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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]

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]

To clarify the possible role of interleukin-6 in aggravation of inflammatory responses in diesel exhaust-exposed mice, we compared the infiltration of inflammatory cells and the production of chemokines between interleukin-6-deficient and wild-type mice following 0, 1.0, or 3.0 mg diesel particles/m3 diesel exhaust inhalation for 4 weeks. Exposure to diesel exhaust significantly increased the number of inflammatory cells and the amount of CCL17 and CXCL3 in bronchoalveolar lavage fluids from wild-type mice, but not in interleukin-6-deficient mice. These findings suggest that interleukin-6 plays a critical role in airway inflammatory responses induced by diesel exhaust inhalation. [ABSTRACT FROM AUTHOR]

While recent human studies suggested adverse neurobehavioral outcomes of low-level exposure to mercury vapor (Hg0) as found among those having dental amalgam fillings and dental personnel, past animal experiments only dealt with exposure at much higher mercury concentrations. The present study aimed to examine neurobehavioral effects of prolonged, low-level Hg0 exposure in mice and to evaluate the protective role of metallothionein-I,II (MT-I,II) against Hg0-induced neurotoxicity, using a knock-out strain of mice. Adult female metallothionein-I,II-null (MT-null) and wild-type OLA129/C57BL6 mice were exposed to 0.06 mg/m3 of Hg0 for 8 h per day for 23 weeks. Neurobehavioral effects were evaluated at 12 and 23 weeks of exposure using open-field test and passive avoidance test. Subcellular distribution of mercury and the induction of MT were also assessed. The Hg0 exposure resulted in significantly enhanced locomotion in the open-field test and poorer performance in the passive avoidance test at a brain Hg concentration less than 1 ppm. These effects were slightly exaggerated in MT-null mice, which showed less induction of MT, lower brain Hg concentration, and lower calculated concentration of MT-unbound cytosolic Hg. The results showed, for the first time, that a concentration of Hg0 relevant to human exposure level could cause neurobehavioral effects in adult mice. The higher susceptibility of MT-null mice suggested that MT-I,II have protective roles in the metal-induced neurobehavioral toxicity, which cannot be entirely explained by kinetic mechanisms, thus suggesting an involvement of nonkinetic mechanisms. [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]

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]

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: 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]

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: 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]

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]

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]

Although cadmium causes hepatotoxicity, its molecular mechanism is unclear. In the present study, transcriptional responses in the liver of C57BL/6J mice given 50 ppm cadmium as a drinking water for 30 days were evaluated with DNA microarray. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were not elevated following the administration of cadmium. Cadmium increased the expressions of 2 genes and reduced those of 15 genes in the liver of mice before the leading to hepatotoxicity. [ABSTRACT FROM AUTHOR]

DNA microarray containing 30,000 genes was used to monitor the transcriptional response of the neonatal brain after cadmium (Cd) exposure. C57BL/6J pregnant mice were exposed to Cd (10 ppm) during gestation and lactation via drinking water. In a comparison between the Cd-exposed neonatal brain and control, three genes including transferrin receptor (Tfrc) were up-regulated and one gene was down-regulated. [ABSTRACT FROM AUTHOR]

In order to elucidate the effect of metallothionein (MT)-III on hepatic gene expression altered by cadmium (Cd), we examined gene expression patterns in the liver of MT-III null mice and wild-type mice after Cd injection using a DNA microarray containing 35,852 genes. In a comparison between Cd-injected MT-III null mice and Cd-injected wild-type mice, 9 genes were found to be up-regulated and 28 genes-including serum amyloid A1 (SAA-1) and SAA-2-were down-regulated. [ABSTRACT FROM AUTHOR]