Metallothionein-3 is expressed in the brain and various peripheral organs of the rat.

Metallothionein-3 (MT-3), also known as growth inhibitory factor (GIF), was originally identified in the brain. An essential step in elucidating the potential roles of MT-3 is to evaluate its expression levels in organs other than the brain. In this present study, we carried out RT-PCR, Western blot and immunohistochemical analyses to quantify MT-3 mRNA and its protein in the cerebrum, eye, heart, kidney, liver, prostate, testis, tongue, and muscle in male Wistar rats. MT-3 mRNA was detected in the cerebrum, the dorsolateral lobe of the prostate, testis, and tongue. Using a monoclonal anti-MT-3 antibody, we detected MT-3 in the cerebrum, the dorsolateral lobe of the prostate, testis, and tongue as a single band on an immunoblot. Immunohistochemical staining showed MT-3 in some astrocytes in the deep cortex, ependymal cells, and choroidal cells in the cerebrum. MT-3 was also detected in some cells of the glomerulus and the collective tubules in the kidney, some cells in the glandular epithelium of the dorsolateral lobe of the prostate, some Sertoli cells and Lydig cells in the testis, and taste bud cells in the tongue. Although MT-3 immunopositivity was obviously demonstrated in the kidney by the immnunohistochemical method, the expression of MT-3 was not fully detectable by RT-PCR and Western blot analyses. Interestingly, only a subset of cells showed positivity for MT-3, not all cells in all tissues. The localization of MT-3 in peripheral organs outside the brain suggests that MT-3 has roles in these tissues besides its role in growth inhibition of neurites.

Role of metallothionein in murine experimental colitis.

Metallothioneins (MTs) are a family of cysteine-rich low molecular-weight proteins that can act as reactive oxygen species scavengers. Although it is known that the induction of MT expression suppresses various inflammatory disorders, the role of MTs in intestinal inflammation remains unclear. In this study, we investigated the effects of dextran sulfate sodium (DSS) administration in mice with targeted deletions of the MT-I/II genes. Acute colitis was induced by 2% DSS in male MT-I/II double knockout (MT-null) and C57BL/6 (wild-type) mice. The disease activity index (DAI) was determined on a daily basis for each animal, and consisted of a calculated score based on changes in body weight, stool consistency and intestinal bleeding. Histology, colon length, myeloperoxidase (MPO) activity and colonic mRNA expression and the concentration of inflammatory cytokines were evaluated by real-time-PCR and enzyme-linked immunosorbent assay (ELISA). The localization of MTs and macrophages was determined by immunohistological and immunofluorescence staining. To investigate the role of MTs in macrophages, peritoneal macrophages were isolated and their responses to lipopolysaccharide were measured. Following DSS administration, the DAI score increased in a time-dependent manner and was significantly enhanced in the MT-I/II knockout mice. Colonic MPO activity levels and inflammatory cytokines [tumor necrosis factor (TNF)-α, interferon (IFN)-γ and interleukin (IL)-17] production increased following DSS administration, and these increases were significantly enhanced in the MT-I/II knockout mice compared with the wild-type mice. MT-positive cells were detected in the lamina propria and submucosal layer by immunohistochemical and immunofluorescence staining, and were mainly co-localized in F4/80-positive macrophages. The production of inflammatory cytokines (TNF-α, IFN-γ and IL-17) from isolated peritoneal macrophages increased following lipopolysaccharide stimulation, and these increases were significantly enhanced in the macrophages obtained from the MT-I/II knockout mice. These data indicate that MTs play an important role in the prevention of colonic mucosal inflammation in a mouse model of DSS-induced colitis, thus suggesting that endogenous MTs play a protective role against intestinal inflammation.

Transcriptome of tributyltin-induced apoptosis of the cultured rat mesencephalic neural stem cells.

Exposure to environmental neurotoxic chemicals both in utero and during the early postnatal period can cause neurodevelopmental disorders. To evaluate the disruption of neurodevelopmental programming, we previously established an in vitro neurosphere assay system, using rat mesencephalic neural stem cells (mNSC). Here, we examined the developmental neurotoxicity of tributyltin (TBT) in an in vitro neurosphere assay. A neurosphere was driven from rat E16 mesencephalon and seeded in a poly-l-ornithine/laminin-coated plate. Exposure to TBT increased the number of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL)-positive cells in time-dependent and dose-dependent manner: it was significantly detectable with treatment of 100pM TBT for 90min, or of 1µM TBT for 30min. Disruption of mitochondrial membrane potential and activation of caspase-3/7 were concomitantly observed. Furthermore, DNA microarray analyses using Affymetrix GeneChip revealed that as early as 0.5h after exposure to the metal (1µM), the expression levels of 71 genes were increased by more than 2-fold, whereas those of 8 genes were decreased by 2-fold or less: it was notably altered in expression of Ca(2+)-mobilizing-related genes, and retinoic-acid signal-related genes, as well as bifunctional apoptosis-related genes. The levels of gene expression of Wnt family were also significantly changed. Thus, we established transcriptome of TBT-induced apoptosis of mNSC. This would help to evaluate developmental neurotoxicity of TBT in vivo, contributing to the risk assessment methods based on infant physiology.

Involvement of metallothionein (MT) as a biological protective factor against carcinogenesis induced by benzo[a]pyrene (B[a]P).

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.

Protective role of metallothionein in benzo[a]pyrene-induced DNA damage.

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.

Role of metallothionein as a protective factor against radiation carcinogenesis.

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.

Differences in gene expression and benzo[a]pyrene-induced DNA adduct formation in the liver of three strains of female mice with identical AhRb2 genotype treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin and/or benzo[a]pyrene.

To search for genes whose products modify aryl hydrocarbon receptor (AhR)-dependent toxicity caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), gene expression profiles in the liver were surveyed using microarrays 24 h after the administration of TCDD to three strains of female mice, BALB/cAnN (BALB), C3H/HeN (C3H) and CBA/JN (CBA) all of identical AhR genotype. The BALB/cAnN strain had a more marked induction of a number of glutathione S-transferase (GST) sub-families, particularly the GSTmicro gene family, compared with the other two strains. To assess the effects of GSTs induction to metabolize carcinogens, TCDD (40 microg kg(-1)) was administered to BALB and CBA strains, followed 24 h later by an i.p. injection of low or high dose of benzo[a]pyrene (B[a]P, 50 or 200 mg kg(-1)). The 32P-postlabelling analysis showed that administration of TCDD alone failed to induce DNA adduct formation in both BALB and CBA strain mouse livers. The low dose of B[a]P alone produced DNA adduct in the liver of both strains to a similar extent. Treatment with TCDD 24 h before the low dose of B[a]P suppressed the formation of B[a]P-induced DNA-adduct more markedly in the BALB strain compared with the CBA strain. Taken together, these findings show that TCDD treatment causes strain-specific alterations in gene expression and B[a]P-induced DNA adduct formation in the liver of female mice of the same AhRb2 genotype. Furthermore, it suggests that TCDD-treated female mice of the BALB strain may have genes whose products modify the toxicity of B[a]P as evidenced by TCDD-induced alterations in B[a]P-DNA adduct formation.

Protective role of metallothionein in bone marrow injury caused by X-irradiation.

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.

Metallothionein is a crucial protective factor against Helicobacter pylori-induced gastric erosive lesions in a mouse model.

Infection with the gastric pathogen Helicobacter 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-kappaB and expression of NF-kappaB-mediated chemokines such as macrophage inflammatory protein-1alpha and monocytes chemoattractant protein-1 in gastric cells were markedly higher in MT-null mice than in wild-type mice. These results suggest that MT in the gastric mucosa might play an important role in the protection against H. pylori-induced gastric ulceration.

Exposure of mouse preimplantation embryos to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters the methylation status of imprinted genes H19 and Igf2.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an extremely toxic, persistent environmental contaminant that disrupts normal development in laboratory animals. In our earlier study, we found that exposure of preimplantation embryos to TCDD markedly induced cytochrome P4501A1 mRNA at the blastocyst stage. In the present study, to determine whether exposure of preimplantation embryos to TCDD affects fetal growth, we exposed preimplantation embryos to TCDD from the 1-cell stage to the blastocyst stage and then transferred them to unexposed recipient mice. On Embryonic Day 14, the fetuses exposed to TCDD during the preimplantation stage weighed less than the fetuses in the unexposed control group. Real-time reverse transcription-polymerase chain reaction analysis revealed that exposure of preimplantation embryos to TCDD tended to decrease the expression levels of the imprinted genes H19 and Igf2 (insulin-like growth factor 2 gene). Use of bisulfite genomic sequencing demonstrated that the methylation level of the 430- base pair H19/Igf2 imprint control region was higher in TCDD- exposed embryos and fetuses than in the controls, and methyltransferase activity was also higher in the TCDD-exposed embryos than in the controls. To our knowledge, the present study is the first to provide evidence that TCDD exposure at the preimplantation stage alters the genomic DNA methylation status of imprinted genes, influences the expression level of imprinted genes, and affects fetal development.

Ultrastructural demonstration of mercury granules in the placenta of metallothionein-null pregnant mice after exposure to mercury vapor.

The placenta plays an important role in the regulation of maternal to fetal transfer of toxic substances, including nonessential metals. Metallothioneins (MTs), which are known to have protective effects against heavy metal toxicity, exist in the placenta, but the exact localization of placental MTs (both MT-I and MT-III) and their physiological role in the placenta exposed to mercury are unclear. The present study was performed to examine the localization of MTs and mercury granules in the placenta of mice exposed to mercury vapor. On gestational day 16, MT-I & II-null and wild-type mice were exposed to mercury vapor at 4.9 to 5.9 mg/m3 for 2 hours. At 24 and 48 hours after exposure, the placentas were examined for mercury distribution (autometallography), MT immunoreactivity, and MT mRNA expression (in situ hybridization). No histological changes were observed in the placentas of either MT-null or wild-type mice. Mercury deposition was demonstrated along the boundary between the junctional zone and the labyrinth zone, as well as in the yolk sac, maternal decidual cells, and labyrinth trophoblasts of both MT-null and wild-type mice. MT-I & -II immunoreactivity, which was confined to wild-type mice, was demonstrated in the yolk sac and decidual cells; mercury was also shown in both structures, suggesting that mercury granules were bound to MTs. MT-III mRNA expression was observed in the yolk sac, decidual cells, and spongiotrophoblasts in both MT-null and wild-type mice. There was, however, no evidence of MT at the boundary between the junctional and labyrinth zones, where substantial mercury deposits were demonstrated. These results suggest that placental MTs and the other unknown molecules may be related to the barrier to the placental transfer of mercury.

Metallothionein deficiency enhances skin carcinogenesis induced by 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate in metallothionein-null mice.

To clarify the physiological role(s) of metallothionein (MT) in carcinogenesis, we studied the susceptibility of MT-null mice to chemically mediated carcinogenesis in the 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced two-stage carcinogenesis model. The MT-null mice were subjected to a single topical application of DMBA (50 or 100 micro g/mouse) and, 1 week later, to promotion with TPA (10 micro g/mouse) twice a week for 20 weeks. At week 21, nearly all of the MT-null mice developed tumors in the skin, in contrast to only 10-40% of wild-type mice. No tumors were observed in MT-null or wild-type mice that were administered TPA alone. By using the PCR-restriction fragment length polymorphism and PCR-single strand conformation polymorphism methods, we found a transversion of A182 to T in codon 61 of c-Ha-ras in the papilloma tissue of MT-null mice and wild-type mice but failed to find any mutations in the c-Ki-ras and c-N-ras genes. In two-stage skin carcinogenesis induction by DMBA/TPA, p53 and p21(WAF1/Cip1) expression levels were found to be increased in MT-null mice compared with wild-type mice. As to an earlier change at the promotion stage triggered by TPA application, MT-null mice were found to have both hyperplasia of the epithelium and a marked degree of inflammation in the basal layer, indicating that the induced as well as endogenous MT acted as a protective factor against tumorigenesis. In conclusion, the present study has demonstrated that MT has antitumorigenic potential in both the initiation and promotion stages of the two-stage chemical skin carcinogenesis model.