MT-null Mice Research Articles

Metallothionein I and II Protect against Zinc Deficiency and Zinc Toxicity in Mice

Metallothionein (MT)-bound zinc accumulates when animals are exposed to excess zinc and is depleted under conditions of zinc deficiency, suggesting that MT serves as a means of sequestering excess zinc as well as a zinc reservoir that can be utilized when zinc is deficient. To examine the importance of MT for these processes, mice with null alleles of both MT I and MT II genes were created and the zinc concentration and histological appearance of multiple organs assessed. At birth, the hepatic zinc concentration of these MT-null mice was lower than that of wild-type controls (0.27 ± 0.02 vs. 0.65 ± 0.11 µmol zinc/g tissue, P < 0.05). During the next 3 wk of suckling zinc-replete (95 µg zinc/g diet) dams, the hepatic zinc concentration of controls fell to 0.42 ± 0.04 µmol/g but was unchanged in the MT-null mice (0.28 ± 0.04 µmol/g). The most prominent histological anomaly observed at 3 wk of age was the presence of swollen Bowman's capsules in the kidneys of MT-null mice. When nursing MT-null dams were fed a severely zinc-deficient (1.5 µg/g) diet, kidney development in the MT-null pups was retarded as indicated by the retention of the nephrogenic zone and incomplete tubule development. We suggest that the lack of a hepatic reservoir of zinc jeopardizes the developing kidney in the MT-null mice. In addition to being more sensitive to dietary zinc restriction, MT-null mice are more sensitive to zinc toxicity. When adult mice were challenged with a ramping dose of zinc up to a total of 3700 µmol zinc/kg body weight, MT-null mice had a greater incidence of pancreatic acinar cell degeneration compared with control mice despite accumulating less zinc (2.72 ± 0.46 vs. 1.23 ± 0.52 µmol zinc/g pancreas, control and MT-null, respectively, P < 0.05). The results of these experiments suggest that MT I and MT II can protect against both zinc deficiency and zinc toxicity.

Protective Effects of Metallothionein on Isoniazid and Rifampicin-Induced Hepatotoxicity in Mice

Isoniazid (INH) and Rifampicin (RFP) are widely used in the world for the treatment of tuberculosis, but the hepatotoxicity is a major concern during clinical therapy. Previous studies showed that these drugs induced oxidative stress in liver, and several antioxidants abated this effect. Metallothionein (MT), a member of cysteine-rich protein, has been proposed as a potent antioxidant. This study attempts to determine whether endogenous expression of MT protects against INH and RFP-induced hepatic oxidative stress in mice. Wild type (MT+/+) and MT-null (MT−/−) mice were treated intragastrically with INH (150 mg/kg), RFP (300 mg/kg), or the combination (150 mg/kg INH +300 mg/kg RFP) for 21 days. The results showed that MT−/− mice were more sensitive than MT+/+ mice to INH and RFP-induced hepatic injuries as evidenced by hepatic histopathological alterations, increased serum AST levels and liver index, and hepatic oxidative stress as evidenced by the increase of MDA production and the change of liver antioxidant status. Furthermore, INH increased the protein expression of hepatic CYP2E1 and INH/RFP (alone or in combination) decreased the expression of hepatic CYP1A2. These findings clearly demonstrate that basal MT provides protection against INH and RFP-induced toxicity in hepatocytes. The CYP2E1 and CYP1A2 were involved in the pathogenesis of INH and RFP-induced hepatotoxicity.

Protective effect of cepharanthin on cisplatin-induced renal toxicity through metallothionein expression

AimsCisplatin (CDDP) is a potent anticancer agent, but severe renal toxicity can limit its use. We investigated the protective effect of cepharanthin (CE), a biscoclaurin alkaloid, on the renal toxicity of CDDP. Main methodsMice were given CDDP along with CE. Effects of CE on CDDP toxicity were investigated by assaying markers of renal toxicity together with MT expression, and by histopathological examination of the kidney. MT-null mice were also examined. Key findingsCE induced expression of metallothionein (MT). Pre-administration of CE attenuated an increase in blood urea nitrogen (BUN) concentrations after the CDDP injection. A histochemical analysis demonstrated protection against CDDP-induced necrocytosis of kidney tissues by CE. The protective effect of CE did not occur in the MT-null mice. SignificancePretreatment with CE may reduce the renal toxicity of CDDP through expression of MT.

Preventive effects of metallothionein against DNA and lipid metabolic damages in dyslipidemic mice under repeated mild stress

The effects of repeated mild stress on DNA and lipid metabolic damages in multiple organs of dyslipidemic mice, and the preventive role of metallothionein (MT) were investigated. Female adult wild-type and MT-null mice fed high-fat diet (HFD) or standard diet (STD) were repeatedly subjected to fasting or restraint for three weeks. The liver, pancreas, spleen, bone marrow and serum samples were taken for evaluating DNA damage, MT, glutathione (GSH), corticosterone, carnitine and adiponectin. Body weights of restraint groups were reduced with the intensity of stress increased, even if the energy intakes were higher than those of STD group. Hepatic GSH levels were reduced in HFD control group and were further reduced in stress groups, especially in restraint groups, while the hepatic MT and serum corticosterone levels were increased in concert with the intensity of stress. Cellular DNA damages were generally increased by the restraint stress, especially in MT-null mice. Hepatic carnitine levels of MT-null mice were markedly lower than those of wild-type mice. The data suggest that MT plays a preventive role by acting as an antioxidant in corporation with GSH decreased by repeated stress and that MT may be an essential factor for inducing carnitine under the stress.

Protective effect of naringenin against acetaminophen-induced acute liver injury in metallothionein (MT)-null mice

Naringenin is a natural flavonoid aglycone of naringin that has been reported to have a wide range of pharmacological properties, such as antioxidant activity and free radical scavenging capacity. This study was designed to examine the hepatoprotective effect of naringenin against acetaminophen (250 mg kg(-1), sc) in metallothionein (MT)-null mice. 42 SPF MT-knockout mice were used. Naringenin (200, 400, and 800 mg kg(-1), ig) was administered for 4 days before exposure to acetaminophen (250 mg kg(-1), sc). Liver injury was measured by serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH), as well as liver malondialdehyde (MDA). The glutathione-to-oxidized glutathione ratio (GSH/GSSG) was also assessed. The evidence of liver injury induced by acetaminophen included not only a significant increase in the levels of serum ALT, AST, LDH and liver MDA, and also a significant decrease in GSH/GSSG. Pretreatment of mice with naringenin at 400 and 800 mg kg(-1) reversed the altered parameters. Such reversal effects were dose-dependent: ALT decreased 78.62% and 98.03%, AST decreased 88.35% and 92.64%, LDH decreased 76.54% and 81.63%, MDA decreased 48.59% and 66.27% at a dose of 400 and 800 mg kg(-1) respectively; GSH/GSSG increased 22.57% and 16.93% at a dose of 400 and 800 mg kg(-1) respectively. Histopathological observation findings were also consistent with these effects. Together, this study suggests that naringenin can potentially reverse the hepatotoxic damage of acetaminophen intoxication in MT-null mice.

Metallothionein-induced zinc partitioning exacerbates hyperoxic acute lung injury

Hypozincemia, with hepatic zinc accumulation at the expense of other organs, occurs in infection, inflammation, and aseptic lung injury. Mechanisms underlying zinc partitioning or its impact on extrahepatic organs are unclear. Here we show that the major zinc-binding protein, metallothionein (MT), is critical for zinc transmigration from lung to liver during hyperoxia and preservation of intrapulmonary zinc during hyperoxia is associated with an injury-resistant phenotype in MT-null mice. Particularly, lung-to-liver zinc ratios decreased in wild-type (WT) and increased significantly in MT-null mice breathing 95% oxygen for 72 h. Compared with female adult WT mice, MT-null mice were significantly protected against hyperoxic lung injury indicated by reduced inflammation and interstitial edema, fewer necrotic changes to distal airway epithelium, and sustained lung function at 72 h hyperoxia. Lungs of MT-null mice showed decreased levels of immunoreactive LC3, an autophagy marker, compared with WT mice. Analysis of superoxide dismutase (SOD) activity in the lungs revealed similar levels of manganese-SOD activity between strains under normoxia and hyperoxia. Lung extracellular SOD activity decreased significantly in both strains at 72 h of hyperoxia, although there was no difference between strains. Copper-zinc-SOD activity was ~4× higher under normoxic conditions in MT-null compared with WT mice but was not affected in either group by hyperoxia. Collectively the data suggest that genetic deletion of MT-I/II in mice is associated with compensatory increase in copper-zinc-SOD activity, prevention of hyperoxia-induced zinc transmigration from lung to liver, and hyperoxia-resistant phenotype strongly associated with differences in zinc homeostasis during hyperoxic acute lung injury.

Metallothionein deficiency exacerbates chronic inflammation associated with carcinogenesis in stomach of mice infected with &lt;i&gt;Helicobacter pylori&lt;/i&gt;

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.

Early life inorganic lead exposure induces testicular teratoma and renal and urinary bladder preneoplasia in adult metallothionein-knockout mice but not in wild type mice

Inorganic lead compounds are carcinogenic in animals and have carcinogenic potential in humans. In mice, lead (Pb) is a transplacental carcinogen in the kidney. Metallothionein (MT) is a metal-binding protein that can reduce the toxicity of various metals, including Pb, either by direct sequestration or as an antioxidant for metals that generate reactive oxygen species. Although MT appears to reduce Pb carcinogenicity in adult mice it is unknown how MT deficiency may affect Pb carcinogenicity from early life exposure. Thus, groups ( n = 10) of pregnant MT-I/II double knockout (MT-null) or 129/SVJ MT wild type (WT) mice were exposed to Pb acetate in the drinking water (0, 2000, 4000 ppm Pb) from gestation day 8 through birth and during lactation. Maternal drinking water Pb exposure continued to wean at 4 weeks of age and the male offspring were then directly exposed to Pb until 8 weeks of age and observed until 2 years old. High dose (4000 ppm) but not low dose (2000 ppm) Pb reduced survival in the latter part of the study in both MT-null and WT mice. In MT-null mice, but not WT, early life Pb exposure caused a dose-related increase in testicular teratomas, to a maximum incidence of 28% compared to control (4%). Pb-induced renal cystic hyperplasia, considered preneoplastic, was a prominent occurrence in MT-null mice but nearly absent in WT mice. Pb dose-related increases in renal cystic hyperplasia occurred in adult MT-null with early life exposure with maximal incidence of 52%. Pb-treated MT-null mice also showed dose-related increases in urinary bladder hyperplasia with occasional papilloma that were absent in WT mice. Thus, MT deficiency made mice more sensitive to early life Pb exposure with regard to testes tumors, and renal and urinary bladder preneoplastic lesions.

Potential role of alpha-synuclein and metallothionein in lead-induced inclusion body formation.

Lead (Pb) produces aggresome-like inclusion bodies (IBs) in target cells as a toxic response. Our prior work shows metallothionein (MT) is required for this process. We used MT-I/II double knockout (MT-null) and parental wild-type (WT) cell lines to further explore the formation process of Pb-induced IBs. Unlike WT cells, MT-null cells did not form IBs after Pb exposure. Western blot of cytosol showed soluble MT protein in WT cells was lost during Pb exposure as IBs formed. Transfection of MT-I into MT-null cells allowed IBs formation after Pb exposure. Considering Pb-induced IBs may be like disease-related aggresomes, which often contain alpha-synuclein (Scna), we investigated Scna expression in cells capable (WT) and incapable (MT-null) of producing IBs after Pb exposure. Scna protein showed poor basal expression in MT-null cells. Pb exposure increased Scna expression only in WT cells. MT transfection increased Scna transcript to WT levels. In WT or MT-transfected MT-null cells, Pb-induced Scna expression rapidly increased and then decreased over 48 h as Pb-induced IBs were formed. A direct interaction between Scna and MT was confirmed ex vivo by antibody pulldown assay where the proteins coprecipitated with an antibody to MT. Pb exposure caused increased colocalization of MT and Scna proteins with time only in WT cells. In WT mice after chronic Pb exposure Scna was localized in renal cells containing forming IBs, whereas MT-null mice did not form IBs. Thus, Scna could be component of Pb-induced IBs and, with MT, may play a role in IBs formation.

Tissue-dependent preventive effect of metallothionein against DNA damage in dyslipidemic mice under repeated stresses of fasting or restraint

Aims To investigate the effect of repeated stress on DNA damage in seven organs of dyslipidemic mice, and the preventive role of metallothionein (MT). Main methods Female adult 129/Sv wild-type and MT-null mice fed high-fat diet (HFD) were repeatedly subjected to mild stress of fasting or restraint in weeks 2 to 4 of 4-week study period. Serum cholesterol level, DNA damage in the liver, pancreas, spleen, bone marrow, kidney, lung and gastric mucosa, and other parameters were determined. Key findings Body weights were increased in both types of mice fed HFD compared to those fed standard diet (STD), and further increased by 12 h-fasting, while they were markedly decreased by 1–3 h-restraint. Fasting accelerated accumulation of fat in the liver, and increase in serum cholesterol of both types of mice fed HFD. Feeding of HFD increased DNA damage in the pancreas, spleen and bone marrow of both types of mice, compared with those fed STD. In the wild-type mice fed HFD, 24 h-fasting increased DNA damage in the liver and spleen, while restraint increased the damage in the liver, pancreas, spleen and bone marrow. DNA damage in the cells of organs was markedly increased in the MT-null mice. Specifically, damage in the liver, pancreas, spleen and bone marrow was greatly increased with the intensity of stress increased, and the damage was much greater in the restraint mice than in the fasting mice. Significance MT plays a tissue-dependent preventive role against DNA damage in various murine organs induced by repeated stress.

Methylmercury Pause: Study Suggests Long Latency for Neurotoxicity

Vol. 116, No. 6 EnvironewsOpen AccessMethylmercury Pause: Study Suggests Long Latency for Neurotoxicity Julia R. Barrett Julia R. Barrett Search for more papers by this author Published:1 June 2008https://doi.org/10.1289/ehp.116-a259aAboutSectionsPDF ToolsDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InReddit Methylmercury (MeHg) easily crosses the blood–brain barrier and accumulates in the central nervous system, where it is demethylated to inorganic mercury. Chronic perinatal exposure to environmentally relevant levels of MeHg is associated with the occurrence later in childhood of neurobehavioral problems such as impaired attention and fine motor function. Animal studies confirm this association, but epidemiologic evidence is mixed despite extensive study. Moreover, MeHg toxicity and the period of time before effects appear are not completely understood, as few studies have been conducted beyond the first months or years of life in either animals or humans. Researchers now demonstrate in a mouse model that effects from early exposure to methylmercury can occur years after early-life mercury levels in the brain have declined [EHP 116:746–751; Yoshida et al.].In the current study, investigators used two strains of mice—the wild-type C57BL strain and the genetically manipulated metallothionein (MT)-null strain. The latter was used to examine potential genetic susceptibility to the toxic effects of MeHg exposure, as MT-null mice do not produce metallothionein-I and II proteins that can bind metals and protect against their toxic effects. Mice were exposed through diet to low levels of MeHg (5 μg/g diet) from the first day of pregnancy through the tenth day after birth. Offspring of the treated mice were weaned at 28 days. At 12 and 52 weeks (roughly comparable to young adulthood and middle age in humans), the offspring underwent behavioral tests of their locomotor activity and learning ability. All animals were weighed biweekly, and mercury concentrations in the brains, livers, and kidneys were measured for 10-day-old mice and for the group tested at 12 weeks.In 10-day-old exposed mice, mercury concentrations in the brain were 0.5 μg Hg/g body weight or lower, with no significant differences observed between exposed wild-type mice and MT-null mice. At 13 weeks, concentrations of mercury in the brain of exposed groups were similar to those of the unexposed groups. Except for one activity measure in female MT-null mice, exposure to MeHg did not significantly affect behavioral test responses at 12 weeks. At 52 weeks, however, investigators observed significant effects in all behavioral test responses, with MT-null mice being slightly more affected. After 28 weeks, wild-type male and all MT-null mice exposed to MeHg weighed significantly less than control mice, which may signal an emerging toxic effect.The authors demonstrate a long latency period after perinatal exposure to low levels of MeHg and show that this period may be influenced by genetic susceptibility, given the stronger effect of MeHg exposure in MT-null mice. The existence of a latency period suggests that a slow process, such as aging, plays a role in MeHg toxicity, although the actual damage occurs much earlier in life.FiguresReferencesRelatedDetails Vol. 116, No. 6 June 2008Metrics About Article Metrics Publication History Originally published1 June 2008Published in print1 June 2008 Financial disclosuresPDF download License information EHP is an open-access journal published with support from the National Institute of Environmental Health Sciences, National Institutes of Health. All content is public domain unless otherwise noted. Note to readers with disabilities EHP strives to ensure that all journal content is accessible to all readers. However, some figures and Supplemental Material published in EHP articles may not conform to 508 standards due to the complexity of the information being presented. If you need assistance accessing journal content, please contact [email protected]. Our staff will work with you to assess and meet your accessibility needs within 3 working days.

Metallothionein is a crucial protective factor againstHelicobacter 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.

Endoplasmic Reticulum Stress and Metallothionein

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The Role of Zinc and Metallothionein in the Dextran Sulfate Sodium-Induced Colitis Mouse Model

Zinc (Zn) and its binding protein metallothionein (MT) have been proposed to suppress the disease activity in ulcerative colitis. To determine the role of Zn and MT in the dextran sulfate sodium (DSS)-induced model of colitis in mice, a DSS dose-response study was conducted in male C57BL/6 wild-type (MT+/+) and MT-null (MT-/-) mice by supplementing 2%, 3%, and 4% DSS in the drinking water for 6 days. In the intervention study, colitis was induced with 2% DSS, Zn (24 mg/ml as ZnO) was gavaged (0.1 ml) daily, concurrent with DSS administration, and the disease activity index (DAI) was scored daily. Histology, MT levels, and myeloperoxidase (MPO) activity were determined. DAI was increased (P<0.05) by 16% and 21% with 3% and 4% concentrations of DSS, respectively, compared to 2%, evident after 5 days of DSS administration. MPO activity was increased in MT+/+ compared to MT-/- mice and those receiving DSS. Zn administration had a 50% (P<0.05) lower DAI compared to DSS alone. Zn partially prevented the distal colon of MT+/+ by 47% from DSS-induced damage compared to MT-/- mice. MT did not prevent DSS-induced colitis and Zn was partially effective in amelioration of DSS-induced colitis.

Endoplasmic Reticulum Stress and Metallothionein

Much attention has been paid to lifestyle-related diseases including type 2 diabetes mellitus, cardiovascular disease, hypertension, and hyperlipidemia because the incidence rates of these diseases are increasing in developed countries. Elucidation of factors contributing to the development of obesity and insulin resistance is needed. Metallothionein (MT), a ubiquitous metal-binding protein, is induced not only by heavy metals but also by various kinds of stresses. Endoplasmic reticulum (ER) stress is caused by accumulation of misfolded proteins in ER. Recently, increased ER stress by obesity and impairment of insulin action by ER stress have been reported. Exposure to ER stress increased induction of MT synthesis, and an enhanced response to ER stress evaluated as expression of Bip/GRP78mRNA was observed in the liver of MT-null mice, suggesting that MT attenuates expression of ER stress. MT may prevent ER stress and thereby modulate the development of obesity and insulin resistance. A possible role of metallothionein in response reaction for ER stress is discussed.

Genome-level expression profiles in pediatric septic shock indicate a role for altered zinc homeostasis in poor outcome

Human septic shock involves multiple genome-level perturbations. We have conducted microarray analyses in children with septic shock within 24 h of intensive care unit admission, using whole blood-derived RNA. Based on sequential statistical and expression filters, there were 2,482 differentially regulated gene probes (1,081 upregulated and 1,401 downregulated) between patients with septic shock (n = 42) and controls (n = 15). Both gene lists encompassed several biologically relevant gene ontologies and canonical pathways. Notably, many of the genes downregulated in the patients with septic shock, relative to the controls, participate in gene ontologies related to metal or zinc homeostasis. Comparison of septic shock survivors (n = 33) and nonsurvivors (n = 9) demonstrated differential regulation of 63 gene probes. Among the 63 gene probes differentially regulated between septic shock survivors and nonsurvivors, two isoforms of metallothionein (MT) demonstrated increased expression in the nonsurvivors. Consistent with the ability of MT to sequester zinc in the intracellular compartment, nonsurvivors had lower serum zinc levels compared with survivors. In a corroborating study of murine sepsis, MT-null mice demonstrated a survival advantage compared with wild-type mice. These data represent the largest reported cohort of pediatric patients with septic shock that has undergone genome-level expression profiling based on microarray. The data are biologically plausible and demonstrate that genome-level alterations of zinc homeostasis may be prevalent in clinical pediatric septic shock.

The role of zinc (Zn) and metallothionein (MT) in dextran sulfate sodium (DSS)‐induced colitis in MT‐null (MT−/−) and wild‐type mice

Introduction: Zinc (Zn) and its binding protein metallothionien (MT) have been proposed to suppress the disease activity of ulcerative colitis. The aim of the present is to determine the role of Zn and MT in the dextran sulfate sodium (DSS)-induced model of colitis in mice. Methods: A DSS dose-response study was conducted in male C57BL/6 wild-type (MT+/+) and MT-null (MT−/−) mice by supplementing 2%, 3% and 4% DSS in the drinking water for 6 d. In the intervention study, colitis was induced by 2% DSS, Zn (24 mg/ml as ZnO) was gavaged (0.1 ml) daily, concurrent with DSS administration, and the disease activity index (DAI) scored daily. Histology, MT levels and myeloperoxidase (MPO) activity were determined. Results: DAI was increased (p<0.05) by 16% and 21% with 3% and 4% concentrations of DSS, respectively compared to 2%, evident after 5 d of DSS administration. MPO activity was increased in MT+/+ compared to MT−/− mice and those receiving DSS. Zn administration had a 50% (p<0.05) lower DAI compared to DSS alone. Zn partially prevented the distal colon of MT+/+ by 47% from DSS-induced damaged compared to MT−/− mice. Conclusions: MT did not prevent DSS-induced colitis and Zn was partially effective in amelioration of DSS-induced colitis.

Hepatic Responses to Dietary Stress in Zinc- and Metallothionein-Deficient Mice

Metallothionein (MT) and zinc are both reported to be protective against oxidative and inflammatory stress and may also influence energy metabolism. The role of MT in regulating intracellular labile zinc, thus influencing zinc (Zn)-modulated protein activity, may be a key factor in the response to stress and other metabolic challenges. The objective of this study was to investigate the influence of dietary zinc intake and MT on hepatic responses to a pro-oxidant stress and energy challenge in the form of a high dietary intake of linoleic acid, an omega-6 polyunsaturated fatty acid. Male MT-null (KO) and wild-type (WT) mice, aged 16 weeks, were given semisynthetic diets containing 16% fat and either 5 (marginally zinc-deficient [ZD]) or 35 (zinc-adequate [ZA]) mg Zn/kg. For comparison, separate groups of KO and WT mice were given a rodent chow diet containing 3.36% fat and 86.6 mg Zn/kg. After 4 months on these diets, the body weights of all mice were equal, but liver size, weight, and lipid content were much greater in the animals that consumed semisynthetic diets compared to the chow diet. The increase in liver size was significantly lower in ZA but not ZD KO mice, compared with WT mice. Principally, MT appears to affect the diet-induced increase in liver tissue but it also influences the concentration of hepatic lipid. Plasma levels of C-reactive protein (CRP), a marker of inflammation, were increased by zinc deficiency in WT mice, suggesting that marginal zinc deficiency is proinflammatory. CRP was unaffected by zinc deficiency in KO mice, indicating a role for MT in modulating the influence of zinc. Neither zinc nor MT deficiency affects the level of soluble liver proteins, as determined using two-dimensional (2D) gel proteomics. This study highlights the close association between zinc and MT in the manifestation of stress responses.

Hypersusceptibility to cisplatin carcinogenicity in metallothionein‐I/II double knockout mice: Production of hepatocellular carcinoma at clinically relevant doses

Metallothionein (MT) is a high-affinity metal binding protein thought to mitigate the toxicity of various metals. Cisplatin is a widely used cancer chemotherapeutic that is a rodent carcinogen and may have carcinogenic potential in humans. MT seems to reduce cisplatin toxicity by binding the metal compound but how MT deficiency might impact the carcinogenic effects of cisplatin is unknown. Thus, groups (n = 25) of male MT-I/II double knockout (MT-null) or MT wild-type (WT) mice were exposed to a single treatment of cisplatin (5 or 10 mg/kg, i.p.), or left untreated (control) and observed over the next 104 weeks. The doses of cisplatin used equate to only a fraction of the total dose used typically in clinical settings. In cisplatin-treated MT-null mice, a dose-related increase in hepatocellular carcinoma (HCC) occurred (control, 0%; 5 mg/kg, 17%; 10 mg/kg, 36%) that was not seen in WT mice. Similarly, liver carcinoma multiplicity (HCC/liver) was increased markedly by cisplatin but only in MT-null mice, indicating the formation of multiple primaries in MT deficient mice. Harderian gland carcinoma incidence was also increased by cisplatin treatment in MT-null mice but not WT mice. Our results indicate that MT-null mice are hypersusceptible to the hepatocarcinogenic effects of cisplatin, and poor MT expression may be a predisposing factor for cisplatin-induced secondary tumors after chemotherapy.

Involvement of Oxidative Stress in the Synthesis of Metallothionein Induced by Mitochondrial Inhibitors

We previously reported that synthesis of metallothionein (MT) was induced by mitochondrial inhibitors such as 2,4-dinitrophenol (DNP) or antimycin A (Kondoh et al., 2001), which are potent inhibitors of mitochondrial respiration. Although the inhibitors are known to be radical generators in mitochondria, the involvement of oxidative stress in the synthesis of MT induced by mitochondrial inhibitors and the biological functions of MT remain obscure. In this study, therefore, we examined the involvement of oxidative stress in MT synthesis induced by mitochondrial inhibitors and the biological functions of MT. In cultured mouse fibroblast cells, the addition of DNP increased both MT concentration and MT mRNA level. Administration of DNP to L-buthionine-SR-sulfoximine (BSO)-pretreated mice increased hepatic lipid peroxidation and induction of MT synthesis. In addition, vitamin E prevented induction of MT synthesis as well as lipid peroxidation in the liver of mice caused by administration of DNP. Administration of mitochondrial inhibitor to mice elevated the levels of lipid peroxidation in the liver and mitochondria, and MT in the liver, indicating the generation of mitochondrial oxidative stress. These data suggest that the induction of MT synthesis by mitochondrial inhibitors is correlated with generation of oxidative stress in mitochondria. Furthermore, the level of DNP-induced alanine aminotransferase (ALT) activity, reflecting hepatic damage, was greater in MT-null mice than in wild-type mice, and intracellular accumulation of reactive oxygen species (ROS) caused by the action of mitochondrial inhibitors was greater in MT-null fibloblast cells than in wild-type cells. The results suggest that MT plays a role as a radical scavenger of intracellular ROS produced in mitochondria. Taken together, the results suggest that mitochondrial oxidative stress induces the synthesis of MT, which may contribute to regulation of mitochondrial ROS production.