Hepatic Metallothionein Expression Research Articles

ZnO-NPs alleviate aflatoxin B1-induced hepatoxicity in ducklings by promoting hepatic metallothionein expression

Aflatoxin B1 (AFB1) is a mycotoxin widely present in animal feed and human food, posing a serious threat to animal and human health. This study was aim to illustrate the mechanism of the protective role of MT against AFB1-induced hepatotoxicity, as well as to explore the feasibility of enhancing the tolerance of poultry to AFB1 by upregulating the expression of hepatic MT. After being exposed to AFB1 (50 ng/kg) primary duckling hepatocytes, the cell viability, the antioxidant index (SOD and GPx) and the mRNA levels of MT downstream genes (PTGR, p53, TrxR, AR and Bcl-2) significantly (p < 0.05) decreased, while the intracellular formation of (AFBO)-DNA adduct content, apoptosis, and MDA content significantly (p < 0.05) increased. Interestingly, overexpression of MT in primary duckling hepatocytes markedly (p < 0.05) reversed the detrimental impact of AFB1 and increased the expression of MT downstream genes. HepG2 cells were applied to study the mechanism how MT works to relieve the hepatic toxicity of AFB1. The ZnO-NPs (20 μg/mL) + AFB1 (20 μg/mL) group significantly (p < 0.05) increased the cell viability, the expression of NRF2, NQO1 and SOD, and expression of MT and MTF-1, as well as significantly (p < 0.05) decreased LDH, ROS and apoptotic rate, comparing with the AFB1 group. While joint treatment with AFB1 and ZnO-NPs, the hepatic toxicity exerted by AFB1 alone was reversed, along with the translocation of MTF-1 from the cytoplasm to the nucleus and upregulated its expression. Duckling trails were further carried out. A total number of 96 1-day-old healthy Cherry Valley commercial ducklings were randomly allocated according to a 2 by 2 factorial arrangement of treatments with the main factors including oral administration of AFB1 (0 vs. 40 μg/kg) and dietary supplementation of ZnO-NPs (0 vs. 60 mg/kg) for 7 days. It showed that AFB1 exposure caused body weight loss (p < 0.05), impaired liver structure and failure in hepatic function (activity of ALT, AST and concentration of TP and GLU) (p < 0.05), and decreases in antioxidant capacity(activity of SOD, CAT and concentration of GSH) (p < 0.05), along with the decrease in hepatic concentration of Zn, increase in expression of apoptosis-related genes and protein CAS3 and mRNA Bcl-2 expression (p < 0.05), and suppressed mRNA levels of antioxidant-related genes MT, SOD1, NRF2, and NQO1 (p < 0.05). In accordance with the cell test, dietary supplementation with ZnO-NPs mitigated the toxicity exerted by AFB1. In conclusion, ZnO-NPs has the protective effects against AFB1-induced hepatocyte injury by activating the expression of MTF-1 and the ectopic induction of MT expression, providing detailed information on the detoxification ability of MT on AFB1.

Zinc Supplementation Prevented Type 2 Diabetes-Induced Liver Injury Mediated by the Nrf2-MT Antioxidative Pathway.

Zinc is an essential trace element that is often reduced under the type 1 diabetic condition. Previous studies demonstrated that zinc deficiency enhanced type 1 diabetes-induced liver injury and that zinc supplementation significantly helped to prevent this. Due to the differences in pathogenesis between type 1 and type 2 diabetes, it is unknown whether zinc supplementation can induce a beneficial effect on type 2 diabetes-induced liver injury. This possible protective mechanism was investigated in the present study. A high-fat diet, along with a one-time dose of streptozotocin, was applied to metallothionein (MT) knockout mice, nuclear factor-erythroid 2-related factor (Nrf) 2 knockout mice, and age-matched wild-type (WT) control mice, in order to induce type 2 diabetes. This was followed by zinc treatment at 5 mg/kg body weight given every other day for 3 months. Global metabolic disorders of both glucose and lipids were unaffected by zinc supplementation. This induced preventive effects on conditions caused by type 2 diabetes like oxidative stress, apoptosis, the subsequent hepatic inflammatory response, fibrosis, hypertrophy, and hepatic dysfunction. Additionally, we also observed that type 2 diabetes reduced hepatic MT expression, while zinc supplementation induced hepatic MT expression. This is a crucial antioxidant. A mechanistic study showed that MT deficiency blocked zinc supplementation-induced hepatic protection under the condition of type 2 diabetes. This suggested that endogenous MT is involved in the hepatic protection of zinc supplementation in type 2 diabetic mice. Furthermore, zinc supplementation-induced hepatic MT increase was unobserved once Nrf2 was deficient, indicating that Nrf2 mediated the upregulation of hepatic MT in response to zinc supplementation. Results of this study indicated that zinc supplementation prevented type 2 diabetes-induced liver injury through the activation of the Nrf2-MT-mediated antioxidative pathway.

Metallothioneins alter macrophage phenotype and represent novel therapeutic targets for acetaminophen-induced liver injury.

Acetaminophen (APAP) intoxication is the foremost cause of drug-induced liver failure in developed countries. The only pharmacologic treatment option, N-acetylcysteine (NAC), is not effective for patients who are admitted too late and/or who have excessive liver damage, emphasizing the need for alternative treatment options. APAP intoxication results in hepatocyte death and release of danger signals, which further contribute to liver injury, in part by hepatic monocyte/macrophage infiltration and activation. Metallothionein (MT) 1 and 2 have important danger signaling functions and might represent novel therapeutic targets in APAP overdose. Therefore, we evaluated hepatic MT expression and the effect of anti-MT antibodies on the transcriptional profile of the hepatic macrophage population and liver injury following APAP overdose in mice. Hepatic MT expression was significantly induced in APAP-intoxicated mice and abundantly present in human livers. APAP intoxication in mice resulted in increased serum transaminase levels, extended necrotic regions on liver histology and induced expression of proinflammatory markers, which was significantly less pronounced in mice treated with anti-MT antibodies. Anti-MT antibody therapy attenuated proinflammatory macrophage polarization, as demonstrated by RNA sequencing analyses of isolated liver macrophages and in LPS-stimulated bone marrow-derived macrophages. Importantly, NAC and anti-MT antibodies were equally effective whereas administration of anti-MT antibody in combination with NAC exceeded the efficiency of both monotherapies in APAP-induced liver injury (AILI). We conclude that the neutralization of secreted MTs using a monoclonal antibody is a novel therapeutic strategy as mono- or add-on therapy for AILI. In addition, we provide evidence suggesting that MTs in the extracellular environment are involved in macrophage polarization.

Type I Interferons Ameliorate Zinc Intoxication of Candida glabrata by Macrophages and Promote Fungal Immune Evasion.

SummaryHost and fungal pathogens compete for metal ion acquisition during infectious processes, but molecular mechanisms remain largely unknown. Here, we show that type I interferons (IFNs-I) dysregulate zinc homeostasis in macrophages, which employ metallothionein-mediated zinc intoxication of pathogens as fungicidal response. However, Candida glabrata can escape immune surveillance by sequestering zinc into vacuoles. Interestingly, zinc-loading is inhibited by IFNs-I, because a Janus kinase 1 (JAK1)-dependent suppression of zinc homeostasis affects zinc distribution in macrophages as well as generation of reactive oxygen species (ROS). In addition, systemic fungal infections elicit IFN-I responses that suppress splenic zinc homeostasis, thereby altering macrophage zinc pools that otherwise exert fungicidal actions. Thus, IFN-I signaling inadvertently increases fungal fitness both in vitro and in vivo during fungal infections. Our data reveal an as yet unrecognized role for zinc intoxication in antifungal immunity and suggest that interfering with host zinc homeostasis may offer therapeutic options to treat invasive fungal infections.

Reduced hepatic metallothionein expression in first trimester fetuses in response to intrauterine smoking exposure: a consequence of low maternal zinc levels?

Does maternal smoking in early pregnancy affect metallothionein 1 and 2 (MT1 and MT2) mRNA and protein expression in first trimester placenta or embryonic/fetal liver? In the first trimester, MT protein expression is seen only in liver, where smoking is associated with a significantly reduced expression. Zinc homeostasis is altered by smoking. Smoking induces MT in the blood of smokers properly as a result of the cadmium binding capacities of MT. In term placenta MT is present and smoking induces gene and protein expression (MT2 in particular), but the MT presence and response to smoking have never been examined in first trimester placenta or embryonic/fetal tissues. Cross sectional study where the presence of MT mRNA and protein was examined at the time of the abortion. The material was collected with informed consent after surgical intervention and frozen immediately. For protein expression analysis, liver tissue originating from smoking exposed n = 10 and unexposed n = 12 pregnancies was used. For mRNA expression analyses, placental tissue originating from smokers n = 19 and non-smokers n = 23 and fetal liver tissue from smoking exposed n = 16 and smoking unexposed pregnancies n = 13, respectively, were used. Tissues were obtained from women who voluntarily and legally chose to terminate their pregnancy between gestational week 6 and 12. Western blot was used to determine the protein expression of MT, and real-time PCR was used to quantify the mRNA expression of MT2A and eight MT1 genes alongside the expression of key placental zinc transporters: zinc transporter protein-1 (ZNT1), Zrt-, Irt-related protein-8 and -14 (ZIP8 and ZIP14). A significant reduction in the protein expression of MT1/2 in liver tissue (P = 0.023) was found by western blot using antibodies detecting both MT forms. Overall, a similar tendency was observed on the mRNA level although not statistically significant. Protein expression was not present in placenta, but the mRNA regulation suggested a down regulation of MT as well. A suggested mechanism based on the known role of MT in zinc homeostasis could be that the findings reflect reduced levels of easily accessible zinc in the blood of pregnant smokers and hence a reduced MT response in smoking exposed fetal/embryonic tissues. Smoking was based on self-reports; however, our previous studies have shown high consistency regarding cotinine residues and smoking status. Passive smoking could interfere but was found mainly among smokers. The number of fetuses was limited, and other factors such as medication and alcohol might affect the findings. Information on alcohol was not consistently obtained, and we cannot exclude that it was more readily obtained from non-users. In the study, alcohol consumption was reported by a limited number (less than 1 out of 5) of women but with more smokers consuming alcohol. However, the alcohol consumption reported was typically limited to one or few times low doses. The interaction between alcohol and smoking is discussed in the paper. Notably we would have liked to measure zinc status to test our hypothesis, but maternal blood samples were not available. Zinc deficiency-in particular severe zinc deficiency-can affect pregnancy outcome and growth. Our findings indicate that zinc homeostasis is also affected in early pregnancy of smokers, and we know from pilot studies that even among women who want to keep their babies, the zinc status is low. Our findings support that zinc supplements should be considered in particular to women who smoke. We thank the Department of Biomedicine for providing laboratory facilities and laboratory technicians and the Lundbeck Foundation and Læge Sofus Carl Emil Friis og Hustru Olga Doris Friis Legat for financial support. The authors have no competing interests to declare. N/A.

Metallothionein Expression in Horses With Chronic Liver Disease and Its Correlation With Ki-67 Immunoreactivity.

Chronic liver disease is an important cause of illness in horses, and treatment is mainly supportive. Research into new treatment modalities for humans has shown promising data regarding metallothionein (MT), which has been shown to possess regenerative, antifibrotic, and anti-inflammatory properties. This study aimed to examine the relationship between hepatic MT expression and the histopathologic markers of hepatic inflammation, fibrosis and bile duct proliferation, as well as cellular regeneration in 77 selected cases of chronic liver disease in horses. We hypothesized that higher MT expression would be associated with increased heptocellular proliferation and decreased fibrosis, inflammation, and bile duct proliferation. Hepatocellular MT expression was evaluated with immunohistochemistry. Additionally, cellular regeneration was evaluated with immunohistochemistry for Ki-67, a protein expressed during all active stages of the cell cycle. The severity of inflammation and fibrosis was scored, and bile duct proliferation was assessed by counting bile duct profiles. MT expression was observed in 73 of 77 (94.8%) cases of chronically diseased livers. Ki-67 expression was seen in resident Kupffer cells ( n = 42, 54.6%), lymphocytes ( n = 39, 50.7%), bile duct epithelium ( n = 10, 13.0%), and hepatocytes ( n = 8, 10.4%). MT expression was significantly associated with Ki-67 staining in bile duct epithelium and Kupffer cells. Additionally, median MT expression was higher in cases containing lymphocytic infiltrates as compared with cases with no lymphocytic infiltrate ( P < .05). These findings are the first known report of MT expression within chronic equine hepatic disease.

Zinc is a potent and specific inhibitor of IFN-\u03bb3 signalling

Lambda interferons (IFNL, IFN-λ) are pro-inflammatory cytokines important in acute and chronic viral infection. Single-nucleotide polymorphisms rs12979860 and rs8099917 within the IFNL gene locus predict hepatitis C virus (HCV) clearance, as well as inflammation and fibrosis progression in viral and non-viral liver disease. The underlying mechanism, however, is not defined. Here we show that the rs12979860 CC genotype correlates with increased hepatic metallothionein expression through increased systemic zinc levels. Zinc interferes with IFN-λ3 binding to IFNL receptor 1 (IFNLR1), resulting in decreased antiviral activity and increased viral replication (HCV, influenza) in vitro. HCV patients with high zinc levels have low hepatocyte antiviral and inflammatory gene expression and high viral loads, confirming the inhibitory role of zinc in vivo. We provide the first evidence that zinc can act as a potent and specific inhibitor of IFN-λ3 signalling and highlight its potential as a target of therapeutic intervention for IFN-λ3-mediated chronic disease.

Molecular cloning, characterization and expression analysis of metallothionein in the liver of the teleost Acrossocheilus fasciatus exposed to cadmium chloride

Metallothionein (MT) has a characteristic molecular structure with a cysteine-rich content. This unique structure provides metal-binding and redox capabilities and promoting metal homeostasis and detoxification in living animals. In order to evaluate the effects of cadmium (Cd) on hepatic MT expression in the liver of Acrossocheilus fasciatus, we obtained the complete cDNA of the A. fasciatus liver MT for the first time. The MT cDNA contains a 605-bp sequence, which codes for 60 amino acids. Protein alignment showed that the similarity between MT protein sequences of A. fasciatus and those of other vertebrates (especially teleosts) was very high and a cysteine residue structure was also conserved. MT was detected in the liver, kidney, gill, testis, muscle, spleen, heart and brain tissues of A. fasciatus by tissue-specific expression analysis. After Cd exposure, Cd/hemoglobin saturation assay, immunohistochemistry and reverse-transcription quantitative PCR (RT-qPCR) were used to describe MT expression in liver tissue. These techniques indicate a sensitive response by liver MT to Cd exposure. The results suggest that A. fasciatus MT may play an important role in the detoxification processes in the liver, and also would be a useful biomarker for monitoring metal pollution in aquatic environments. In addition, A. fasciatus could be regarded as one candidate for a model species for bony fishes in ecotoxicology.

Zinc treatment prevents type 1 diabetes-induced hepatic oxidative damage, endoplasmic reticulum stress, and cell death, and even prevents possible steatohepatitis in the OVE26 mouse model: Important role of metallothionein

Whether zinc is able to improve diabetes-induced liver injury remains unknown. Transgenic type 1 diabetic (OVE26) mice develop hyperglycemia at 3 weeks old; therefore therapeutic effect of zinc on diabetes-induced liver injury was investigated in OVE26 mice. Three-month old OVE26 and age-matched wild-type mice were treated by gavage with saline or zinc at 5mg/kg body-weight every other day for 3 months. Hepatic injury was examined by serum alanine aminotransferase (ALT) level with liver histopathological and biochemical changes. OVE26 mice at 6 months old showed significant increases in serum ALT level and hepatic oxidative damage, endoplasmic reticulum stress and associated cell death, mild inflammation, and fibrosis. However, all these hepatic morphological and functional changes were significantly prevented in 3-month zinc-treated OVE26 mice. Mechanistically, zinc treatment significantly increased hepatic metallothionein, a protein with known antioxidant activity, in both wild-type and OVE26 mice. These results suggest that there were significantly functional, structural and biochemical abnormalities in the liver of OVE26 diabetic mice at 6 months old; however, all these changes could be prevented with zinc treatment, which was associated with the upregulation of hepatic metallothionein expression.

Effects of antihypertensive and triglyceride-lowering agents on hepatic copper concentrations in rats with fatty liver disease.

Copper deficiency had been suggested to link between fructose-enriched diet (FED) and the development of non-alcoholic fatty liver disease (NAFLD). In this study, we characterized changes in hepatic copper concentrations and hepatic oxidative milieu, in rats with the metabolic syndrome and NAFLD as a result of FED with pharmacological manipulations to reduce blood pressure or plasma triglycerides. Changes in plasma and hepatic copper concentrations were correlated with changes observed in the immunohistochemical hepatic expression of copper-zinc-superoxide dismutase (CuZnSOD; SOD1), metallothionein (MT) and nitrotyrosine (NITT). FED administration was associated with a 2.2-fold reduction in hepatic copper concentrations, a decrease in the hepatic SOD1 expression, disappearance of the hepatic MT expression and increase in the hepatic NITT expression. Bezafibrate administration restored the hepatic copper concentrations and the hepatic SOD1 expression to levels that were observed in the control rats. A significant positive correlation between hepatic copper concentrations and the values of hepatic SOD1 expression of each animal included in this study was found. Administration of either captopril or bezafibrate increased hepatic MT expression, however, to levels that were lower than those observed in the control group. Administration of either amlodipine, or captopril or bezafibrate to the FED rats, had no effect on hepatic NITT expression. NAFLD development in FED rats is associated with a decrease in hepatic copper concentrations that is associated with a decrease in the hepatic SOD1 expression. Bezafibrate administration increases hepatic copper concentrations and restores the hepatic SOD1 expression.

The yellow catfish, Pelteobagrus fulvidraco (Siluriformes) metallothionein cDNA: molecular cloning and transcript expression level in response to exposure to the heavy metals Cd, Cu, and Zn

Metallothionein (MT) has been used extensively as a potential molecular biomarker to detect heavy metal pollution in aquatic organisms. In order to investigate the modulation effect of heavy metals and to establish suitable biomarkers for the monitoring of heavy metal pollution, Pelteobagrus fulvidraco metallothionein gene was characterized as the first report in the family Bagridae. Pf-MT transcript was detected at high levels in liver, gonad, kidney, and brain compared to other tissues. A time-course study in response to waterborne Cd (5ppm) revealed that a significant increase in the Pf-MT transcript abundance was observed at 6h in gill, kidney, and liver. These elevated levels were kept for 96h, implying that Cd distributed fast into different organs and was involved in the tissue-specific induction pattern. We observed a significant Pf-MT transcript increase in liver tissues at 48h, followed by gill at 12h and intestine at 48h after Cd exposure. This indicates hepatic MT expression as a potential biomarker of acute Cd exposure in this species. Cd-binding ability of recombinant Pf-MT protein provided evidence for sensitivity to Cd and other heavy metal exposure. In the case of Zn exposure (1ppm), a significant increase in Pf-MT transcript abundance was observed at 12h, and a peak induction level reaching sixfold at 24h was kept until 48h, showing similar transcript induction patterns with Cd. A high level of Pf-MT mRNA after exposure to Cu (1ppm) was observed at 12h that gradually increased until 96h with a 12-fold induction, revealing a long-lasting induction and somewhat dissimilar pattern compared to other metals in liver. Our results demonstrate that Pf-MT can be induced by heavy metals in a tissue-specific and metal-specific manner and plays probably a conserved role in metal detoxification. This study provides new information on P. fulvidraco metallothionein gene for the use of biomarkers indicating metal pollution in fish.

Involvement of Cytokines in the Hepatic Metallothionein Expression by α-Hederin

Alpha-hederin, a triterpenoid saponin, has been reported to induce hepatic metallothionein (MT). However, the mechanism underlying its effects is unknown. This study investigated the effects of alpha-hederin on the regulation of MT expression in an in vitro model using the murine hepatoma cell line, Hepa-1c1c7, and the murine macrophage cell line, RAW 264.7. Alpha-hederin that was added directly to Hepa-1c1c7 cells had no effect on MT induction. However, MT and its mRNA levels increased markedly when the Hepa-1c1c7 cells were cultured with the alpha-hederin-treated, conditioned medium from the RAW 264.7 cells. Co-treating the RAW 264.7 cells with alpha-hederin and pentoxifylline, a TNF-alpha synthesis inhibitor, resulted in decreased effects of alpha-hederin on MT induction. In the alpha-hederin-exposed RAW 264.7 cell cultures, production and mRNA levels of TNF-alpha and IL-6 were increased. Accordingly, it was found that the MT induction activity was inhibited when antibodies to TNF-alpha and/or IL-6 were added to the alpha-hederin-treated, conditioned medium from the RAW 264.7 cells. These results suggest that the upregulation of MT expression by alpha-hederin is mediated by TNF-alpha and IL-6.

Involvement of cytokines in the hepatic expression of metallothionein by ursolic acid

Ursolic acid (UA), a pentacyclic triterpene acid, is reported to have inducing activity of hepatic metallothionein (MT) which responsible for the detoxification of heavy metals; however, the mechanism underlying its effects is poorly understood. To further determine the underlying mechanism of UA, this study investigated the effects of UA on the induction of hepatic MT expression in an in vitro model, using murine hepatoma cell line Hepa-1c1c7 and murine macrophage cell line RAW 264.7 cell cultures. The UA added directly to Hepa-1c1c7 cells had no effect on MT induction. However, MT and its mRNA levels were markedly increased when Hepa-1c1c7 cells were cultured with UA-treated conditioned media from RAW 264.7. Concomitant treatment with UA and pentoxifylline, a TNF-α synthesis inhibitor, to RAW 264.7 cells decreased the effects of UA on the MT induction. In UA-exposed RAW 264.7 cell cultures, TNF-α and IL-6 production and TNF-α and IL-6 mRNA levels increased. When antibodies to TNF-α or/and IL-6 were added to UA-treated conditioned media from RAW 264.7, the MT induction activity was inhibited. These results demonstrate that UA induces hepatic MT expression through TNF-α and IL-6 released from UA-activated macrophages, which may be the mechanism, whereby UA elicits its biological effects.

Hepatic metallothionein in patients with chronic hepatitis C: relationship with severity of liver disease and response to treatment

Objectives Reactive oxygen species may be involved in the pathogenesis of chronic hepatitis C virus infection. Metallothionein (MT) is an essential protein for the protection of cells against reactive oxygen species. The aim of this prospective study was to assess the influence of the hepatic level and cellular distribution of MT in hepatitis C virus (HCV) infection and in the liver disease outcome. Methods In liver biopsy samples of 32 patients with chronic HCV infection and of 12 control subjects, quantification of MT was performed by radioimmunoassay, MT, interleukin (IL)-1 and -6, and tumor necrosis factor (INF)–α mRNA by reverse transcription–polymerase chain reaction (PCR) and cellular distribution by immunohistochemistry. Results In HCV-infected patients, MT liver protein level was 3-fold lower than in control specimens. A significant inverse linear regression between MT protein or mRNA expression and the Histological Activity Index, the necroinflammatory grade, and the stage of fibrosis was observed. MT immunostaining was located in the nucleus and cytoplasm in hepatocytes of control subjects, whereas it was mainly cytoplasmic in HCV-infected patients. Before interferon (IFN) therapy, the hepatic MT level in patients that were nonsustained responders was half that of sustained responders. Intrahepatic IL-6 and MT were simultaneously down-regulated, but no correlation was found between MT and intrahepatic cytokine mRNA expression in patients with chronic HCV infection. Conclusions This study shows that hepatic MT expression could reflect the severity of chronic HCV infection and could be one of the factors associated with a favorable clinical outcome in the response to interferon therapy.

Arctic char (Salvelinus alpinus) metallothionein: cDNA sequence, expression, and tissue‐specific inhibition of cadmium‐mediated metallothionein induction by 17β‐estradiol, 4‐OH‐PCB 30, and PCB 104

AbstractIn this study, we haveexamined the basallevel expression and tissue‐specificexpressionpatternsof metallothionein (MT) in Arctic char following metal and E2 (17β‐estradiol) treatment. To study the gene regulation in Arctic char, the two MT isoforms were isolated from a lambda‐ZAP hepatic cDNA library and characterized. Determination of basal MT mRNA and MT expression for 10 different tissues revealed a lack of correlation between MT mRNA and MT levels. The inducibility of MT mRNA and the correlation to resulting MT levels were then determined for liver and kidney. We found a more rapid and stronger induction of MT mRNA in liver than in kidney at day 1 and 3 postinjection, whereas the MT protein quantification showed higher MT levels in kidney than in liver at days 3 and 7 postinjection. These discrepancies indicate that differences in metal handling or posttran‐scriptional regulation of MT exists between tissues. Whereas metals induce MT synthesis, E2 inhibit the hepatic MT expression. To examine the tissue specificity of this inhibition, we determined the effect of 17pj‐estradiol (E2) and two estrogenic PCBs (4′‐OH‐PCB 30 and PCB 104) on Cd‐mediated MT induction in liver and kidney. Although E2 and the estrogenic PCBs inhibited cadmium‐mediated hepatic MT induction, these compounds did not interfere with renal MT induction.

Relationship between expression of hepatic metallothionein and sublethal stress in channel catfish following acute exposure to copper sulphate

Copper sulphate is frequently used as a general biocide in the aquacultural industry. The acute effects of copper sulphate on sublethal endpoints of stress and tissue damage in channel catfish have not been explored previously. Consequently, the purpose of this study was to examine the effects of a therapeutically relevant dose of copper sulphate on plasma cortisol, hepatic metallothionein (MT) expression, hepatic copper content and plasma concentrations of aspartate aminotransferase (AST). Exposure of prespawn juvenile catfish to 1.7 mg/l as copper sulphate (326±9.4 μg/l copper; 175–180 mg/l CaCO 3; pH 7.45) led to time-dependent increases in hepatic MT expression, hepatic copper content as well as plasma cortisol concentrations. Exposure to a 1 h confinement stress led to a redistribution of copper to the liver but no significant increases in hepatic MT or plasma cortisol after 23 h. Neither stress nor copper exposure caused liver damage as measured by AST. The parallel expression of hepatic MT with plasma cortisol and hepatic copper residues suggests that MT may serve as a useful indicator of acute stress and acute exposure to copper sulphate in channel catfish.

Effect of arsenite, arsenate, and the herbicide monosodium methyl arsonate (MSMA) on hepatic metallothionein expression and lipid peroxidation in channel catfish

Arsenic exerts its toxicity by the generation of reactive oxygen intermediates which cause lipid peroxidation and cellular damage. Metallothioneins (MTs) have been shown to be induced by oxidative stress and act as scavengers of reactive oxygen intermediates. Thus, hepatic MT was examined in channel catfish treated with the herbicide monosodium methyl arsonate (MSMA) and compared to equal doses of trivalent and pentavalent arsenic. Fish were exposed to 0.01, 0.1, and 1.0 mg/L of each compound for 1 week by static renewal. Hepatic MT was measured by the cadmium/hemoglobin ( Cd Hb ) saturation assay, ELISA using antibodies raised against the first 10 amino acids of piscine MT, and Northern blot analysis using a cDNA encoding winter flounder hepatic MT. Cd Hb and ELISA measurements of low molecular weight fractions from the hepatic cytosolic component of fish exposed to MSMA revealed a dose dependent increase in MT. MTs and MT mRNA of fish receiving the 1.0 mg/L dose were significantly induced vs control. Responses to arsenate exposure were more variable, but showed a trend toward a dose-dependent induction of MT and MT mRNA. MT mRNA and protein also showed a dose-dependent increase with arsenite exposure with no significant differences with untreated animals. Hepatic lipid peroxidation (as determined by TBARS) and glutathione was unaltered by any of the arsenical treatments. Thus, the lack of correlation between oxidative stress and MT expression suggest MT may not be a reliable indicator of oxidative stress. In addition, the induction of hepatic MT by various forms of As does not appear to be mediated through an oxidative stress mechanism in the liver.

Characterization of hepatic metallothionein expression in channel catfish (Ictalurus punctatus) by reverse transcriptase polymerase chain reaction

Measurements of metallothioneins (MTs) can be labour intensive and require expensive probes and or analytical instrumentation. In contrast, the reverse transcriptase polymerase chain reaction (RT-PCR0 has provided a relatively sensitive and simple mechanism to measure expression of MT in sentinel species in aquatic environments. The objectives of this study were to characterize expression of hepatic MT in a commonly occurring fish species of the southeastern United States, the channel catfish (Ictalurus punctatus). Expression of hepatic MT was assessed following exposure to several metals found naturally and in excess due to anthropogenic inputs. MT expression by RT PCR was confirmed by Southern blot with a cDNA probe (552 bp) encoding MT and the complete 3 untranslated region from the common carp (Cyprinus carpio) and a 388 bp cDNA probe encoding 49 bp of the 5′ untranslated region, the entire MT protein, and the complete 3′ untranslated region of winter flounder (Pleuronectes americanus). Each probe recognized a single band of approximately 550 bp which corresponded to the RT-PCR product from hepatic RNA of channel catfish 24 h after an intraperitoneal injection of cadmium chloride (10 mg kg-1). Catfish were also injected with intraperitoneal administrations of zinc sulphate (10 mg kg-1) and copper sulphate (2 mg kg-1). After 24 h, zinc increased MT expression 16-fold over controls whereas cadmium or copper increased levels 13 and nearly 6-fold, respectively. A 1-week exposure to water borne 1 mg l-1 sodium arsenite or monosodium methylarsonate (MSMA) induced hepatic MT expression nearly two fold, whereas water borne exposure to the same dose of sodium arsenate failed to increase MT levels. To examine the effect of dietary sub chronic methylmercury exposure on hepatic MT expression, Japanese medaka (Oryzias latipes), were lethally injected with solutions of methylmercuric chloride to provide a 0.1 mg kg-1 daily dose to catfish. After 30 days, hepatic MT expression, condition factor and liver somatic indices were unchanged, although hepatic and muscle residues of total mercury were significantly increased following treatment. The results of this study show that MT expression can vary in aquatic organisms in response to different metal treatments and may be utilized as a biomarker of exposure and possibly effect in the channel catfish.

Induction and characterization of hepatic metallothionein expression from cadmium‐induced channel catfish (Ictalurus punctatus)

AbstractTwo low‐molecular‐weight proteins of approximately 10,000 daltons (Da) were isolated from the hepatic tissue of catfish intraperitoneally injected with CdCl2 (5 mg/kg). Each protein was significantly induced following Cd treatment. Bound Cd, co‐eluted from DEAE anion‐exchange HPLC with rabbit hepatic metallothioneins I and II, did not possess UV absorption indicative of aromatic amino acids and corresponded to the expression of two mRNA transcripts of approximately 400 base pairs, which hybridized to a full‐length cDNA probe to winter flounder metallothionein (MT). Because these data indicated that these two low molecular weight proteins were hepatic MT isoforms, induction of MT mRNA expression was studied in livers of channel catfish exposed to aqueous CdCl2 utilizing the ribonuclease protection assay. Following a 7‐d exposure to 10, 50, and 100 μg/L CdCl2, expression of two MT mRNAs increased in a dose‐dependent manner (MT‐a, r2 = 0.95; MT‐b, r2 = 0.98). Each transcript was maximally induced 48 h after exposure to 100 μg/L CdCl2 and then gradually declined to approximately 20% above controls after 96 h and maintained this level of expression for 7 d. Although MT expression is rapidly induced in channel catfish by Cd, and MT mRNA measurement is a sensitive indicator of MT induction, the use of hepatic MT of channel catfish may not be a sensitive bioindicator of short‐term, low‐level aqueous exposures of Cd.