater in the MT-I/II null mice than in the wild-type mice. Thus, the present results suggest that MT plays a protective role against carcinogenesis and oxidative DNA damage caused by X-irradiation. [ABSTRACT FROM AUTHOR]

Abstract: The purpose of the present study was to examine the in vitro effects of low-dose cadmium (Cd) on developing cortical cells. The cortical cells removed from fetuses (embryonic day 15) were treated with 10nM of Cd for 24h. The effects of Cd on dendritic and synaptic development were immunocytochemically observed with anti-microtubule associated protein-2 (MAP2) and anti-synapsin I antibodies, respectively. Administration of Cd suppressed dendritic as well as synaptic development at 10nM. By two-dimensional gel electrophoresis and liquid chromatography–tandem mass spectrometric (LC/MS/MS) analysis, we identified three proteins with different expression after Cd-treatment; dihydropyrimidinase-related protein 2 (DRP-2/CRMP-2), 14-3-3-epsillon and calmodulin (CaM). Though the number of identified proteins was small, these proteins are known to be involved in neuronal development. The present study demonstrated the morphological effects as well as affected proteins in Cd-treated cortical cells, providing tools and insights in elucidating mechanisms how low-dose Cd distorts brain development. [Copyright &y& Elsevier]

Abstract: In previous report, we have shown that trichloroethylene (TCE) increases histamine release and inflammatory cytokine production from antigen-stimulated mast cells. In this study, we examined the enhancing effect of a small amount of TCE ingestion from drinking water on antigen-stimulated allergic responses. After exposure of Wistar rats to TCE ingestion for 2 or 4 weeks, we performed a passive cutaneous anaphylaxis (PCA) reaction. TCE ingestion for 2 and 4 weeks enhanced PCA reaction in a dose-dependent manner. On histological examination, TCE ingestion for 2 weeks exacerbated inflammation characterized by infiltration of lymphocytes and accumulation of mast cells around the vessel in the skin. After TCE ingestion for 4 weeks, the mesenteric lymph nodes (MLNs) showed increase of the size and wet weight, and germinal centers changed distinctly. The interleukin-4 (IL-4) mRNA levels on spleen, MLNs and leukocytes were increased. Moreover, serum total IgE levels of TCE ingestion increased in a time-dependent manner. Our results suggest that TCE ingestion induces pro-inflammatory responses and causes Th1/Th2-type helper T-cell imbalance. And more, a small amount of TCE ingestion may lead to the initiation and acceleration of type I allergic reaction. [Copyright &y& Elsevier]

Abstract: Previously, we observed that tetrachloroethylene (perchloroethylene, PCE) increased histamine release and inflammatory mediator production from antigen-stimulated mast cells. In this study, we examined the enhancing effect of low concentrations of PCE in drinking water on antigen-stimulated allergic responses. After exposure of Wistar rats to PCE in drinking water for 2 or 4 weeks, we performed a passive cutaneous anaphylaxis (PCA) reaction. PCE exposure for 4 weeks enhanced PCA reaction in a dose-dependent manner. In pathological studies, PCE exposure for 2 weeks exacerbated inflammation characterized by infiltration of lymphocytes and accumulation of mast cells around the vessel. Non-purified mast cells (NPMCs) from rats treated with 1mg/L PCE in drinking water for 2 weeks increased antigen-stimulated histamine release. Furthermore, the leukocytes of rats treated with PCE in drinking water for 4 weeks showed increased interleukin (IL)-4 expression. The mechanism of enhancing the PCA reaction is assumed to be that PCE increases IL-4 production and PCE causes T helper (Th) 1/Th2-type helper T-cell imbalance and increases histamine release from excessively accumulated mast cells. The results suggest that the intake of PCE in drinking water, even at a low concentration, leads to the initiation and acceleration of allergic diseases. [Copyright &y& Elsevier]

In order to elucidate the role of metallothionein (MT) in preventing the adverse effects of X-ray irradiation, we examined the susceptibility of MT-I/II null mice to bone marrow injury caused by X-irradiation and effects of pretreatment with MT-inducing metals on X-ray injury. Eight-week-old male mice were exposed to a single bout of whole-body X-irradiation at a dose between 0.1 and 6.0 Gy. The numbers of leukocytes, reticulocytes with micronuclei (MNRET) in the blood, and polychromatic erythrocytes with micronuclei (MNPCE) in the bone marrow were determined 24 hr after X-irradiation. X-irradiation significantly decreased the total number of leukocytes in MT-I/II null mice and wild-type mice in a dose-dependent manner, but the total number of leukocytes was significantly lower in MT-I/II null mice than in wild-type mice at a low dose of irradiation, between 0.1 and 1.0 Gy. X-irradiation (0.1 and 0.5 Gy) significantly increased the appearance of MNRET and MNPCE in both strains, but the increase was greater in the MT-I/II null mice than in the wild-type mice. Additional groups of mice were pre-administered bismuth nitrate or zinc sulfate to induce MT in the bone marrow cells prior to X-irradiation; the X-ray injury was prevented by such treatments in wild-type mice only. Thus, the present results suggest that MT plays a protective role against a low dose of X-ray injury. [ABSTRACT FROM AUTHOR]

Epidemiological and experimental studies have suggested that exposure to metalloid arsenic constitutes a risk factor for vascular disease associated with atherosclerosis. Since in atherosclerosis, types of proteoglycans (PGs) present change depending on the stage, we investigated the effect of 2 chemical forms of inorganic arsenic—a trivalent sodium arsenite and a pentavalent sodium arsenate—on the synthesis of PGs in cultured arterial smooth muscle cells. The results indicate that arsenite but not arsenate, at a noncytotoxic level, inhibits general PG synthesis independent of cell density. Arsenite may be one of the chemical forms of inorganic arsenic that influences the PG composition in blood vessel walls during the progression of vascular disorders such as atherosclerosis. [ABSTRACT FROM AUTHOR]

BACKGROUND: Although a long latency period of toxicity after exposure to methylmercury (MeHg) is known to exist in humans, few animal studies have addressed this issue. Substantiation of delayed MeHg toxicity in animals would affect the risk evaluation of MeHg. OBJECTIVES: Our goal in this study was to demonstrate the existence of a latency period in a rodent model in which the toxicity of perinatal MeHg exposure becomes apparent only later in life. Our study included metallothionein (MT) knockout mice because studies have suggested the potential susceptibility of this strain to the neurodevelopmental toxicity of MeHg. METHODS: Pregnant MT-null and wild-type C57Bl/6J mice were exposed to MeHg through their diet containing 5 μg Hg/g during gestation and early lactation. We examined behavioral functions of the offspring using frequently used paradigms, including open field behavior (OPF), passive avoidance (PA), and the Morris water maze (MM), at ages of 12-13 and 52-53 weeks. RESULTS: At 12 weeks of age, behavioral effects of MeHg were not detected, except for OPF performance in MeHg-exposed MT-null females. At 52 weeks of age, the MeHg-exposed groups showed poorer performance both in PA and MM, and their OPF activity differed from controls. These effects of MeHg appeared exaggerated in the MT-null strain. The brain Hg concentration had leveled off by 13 weeks of age. CONCLUSIONS: The results suggest the existence of a long latency period after perinatal exposure to low-level MeHg, in which the behavioral effects emerged long after the leveling-off of brain Hg levels. Hence, the initial toxicologic event responsible for the late effects should have occurred before this leveling-off of brain Hg. [ABSTRACT FROM AUTHOR]

The main objective of this study, on mice, was to compare the neuroprotective effects of propofol with those of propofol plus disodium edetate (propofol EDTA). We also administered propofol EDTA (0.005% (w/v) EDTA) to mice intravenously, and measured the changes in zinc concentrations occurring after permanent middle cerebral artery occlusion. In the in vivo study, propofol EDTA displayed stronger neuroprotective effects than propofol alone. Furthermore, we examined the neuroprotective effects of EDTA administered alone, and found that EDTA Na significantly reduced the infarct volume. The number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling-positive cells in the ischemic penumbra was reduced more by propofol EDTA than by propofol alone. We performed in the in vitro study in five groups (aerobic, vehicle (control), propofol, EDTA, and propofol plus EDTA). Propofol and EDTA each protected PC12 cells against oxygen–glucose deprivation-induced cell damage, and the effect of propofol was increased by adding EDTA. Because the chelating action of EDTA was a potential causal mechanism, we examined the effect of propofol EDTA on intracerebral zinc homeostasis. When propofol EDTA was given intravenously 10 mins before cerebral ischemia, the zinc concentration decreased significantly in the cortical area, but not in the subcortex. In conclusion, (a) propofol provides neuroprotection against both in vivo and in vitro ischemic damage, and its effects are enhanced when EDTA is added; and (b) EDTA itself protects against ischemic neuronal damage, possibly, owing to its zinc-chelating action.Journal of Cerebral Blood Flow & Metabolism (2008) 28, 354–366; doi:10.1038/sj.jcbfm.9600532; published online 25 July 2007 [ABSTRACT FROM AUTHOR]