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The generation and collection of cigarette smoke (CS) is a prerequisite for any toxicology study on smoking, especially an in vitro CS exposure study. In this study, the effects on blood and vascular function were tested with two widely used CS preparations to compare the biological effects of CS with respect to the CS preparation used. CS was prepared in the form of total particulate matter (TPM), which is CS trapped in a Cambridge filter pad, and cigarette smoke extract (CSE), which is CS trapped in phosphate-buffered saline. TPM potentiated platelet reactivity to thrombin and thus increased aggregation at a concentration of 25~100 μg/mL, whereas 2.5~10% CSE decreased platelet aggregation by thrombin. Both TPM and CSE inhibited vascular contraction by phenylephrine at 50~100 μg/mL and 10%, respectively. TPM inhibited acetylcholine-induced vasorelaxation at 10~100 μg/mL, but CSE exhibited a minimal effect on relaxation at the concentration that affects vasoconstriction. Neither TPM nor CSE induced hemolysis of erythrocytes or influenced plasma coagulation, as assessed by prothrombin time (PT) and activated partial thromboplastin time (aPTT). Taken together, CS affects platelet activity and deteriorates vasomotor functions in vitro . However, the effect on blood and blood vessels may vary depending on the CS preparation. Therefore, the results of experiments conducted with CS preparations should be interpreted with caution.

Metallothioneins (MTs) are proteins that protect cells from toxic agents such as heavy metal ions or reactive oxygen species. MT2A A-5G is a single nucleotide polymorphism in the promoter region of the MT2A gene, and the minor G allele results in lower transcription efficiency. We aimed to elucidate associations between MT2A A-5G and risks of 2 diseases potentially related to lowered MT expression, chronic kidney disease (CKD), and diabetes mellitus (DM), in a community-dwelling population. Study subjects were Nagoya city residents participating in the Japan Multi-Institutional Collaborative Cohort Study (J-MICC) Daiko Study, comprised 749 men and 2,025 women, aged 39-75 years. CKD (>stage 3) and DM were defined by standard guidelines. Associations were evaluated using logistic regression models with adjustments for age, sex and potential confounders in a cross-sectional study, and verified in a 5-year longitudinal study. Odds ratios (OR [95% confidence interval]) were calculated relative to the AA genotype. Serum MT (I + II), Cd and zinc levels were also determined by genotype. The OR of the GG genotype for CKD risk was 3.98 (1.50, 10.58) in the cross-sectional study and 5.17 (1.39, 19.28) in the longitudinal study. The OR of the GA genotype for DM was 1.86 (1.26, 2.75) in the cross-sectional study and 2.03 (1.19, 3.46) in the longitudinal study. MT2A A-5G may be associated with CKD and DM risks. This polymorphism is a promising target for evaluations of CKD and DM risks with possible involvement of low-dose chronic exposure to environmental pollutants., (© The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology.)

Metallothionein (MT) plays a central role in cellular defense against heavy metals and oxidative stress. Since the induction of MT requires the activation of metal response element (MRE)-binding transcription factor-1 (MTF-1) by binding of zinc ions, inorganic zinc is regarded as a typical MT inducer. However, in a previous report, we showed that inorganic zinc could not induce MT in vascular endothelial cells. While it is suggested that endothelial MT presents mechanisms different from those of other cell types, these remain unclear. In this study, we investigated whether the induction of endothelial MT expression involves the Nrf2-ARE pathway using copper(II) bis(diethyldithiocarbamate), termed Cu10, using a culture system of bovine aortic endothelial cells. Cu10 induced MT-1/2 protein expression and increased the expression of mRNAs for MT-1A, MT-1E, and MT-2, MT isoforms expressed in the cells. Cu10 activated not only the MTF-1-MRE, but also the Nrf2-ARE pathway. MTF-1 knockdown resulted in the repression of Cu10-induced MT-1 and -2 expression. Cu10-induced MT-1 expression was down-regulated by Nrf2 knockdown. However, MT-2 expression was not affected by Nrf2 knockdown. These results suggest that the expression of endothelial MT is up-regulated by the Nrf2-ARE pathway as well as by the MTF-1-MRE pathway. Moreover, MT-1 regulation mechanisms differ from that of MT-2. Specifically, the present data support the hypothesis that MT-1 participates in the biological defense system, while MT-2 mainly regulates intracellular zinc metabolism.

Rhus verniciflua Stokes (RVS) exert cardiovascular protective activity by promoting blood circulation, but its active ingredients and underlying mechanism have yet to be identified. This study investigated the vascular effects of RVS, focusing on vasoconstriction and smooth muscle Ca(2+) signaling. RVS heartwood extract attenuated contraction of aortic rings induced by the vasoconstrictors serotonin and phenylephrine, and inhibited the Ca(2+) signaling evoked by serotonin in vascular smooth muscle cells. Subsequent activity-guided fractionation identified fisetin as an active constituent exerting a Ca(2+) inhibitory effect. Fisetin could inhibit major Ca(2+) mobilization pathways including extracellular Ca(2+) influx mediated by the L-type voltage-gated Ca(2+) channel, Ca(2+) release from the intracellular store and store-operated Ca(2+) entry. In accordance with Ca(2+) inhibitory effect, fisetin attenuated vasoconstriction by serotonin and phenylephrine. These results suggest that the anticontractile effect, which is presumably mediated by inhibition of Ca(2+) signaling, may contribute to the improvement of blood circulation by RVS.

Chronic exposure to cadmium (Cd) is known to adversely affect renal function. Our previous studies indicated that Cd induces p53-dependent apoptosis by inhibiting gene expression of the ubiquitin-conjugating enzyme (Ube) 2d family in both human and rat proximal tubular cells. In this study, the effects of Cd on protein expression of p53 and apoptotic signals in the kidney and liver of mice exposed to Cd for 12 months were examined, as well as the effects of Cd on p53 protein levels and gene expression of the Ube2d family in various cell lines. Results showed that in the kidney of mice exposed to 300 ppm Cd for 12 months, there was overaccumulation of p53 proteins in addition to the induction of apoptosis, which was triggered specifically in the proximal tubules. Interestingly, the site of apoptosis was the same as that of p53 accumulation in the proximal tubules. In the liver of mice chronically exposed to Cd, gene expression of the Ube2d family tended to be slightly decreased, together with slight apoptosis without the accumulation of p53 protein. In rat small intestine epithelial (IEC-6) cells, Cd decreased not only the p53 protein level but also gene expression of Ube2d1, Ube2d2 and Ube2d4. In human brain microvascular endothelial cells (HBMECs), Cd did not suppress gene expression of the Ube2d family, but increased the p53 protein level. In human brain astrocytes (HBASTs), Cd only increased gene expression of UBE2D3. These results suggest that Cd-induced apoptosis through p53 protein is associated with renal toxicity but not hepatic toxicity, and the modification of p53 protein by Cd may vary depending on cell type.

Cadmium (Cd) is a toxic heavy metal with a long half-life in humans. It causes disorders of various tissue systems, including the kidney, and is associated with protein aggregation. Our previous study demonstrated Cd-induced suppression of the UBE2D gene family, one of the ubiquitin-conjugating enzyme families. However, the precise role of ubiquitin-coding genes in Cd toxicity remains to be understood. In this study, we investigated the effect of Cd on expression of the ubiquitin-coding genes UBB, UBC, UBA80, and UBA52 in HK-2 human proximal tubular cells. Prior to the appearance of Cd toxicity, the UBB, UBC, and UBA80 expression levels increased following Cd treatment. Knockdown of UBB by siRNA transfection significantly decreased Cd cytotoxicity. Notably, Cd induces ubiquitinated protein levels in HK-2 cells, and knockdown of UBB blocked this process. These results suggest that UBB is involved in Cd-induced increase of protein ubiquitination, and that accumulation of ubiquitinated proteins through increased UBB expression may contribute to Cd toxicity in HK-2 cells.

Purpose: The incidence of blindness is increasing, in part, because of abnormal ocular neovascularization. Anti-VEGF therapies have yielded impressive results; however, they are not a cure for blindness. Recently, metallothioneins (MTs) 1 and 2 have been implicated in the process of angiogenesis. Therefore, we investigated whether MT-1 and MT-2 were also involved in ocular neovascularization., Methods: The concentrations of MT-1 and MT-2 (hereafter MT-1/2) were observed by ELISA. We examined the role of MT-1/2 in ocular neovascularization by using both an oxygen-induced retinopathy (OIR) model and a laser-induced choroidal neovascularization (CNV) model. We investigated the localization of MT-1/2 in retina. Furthermore, we investigated the expression of hypoxia-inducible factor (HIF)-1α and VEGF in OIR. In vitro, we investigated the degradation of HIF-1α., Results: The MT-1/2 were significantly elevated in proliferative diabetic retinopathy patients. Ocular neovascularization, which was induced in both the OIR model and the CNV model, was decreased in MT-1/2 knockout (KO) mice. We confirmed that although MT-1/2 was expressed throughout the murine retina, its expression levels were highest in the endothelial cells. Further, OIR enhanced MT-1/2 expression in the retina. Interestingly, in the OIR model, both HIF-1α and VEGF levels were significantly decreased in the retina of MT-1/2 KO mice. In addition, we found that knockdown of MT-1/2 accelerated ubiquitination of HIF-1α., Conclusions: These results indicate that MT-1/2 are involved in retinal and choroidal neovascularization, and that MT-1/2 might be a new therapeutic target in diseases in which ocular angiogenesis is implicated., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

This study examined the relationship between neurobehavioral changes and alterations in gene expression profiles in the brains of mice exposed to different levels of Hg(0) during postnatal development. Neonatal mice were repeatedly exposed to mercury vapor (Hg(0)) at a concentration of 0.057 mg/m(3) (low level), which was close to the current threshold value (TLV), and 0.197 mg/m(3) (high level) for 24 hr until the 20(th) day postpartum. Behavioral responses were evaluated based on changes in locomotor activity in the open field test (OPF), learning ability in the passive avoidance response test (PA), and spatial learning ability in the Morris water maze (MM) at 12 weeks of age. No significant differences were observed in the three behavioral measurements between mice exposed to the low level of Hg(0) and control mice. On the other hand, total locomotive activity in mice exposed to the high level of Hg(0) was significantly decreased and central locomotion was reduced in the OPF task. Mercury concentrations were approximately 0.4 μg/g and 1.9 μg/g in the brains of mice exposed to the low and high levels of Hg(0), respectively. Genomic analysis revealed that the expression of 2 genes was up-regulated and 18 genes was down-regulated in the low-level exposure group, while the expression of 3 genes was up-regulated and 70 genes was down-regulated in the high-level exposure group. Similar alterations in the expression of seven genes, six down-regulated genes and one up-regulated gene, were observed in both groups. The results indicate that an increase in the number of altered genes in the brain may be involved in the emergence of neurobehavioral effects, which may be associated with the concentration of mercury in the brain. Moreover, some of the commonly altered genes following exposure to both concentrations of Hg(0) with and without neurobehavioral effects may be candidates as sensitive biomarker genes for assessing behavioral effects in the early stages of development.

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

Cadmium is a toxic heavy metal that causes severe clinical symptoms in various tissues including the kidney. In this study, activities of transcription factors were measured to identify what type of transcription factor was affected by cadmium in rat proximal tubular cells (NRK-52E cells) using the protein/DNA binding assay. After treatment of NRK-52E cells with 5 µM CdCl2 for 3 hr, nuclear extracts were used for the protein/DNA binding assay. Among 65 transcription factors, cadmium increased the activities of 6 transcription factors by more than 2.0-fold and decreased those of 15 transcription factors by less than 0.5-fold. These findings may provide new information about novel transcription factors associated with the mechanism of cadmium toxicity.

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.

Cadmium (Cd) is a toxic heavy metal and chronic exposure causes kidney injury. This study used DNA microarray analysis to examine gene expression in the kidney of mice chronically exposed to Cd. Female C57BL/6J mice were fed a 300 ppm Cd-containing diet or a control diet for 12 months. In comparison with control mice, the expression levels of 32 genes, including Hmox1 and Mt2, were elevated more than 2.0-fold, whereas 113 genes, including transport- and ubiquitination-related genes, were reduced less than 0.5-fold.

Metallothionein (MT) is a low molecular weight metal-binding protein induced by endogenous and exogenous stimuli such as cytokines and heavy metals. In 1993 and 1994, two research groups (Choo et al. and Palmiter et al., respectively) produced MT-I/II double-knockout mice (MT-I/II null mice) with null mutations of the MT-I and MT-II genes. Subsequently, MT-I/II null mice have been used to clarify the biological function, physiological role, and pathophysiological relevance of MT by many research groups. Recent studies using MT-I/II null mice to investigate the role of MT in metal toxicity and distribution, oxidative stress, and some disease were reviewed. In addition, several research groups including our laboratory have reported that MT-I/II null mice are highly susceptible to several carcinogenesis caused by 7,12-dimethylbenz[a]anthracene, X-ray, benzo[a]pyrene, N-butyl-N-(4-hydroxybutyl) nitrosamine, lead, and cisplatin. These results suggest that MT is an important protective factor against not only metal toxicity and oxidative stress but also chemical and radiation carcinogenesis. In this review, we present the findings of MT-I/II null mice with regard to the protective role of MT in carcinogenesis and mutagenesis caused by chemical agents and X-ray.

Purpose: Retinal photoreceptor damage is a common feature of ophthalmic disorders, such as age-related macular degeneration and retinitis pigmentosa. Oxidative stress has a key role in these diseases. Metallothioneins (MTs) are a family of cysteine-rich proteins, and various physiologic functions have been reported, including protection against metal toxicity and antioxidative potency. We investigated the functional role of MT-III in light-induced retinal damage., Methods: The expression of retinal MT-I, -II, and -III mRNA was evaluated by real-time reverse-transcription PCR in retina exposed to light. Retinal damage in MT-deficient mice was induced by exposure to white light at 16,000 lux for 3 hours after dark adaptation. Photoreceptor damage was evaluated histologically by measuring the thickness of the outer nuclear layer (ONL) 5 days after light exposure and by electroretinogram recording. In an in vitro experiment, the MT-III siRNAs were tested for their effects on light-induced mouse photoreceptor cell (661W) damage., Results: The mRNAs of the MTs were increased significantly in murine retina after light exposure. The ONL in the MT-III-deficient mice was remarkably thinner compared to light-exposed wild-type (WT) mice, and a- and b-wave amplitudes were decreased; the damage induced in MT-I/-II-deficient mice was comparable to that observed in WT mice. MT-III knockdown by siRNA in 661W exacerbated the cell damage and increased the production of reactive oxygen species in response to light exposure., Conclusions: These findings suggested that MT-III can help protect against light-induced retinal damage compared to MT-I/II. Some of these effects may be exerted by its antioxidative potency.

Cadmium is a nonessential heavy metal and ubiquitous potential environmental pollutant. Although the kidney proximal tubule is an important target for cadmium, the underlying cellular mechanisms of cadmium-induced renal toxicity remain elusive. Numerous studies have demonstrated that cadmium induces apoptotic cell death in various cell types via several apoptotic pathways, including mitochondria-mediated apoptotic cell death. In the epithelial cells of renal proximal tubules, cadmium can also induce apoptotic cell death in vivo and in vitro, which suggests that cell death of the epithelial cells through the apoptotic pathways is one of the key events in cadmium-induced renal toxicity. In this review, based upon the major findings of previous reports related to cadmium and apoptotic cell death, especially in the kidney and kidney proximal tubular cells, we present evidence for the current mechanisms of cadmium-induced renal toxicity via apoptotic cell death.

To examine the contribution of metallothionein (MT) to mercury accumulation in mouse tissues, 129 strain female mice and MT null mice were exposed to metallic mercury vapor at a sub-toxic level, and Hg levels in the brain, kidney and liver were determined on 1, 3 and 7 days after the exposure. After exposure to mercury vapor, significant Hg accumulation was observed in the brains of wild-type and MT-I/II null and MT-III null mice, as well as in the liver and kidneys. No strain difference was observed in the tissue Hg accumulations 24 hr after the exposure except for the kidneys, where the highest accumulation was found in MT-III null mice. Although the brains of MT-III null mice showed slightly higher Hg accumulation than the other two strains, no significant difference was observed except in the cerebrum on Day 7. Gel chromatograms of cerebrum soluble fractions revealed that a significant amount of Hg existed as an MT-bound form in all the mouse strains. On the other hand, MT-bound Hg was found as a minor fraction in soluble fractions of the kidneys and livers in wild-type and MT-III null mice. Despite a significant strain difference in total MT levels in the cerebrum, there was no difference among the three strains in the amount of Hg accumulated in the cerebrum and its distribution rates in MT fractions. The present study demonstrated that brain uptake of Hg(0) and its accumulation as Hg(2+) did not depend on the amount of MT isoform in the tissue, at least in the early phase.

Cadmium (Cd), a harmful metal, exerts severe toxic effects on various tissues such as those in the kidney, liver, lung, and bone. In particular, renal toxicity with damage to proximal tubule cells is caused by chronic exposure to Cd. However, the molecular mechanism underlying chronic Cd renal toxicity remains to be understood. In this review, we present our recent findings since we examined to search for the target molecules involved in the renal toxicity of Cd using toxicogenomics. In NRK-52E rat renal tubular epithelial cells, we found using DNA microarrays that Cd suppressed the expression of the gene encoding Ube2d4, a member of the Ube2d family. The Ube2d family consists of selective ubiquitin-conjugating enzymes associated with p53 degradation. Moreover, Cd suppressed the expressions of genes encoding all Ube2d family members (Ube2d1/2/3/4) prior to the appearance of cytotoxicity in NRK-52E cells. Cd markedly increased p53 protein level and induced p53 phosphorylation and apoptosis in the cells. In vivo studies showed that chronic Cd exposure also suppressed Ube2d family gene expression and induced p53 accumulation and apoptosis in the renal tubules of the mouse kidney. These findings suggest that Cd causes p53-dependent apoptosis due to the inhibition of p53 degradation through the down-regulation of Ube2d family genes in NRK-52E cells and mouse kidney. Thus, the Ube2d family genes may be one of the key targets of renal toxicity caused by Cd.

Metallothionein (MT), a low-molecular-weight protein with a high affinity for divalent heavy metal ions, is involved in many pathophysiological processes, including metal homeostasis, detoxification, cell proliferation and protection against oxidative damage. We previously found that MT in gastric mucosa plays a role in protecting against Helicobacter pylori (H. pylori)-induced gastritis at the early stage of infection. H. pylori-induced chronic gastric inflammation is shown to be associated with gastric carcinogenesis. Thus, to examine whether gastric MT contributes to protection against H. pylori-induced chronic inflammation, we compared histological changes in the gastric mucosa of MT-null and the wild-type mice at 53 weeks after inoculation three times with H. pylori SS1. As a result, we observed disruption of the gastric mucosa in MT-null mice, but not in the wild-type mice, even at the late stage of H. pylori-infection. Evaluation of pathological changes in gastric specimens by the updated Sydney grading system revealed that scores related to chronic inflammation and polymorphonuclear cell activity were higher in infected MT-null mice than those in the wild-type mice. Furthermore, a higher score for metaplasia was also observed in the MT-null stomach. These results suggested that MT might be involved in protecting against H. pylori-induced gastric chronic inflammation associated with carcinogenesis.

Noninvasive growth forming macroscopically dilated cystic pancreatic ducts is a fundamental feature of intraductal papillary mucinous neoplasm (IPMN), from which invasive carcinomas can arise. However, some invasive ductal carcinomas of the pancreas also show a macroscopically cystic feature. We experienced 2 cases of invasive ductal carcinoma of the pancreas tail with noninvasive growth through the main pancreatic duct without dilation at the body side, and with collection of macroscopically cystic carcinomatous glands infiltrating at the spleen side, which resembled some IPMNs and/or IPMN-derived invasive carcinomas. These cases were different from IPMN in that they lacked macroscopic dilatation of the pancreatic ducts, and the macroscopically dilated cystic carcinomatous glands were invasive but not intraductal. The intraductal component of the carcinomas showed papillary growth of neoplastic epithelia with atypia consistent with PanIN-3. Both intraductal and invasive components predominantly showed gastric mucin phenotype (MUC5AC+, MUC6 focally +, MUC2- or MUC2+ in scattered small number of cells). Recognition of these pancreatic carcinoma cases is important in the following 2 points: (1) The presence of such cases should always be kept in mind as differential diagnosis of IPMN or IPMN-derived invasive carcinoma in imaging and pathologic diagnoses. (2) The histogenesis of these cases might be placed in the intermediate between 2 major histogenetic pathways of pancreatic carcinoma, that is, one from microscopic precursors called PanIN and the other from macroscopic precursors of IPMN. These cases can be regarded as invasive carcinomas derived from semimacroscopic extension of the intraductal lesion of the main pancreatic duct., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Cilostazol, an antiplatelet drug, exhibits antiatherogenic effects. The purpose of the present study was to determine the effect of cilostazol on the cytotoxicity of cadmium (Cd) and arsenite (iAs(III)), which involved in the pathogenesis of vascular disorders such as atherosclerosis, in cultured vascular endothelial cells. Cytotoxicity was evaluated by the lactate dehydrogenase leakage assay and morphological observation. Cd (10 µM) -induced cytotoxicity was prevented by pretreatment with cilostazol (30 and 100 µM) and simultaneous treatment with cilostazol (100 µM). On the other hand, iAs(III)-induced cytotoxicity was blocked by pretreatment with cilostazol (30 and 100 µM) but not simultaneous treatment with cilostazol. The mRNA level and the protein level of metallothionein (MT) were significantly increased by cilostazol in the cells. These results suggested, therefore, that pretreatment with cilostazol effectively prevents the cytotoxicity of Cd and iAs(III) in cultured vascular endothelial cells, at least in part through the induction of MT synthesis.

Cadmium (Cd) is a toxic heavy metal that has been shown to induce vascular diseases, such as atherosclerosis. We used DNA microarray to monitor the transcriptional response of human coronary artery endothelial cells (HCAEC) to a non-lethal dose of Cd (10 µM). Out of 35,035 human genes, Cd enhanced the expression of 3 metallothionein (MT)-I subisoform genes, including MT1E, MT1H and MT1B, and reduced the expression of 12 genes, including ISG20 and TK1, 2-fold or greater.

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.

Two research groups produced metallothionein (MT)-I/II knockout mice with null mutation of MT-I and MT-II genes. In 1993, Choo et al. produced MT-I/II knockout mice with a mixed genetic background of 129 Ola and C57BL/6 strains. Palmiter et al. also produced MT-I/II knockout mice with a genetic background of 129/Sv strain in 1994. Subsequently, MT-I/II knockout mice have been used to clarify the biological function and physiological role of MT by many research groups. We were also provided MT-I/II knockout mice from Dr. Choo (Australia). F1 hybrid mice were mated with C57BL/6, and their offspring were back-crossed to C57BL/6 for ten generations. MT-I/II knockout (MT(-/-)) mice and wild-type (MT(+/+)) mice were obtained by mating of those heterozygous (MT(+/-)) mice. We have been investigating the susceptibility of MT-I/II knockout mice to toxicity of harmful factors and some diseases. Our present studies found that MT-I/II knockout mice have an increased sensitivity to harmful metals such as cadmium, mercury, and arsenic, oxidative stress, chemical carcinogenesis and neurodegenerative diseases. These results clearly indicate that MT plays an important role in defense of these toxicities. In this review, we present our findings and summarize recent reports with MT-I/II knockout mice concerning the role of MT as a biological protective factor.

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.

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.

Purpose: The metal binding protein metallothionein (MT) confers drug resistance when MT is induced in tumor tissues. Cisplatin is known to induce MT synthesis in tumor tissues, which may lead to drug resistance. We examined whether a difference in the administration schedule of cisplatin affect the efficiency of MT induction., Materials and Methods: Balb/c nude mice were inoculated with NMB-1 human bladder tumor tissues and injected with cisplatin (total dose of 64 micromol/kg) in a single injection or fractioned daily injections. Tumor MT concentration was determined 24 hours after the last injection of cisplatin by mercury binding assay. The effect of pretreatment with ZnCl2 on antitumor activity of cisplatin was examined in NMB-1 bearing mice., Results: Tumor MT levels increased significantly with the increase in the number of cisplatin injections. Pretreatment of NMB-1 bearing mice with ZnCl2 (200 micromol/kg x 2) caused the same level of MT induction (1.6-fold) as that of fractioned injections of cisplatin (4 x 16 micromol/kg). Pretreatment of NMB-1 bearing mice with ZnCl2 (200 micromol/kg x 2) depressed cisplatin antitumor activity by about 50%., Conclusions: The induction of MT to a moderate extent (1.6-fold) in NMB-1 tumor inoculated in mice conferred cisplatin resistance. This level of MT induction can be achieved by fractioned daily injections of cisplatin but not by a single injection. Therefore, it is preferable to administer cisplatin as a single injection rather than as fractioned injections to achieve effective antitumor activity with minimum MT induction.

Single-strand DNA interruptions (SSIs) are produced during the process of base excision repair (BER). Through biochemical studies, two SSI repair subpathways have been identified: a pathway mediated by DNA polymerase beta (Pol beta) and DNA ligase III (Lig III), and a pathway mediated by DNA polymerase delta/epsilon (Pol delta/epsilon) and DNA ligase I (Lig I). In addition, the existence of another pathway, mediated by Pol beta and DNA Lig I, has been suggested. Although each pathway may play a unique role in cellular DNA damage response, the functional implications of SSI repair by these three pathways are not clearly understood. To obtain a better understanding of the functional relevance of SSI repair by these pathways, we investigated the involvement of each pathway by monitoring the utilization of DNA ligases in cell-free extracts. Our results suggest that the majority of SSIs produced during the repair of alkylated DNA bases are repaired by the pathway mediated by Pol beta and either Lig I or Lig III, although some SSIs are repaired by Pol delta/epsilon and Lig I. At a cellular level, we found that Lig III over-expression increased the resistance of cells to DNA-damaging agents, while Lig I over-expression had little effect. Thus, repair pathways mediated by Lig III may have a role in the regulation of cellular sensitivity to DNA-damaging agents.

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.

We reviewed studies on genotoxicity and carcinogenicity of cadmium (Cd). Salmonella typhimurium and Escherichia coli exposed to Cd did not show mutagenicity, whereas cultured mammalian cells exposed to Cd showed mutation, DNA strand breaks, and chromosomal aberrations. Carcinogenicity tests showed that exposure to Cd increased the occurrence of tumors in testis, lung, prostate, hematopoietic tissues, and injection sites. On the other hand, recent epidemiologic studies are not supportive of earlier observations on the association between Cd and prostate cancer. The US NIOSH data on a possible association between Cd and lung cancer may need reevaluation. No studies which show a positive relationship between oral Cd exposure and carcinogenesis have been reported. All available data suggest that Cd should be reassigned to IARC Group 2A (probably carcinogenic to humans) from the current Group 1.

We have reviewed earlier studies on the possible involvement of cadmium in life-cycle related diseases and the reproductive toxicity of Cd including environmental disrupting actions. Experimental studies have suggested that Cd may be involved in the aggravation of life-cycle related diseases and the occurrence of reproductive toxicity. On the other hand, epidemiological studies did not necessarily support the experimental observations. Thus, we conclude that it is necessary to investigate further to determine whether Cd is responsible for the aggravation of life-cycle related diseases or has the capability to act as an environmental endocrine disrupter in humans.

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 (Hg(0)) vapor. Both strains were exposed to Hg(0) vapor at 5.5-6.7 mg/m(3) for 3 h during late gestation. Twenty-four hours after exposure to Hg(0) 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 Hg(0) 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.