MT-1 mRNA Levels Research Articles

Metallothionein and Heat-Shock Protein 70 Induction in Caged and Wild Fathead Minnows (Pimephales promelas) Exposed to the Ouachita River, Louisiana

The purpose of this study was to explore the changes in mRNA expression levels for metallothionein subtype 2 (MT-2) and heat-shock protein 70 (HSP70) in fathead minnows in response to environmental exposure in a mercury (Hg)-contaminated freshwater ecosystem. It was hypothesized that expression levels of both genes may rise concurrent with the bioaccumulation of Hg and possibly other heavy metals during exposure to the Ouachita River. The experimental design incorporated three distinct populations of fathead minnows: (1) a negative control population of laboratory-raised fathead minnows unexposed to heavy metals or other contaminants, (2) laboratory-raised fatheads placed in cages and exposed to a contaminated ecosystem for 2 wk, and (3) wild-caught (native) fathead minnows captured at the same site where caged fatheads tested positive for Hg bioaccumulation. Study endpoints included growth rates and gross pathology at necropsy. Total Hg levels of the water at the exposure sites as well as in whole fish homogenates were determined by cold vapor atomic absorption spectroscopy (AAS). AAS was also used to assay levels of lead (Pb) and copper (Cu), though these were below detectable limits. Hepatic expression levels of MT and HSP70 mRNA were determined by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). As hypothesized, levels of both transcripts were significantly increased in the caged exposure group and native fish group compared to unexposed control fish. In addition, the native fish group had significantly higher levels of expression for both genes when compared to caged exposed fish.

Diurnal-and sex-related difference of metallothionein expression in mice.

BackgroundMetallothionein (MT) is a small, cysteine-rich, metal-binding protein that plays an important role in protecting against toxicity of heavy metal and chemicals. This study was aimed to define diurnal and sex variation of MT in mice.MethodsAdult mice were maintained in light- and temperature-controlled facilities for 2 weeks with light on at 8:00 and light off at 20:00. The blood, liver, and kidneys were collected every 4 h during the 24 h period. Total RNA was isolated, purified, and subjected to real-time RT-PCR analysis and MT protein was determined by western blot and the Cd/hemoglobin assay.ResultsThe diurnal variations in mRNA levels of MT-1 and MT-2in liver were dramatic, up to a 40-foldpeak/trough ratio. MT mRNA levels in kidneys and blood also showed diurnal variation, up to 5-fold peak/trough ratio. The diurnal variation of MT mRNAs resembled the clock gene albumin site D-binding protein (Dbp), and was anti-phase to the clock gene Brain and Muscle ARNT-like Protein 1 (Bmal1) in liver and kidneys. The peaks of MT mRNA levels were higher in females than in males. Hepatic MT protein followed a similar pattern, with about a 3-fold difference.ConclusionMT mRNA levels and protein showed diurnal- and sex-variation in liver, kidney, and blood of mice, which could impact the body defense against toxic stimuli.

Magnesium and calcium deficiencies additively increase zinc concentrations and metallothionein expression in the rat liver

Mg deficiency increases the concentration of Zn in the liver. We investigated the effect of Mg deficiency on the expression of Zn-regulating factors such as Zn transporters and metallothionein (MT) in the rat liver. Because Ca deficiency alleviates some of the effects of Mg deficiency, we also investigated the interactions associated with Ca and Mg deficiencies. Growing male rats were given a control diet, a Mg-deficient diet, a Ca-deficient diet and a Mg- and Ca-deficient diet for 3 weeks. Mg and Ca deficiencies additively increased the mRNA levels of MT-1 and MT-2, the MT protein concentration and the concentration of Zn in the liver. The hepatic mRNA level of Zip14 increased with Mg deficiency but not with Ca deficiency. The dietary treatments did not affect the mRNA levels of other Zn transporters such as Zip1, Zip5, ZnT1, ZnT5 and ZnT6 in the liver. Ca deficiency was found to decrease the amount of femoral Zn and increase serum Zn concentration. This did not occur in the case of Mg deficiency. These results suggest that Mg deficiency enhances hepatic Zn uptake by the up-regulation of Zip14 expression and increases hepatic Zn concentration, leading to the enhancement of MT expression. Ca deficiency causes a transfer of Zn from the bone to the liver, which increases hepatic Zn concentration and, in turn, up-regulates the expression of MT. Because Mg and Ca deficiencies increase hepatic Zn concentration and increase MT expression by different mechanisms, their effects are additive.

Expressions of heat shock and metallothionein genes in the heart of common carp (Cyprinus carpio): Effects of temperature shock and heavy metal exposure

Heat shock proteins (HSPs) and metallothioneins (MTs) play important roles in protection against environmental stressors. The present study analyzes and compares the regulation of heat shock ( hsp70, hsc70-1 and hsp90alpha ) and metallothionein (MT-1 and MT-2) genes in the heart of common carp, in response to elevated temperature, cold shock and exposure to several heavy metal ions (As 3+ , Cd 2+ and Cu 2+ ), in whole-animal experiments. Among these metals, arsenate proved to be the most potent inducer of the examined stress genes; the hsp90alpha and MT-1 mRNA levels were elevated 11- and 10-fold, respectively, after a 24-h exposure. In contrast, Cd 2+ at 10 mg/L had no impact on the expression of hsp90alpha , and the MT genes also proved to be rather insensitive to Cd 2+ treatment in the heart: only a 2-2.5-fold induction was observed in response to 10 mg/L Cd 2+ . Heat shock resulted in a transient induction of hsp70 (19-fold) and hsp90alpha (15-fold), while elevated temperature had no effect on the expression of the MTs. Direct cold shock induced hsp70 expression (14-fold), while the hsp90alpha (26-fold) and MT-2 (2-fold) expressions peaked after the recovery period following a direct cold shock. The five stress genes examined in this study exhibited a unique, tissue-specific basal expression pattern and a characteristic sensitivity to metal treatments and temperature shocks.

Zinc Antagonizes Homocysteine-Induced Fetal Heart Defects in Rats

It has been suggested that zinc may have a protective role against heart defects during fetal development. We investigated the effects of zinc on the development of fetal cardiac malformations induced by homocysteine. Pregnant Sprague-Dawley rats were randomized into one of five groups: control (C), homocysteine (H), homocysteine + zinc (Z), homocysteine + folic acid (F), or homocysteine + zinc + folic acid (ZF) (each n = 8). Homocysteine (8 nmol/day) was administered intraperitoneally in the H, Z, F, and ZF groups on gestation days (GD) 8, 9, and 10. Zinc (30 mg/kg day), folic acid (30 mg/kg day), or both (30 mg/kg day each) were administered intragastrically daily in the Z, F, and ZF groups, respectively, throughout the pregnancy. In each group, two fetuses were removed on GD 13, 15, 17, and 19 and examined for cardiac malformations; maternal copper/zinc-containing-superoxide dismutase (Cu/Zn-SOD) activity and metallothionein type I (MT-1) mRNA expression were measured simultaneously. The prevalence of cardiac malformations was significantly higher in group H than in group C, and significantly lower in group Z than in group H at the studied time points. Cu/Zn-SOD activity and MT-1 mRNA levels were significantly lower in group H than in group C, and significantly higher in group Z than in group H. Our data suggest that zinc antagonizes homocysteine-induced teratogenic effects on the fetal heart, possibly via the inhibition of excessive peroxidation.

Physiological responses to mercury in feral carp populations inhabiting the low Ebro River (NE Spain), a historically contaminated site

The low Ebro River course (Northeast Spain) is historically affected by mercury pollution due to a chlor-alkali plant operating at the town of Flix for more than a century. River sediments analysed during the last 10 years showed high mercury levels in the river section starting just downstream the factory and spanning some 90 km, down to the river delta. The possible environmental impact was studied by a combination of field and laboratory studies. Mercury concentrations in liver, kidney and muscle of feral carp ( Cyprinus carpio) sampled downstream Flix were one to two orders of magnitude higher than those from carps sampled upstream Flix. Elevated levels of mercury in these samples associated with significant increases on the concentration of reduced glutathione (GSH) in liver and on mRNA expression of two metallothionein genes, MT1 and MT2, in kidney and, partially, in scales, but not in liver. Conversely, no biochemical evidence for oxidative stress or DNA damage was found in these tissues. Non-contaminated carps subjected to intraperitoneal mercury injection resulted in a 20-fold increase of MT1 and MT2 mRNA levels in carp kidney, with minimal changes in liver levels. Our data suggests the coordinate increase of metallothionein mRNA in kidney and of GSH in liver constitutes an excellent marker of exposure to sub-toxic mercury levels in carps. This study also demonstrates that apparently healthy fish populations may exceed the mercury contamination acceptable for human consumption.

Expression of metallothionein mRNAs on mouse cerebellum microglia cells by thimerosal and its metabolites

Effects of thimerosal and its metabolites, ethyl mercury and thiosalicylate, on the expression of metallothionein (MT) mRNAs in mouse cerebellum microglia cell line, C8-B4 cells, were studied. The level of MT-1 mRNA significantly decreased at early hours and recovered time-dependently 24 h after thimerosal was added to the C8-B4 cells. However, MT-2 and MT-3 mRNA expressions did not change from the control group. In contrast, the expression of MT-1 mRNA increased in a mouse neuroblastoma cell line 6 h after incubation with thimerosal. In addition, the level of MT-1 mRNA decreased in C8-B4 cells 6 h after the addition of thiosalicylate, but ethyl mercury induced MT-1 mRNA expression. When cell viability was compared with thimerosal, thiosalicylate, and ethyl mercury, the viability of C8-B4 cells decreased dose-dependently 24 h after either thimerosal or ethyl mercury was added; however, the viability increased dose-dependently until 15 μM thiosalicylate was added. From the present results, it is concluded that the expression of MT-1 mRNA may be mediated by different factors than the expression of MT-2 mRNA in C8-B4 cells. The reduction of MT-1 mRNA level by thiosalicylate may affect the proliferation of C8-B4 cells.

Modification of dietary copper levels on the early stage of tumor-promotion with propylthiouracil in a rat two-stage thyroid carcinogenesis model

To investigate the role of copper (Cu)-related cellular responses on thyroid carcinogenesis, the expression of ceruloplasmin (Cp) and metallothionein (MT)-1/2 were examined in relation to the activities of cell proliferation/apoptosis in the thyroid of rats at an early stage of tumor promotion under different dietary Cu levels. Male F344 rats were initiated with N-bis(2-hydroxypropyl)nitrosamine by single subcutaneous injection at 2800 mg/kg body weight, and 1 week later promoted with 6-propyl-2-thiouracil at 12 ppm in the drinking water for 4 weeks. Animals were fed a diet containing Cu at 0.6, 6 or 60 ppm from the time point of initiator-treatment to create marginally deficient, normal, or non-toxic supplementary levels of Cu. At both 0.6 and 60 ppm, the multiplicity of preneoplastic focal follicular cell hyperplasias (FFCHs) was decreased as compared with 6 ppm Cu, while adenomas also decreased at 0.6 ppm Cu. Both 0.6 and 60 ppm Cu levels revealed decreased Ki-67-immunoreactive proliferating cells in both FFCHs and surrounding follicles accompanied by mRNA downregulation of Cdc2a and Ccnb1, while TUNEL-positive apoptotic cells were unaltered with change of dietary Cu. Both Cp and MT-1/2 were immunolocalized in FFCHs and adenomas, with higher distribution in the latter. At both 0.6 and 60 ppm, the immunoreactivities and/or thyroidal mRNA levels of Cp and MT-1/2 were also decreased. Transcript levels of several antioxidant enzymes were up- or downregulated in the same direction at both Cu levels. Serum levels of thyroid-related hormones were unaltered at both Cu levels, except for non-significant reduction of thyroid-stimulating hormone at 0.6 ppm. These results suggest an involvement of Cp and MT-1/2 on the thyroid tumor promotion that can be suppressed by dietary Cu level through inhibition of cell proliferation associated with altered redox balance.

Expression of Metallothionein-1 (MT-1) mRNA in the Rat Testes and Liver After Cadmium Injection

Metallothioneins (MTs) belong to the family of stress proteins that are present in the majority of living organisms. The MTs play an important task in detoxifying heavy metals. The mammalian scrotal testis is known to be susceptible to cadmium (Cd) exposure. The present work focuses on the MT-1 isoform and aims to ascertain and confirm previous findings to answer whether rodent testes indeed contain MT-1 mRNA, whether its level is increased with Cd injection in liver and testes, and lastly what is the relative difference in the expression of MT-1 mRNA in liver and testes both with and without Cd injection. Adult male Wistar rats weighing 270–290 g received a subcutaneous injection of 4.0 μmol Cd/kg and were sacrificed by cervical dislocation 6 h later. RNA was isolated from testes as well as the liver. There were 2 replicates per treatment for RNA analyses. MT-1 mRNA levels were determined by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis and then assessed by densitometry scanning. The results of RT-PCR clearly demonstrated that the rodent testes express MT-1 mRNA. The densitometry data shows that the expression of MT-1 mRNA increases with Cd treatment in testes. The relative level of MT1-mRNA is greater in the control-liver than in the control-testes. However, upon Cd injection, the level of testes MT-1 mRNA increases 2.16 fold. These results suggest that the testes respond to Cd for at least 6 h post injection through a transcriptional mechanism.

Expression of the melatonin receptor (MT) 1 in benign and malignant human bone tumors

The beneficial effects of melatonin on bone homeostasis have been shown in various diseases. As this indoleamine causes dose-dependent modulation of bone-forming osteoblast and bone-resorbing osteoclast activities by receptor-independent and -dependent pathways, we investigated the expression of G-protein-coupled melatonin receptors (MTs) in malignant and non-malignant human bone lesions. By TaqMan polymerase chain reaction (PCR), we analyzed 30 specimens from osteosarcoma and 11 from benign bone tumors for MT1-mRNA expression. Furthermore, we determined mRNA expression levels of the osteoclast activity-stimulating receptor activator of nuclear factor-kappa B ligand (RANKL) and its counterpart osteoprotegerin (OPG). Although mean MT1-mRNA levels were similar (P = 0.596) in malignant (4.39 +/- 4.98-fold) and benign samples (4.64 +/- 6.81-fold), the highest MT1-mRNA levels (up to 27-fold) were observed in individual osteosarcomas, particularly, in two specimens of patients with local recurrence of the tumor. Moreover, mean RANKL- and OPG-mRNA levels were similar in malignant and benign specimens (RANKL: 7.38 +/- 9.61-fold versus 3.57 +/- 3.11-fold, P = 0.207; OPG: 23.45 +/- 32.76 versus 8.07 +/- 7.23-fold, P = 0.133). Again, highest RANKL- and OPG-mRNA levels (up to 41- and 160-fold, respectively) were observed in individual osteosarcomas. Expression of MT1-mRNA was confirmed in two human osteosarcoma cell lines (HOS, MG63). High expression levels of MT1-mRNA together with low OPG-mRNA were found in both osteosarcoma cell lines, while in normal human osteoblasts and bone marrow stromal cells, high OPG-mRNA levels were associated with low MT1-mRNA levels. These data on the abundant expression of MT1-mRNA in human bone tumors and osteosarcoma cells lines suggest an important role for MT1 in bone pathology.

Induction of Metallothionein Synthesis by Cilostazol in Mice and in Human Cultured Neuronal Cell Lines

In this study, we examined the effect of Cilostazol to induce metallothionein (MT) in vivo and in vitro. Intraperitoneal injection of Cilostazol increased the expression of both MT-1 and MT-2 mRNA and total MT protein in the mouse liver. Cilostazol also augmented MT-1 mRNA levels in the murine brain. In vitro exposure to Cilostazol significantly augmented intracellular MT protein levels in cultured human brain microvascular endothelial cells (HBMEC) and in the neuroblastoma cell line IMR32. Taken together, these findings suggest that Cilostazol is an inducer of MT in the murine liver and brain, and that it has the potential to directly induce MT in cells. The contribution of the anti-oxidative effect of MT to the anti-stroke effect of Cilostazol was discussed.

Acute and prolonged effects of TNF-α on the expression and secretion of inflammation-related adipokines by human adipocytes differentiated in culture

The pro-inflammatory cytokine TNF-alpha has multiple effects on adipocyte function, including the production of adipokines. In this paper, we have examined the acute vs prolonged effects of TNF-alpha on the expression and secretion of key inflammation-related adipokines by human adipocytes. Adipocytes differentiated in culture were treated with TNF-alpha for 1-24 h, mRNA quantitated by real-time polymerase chain reaction (PCR) and secreted adipokines by ELISA. Treatment of adipocytes with TNF-alpha for up to 24 h had little effect on MIF, MT-2 and PAI-1 mRNA levels. TNF-alpha decreased adiponectin, adipsin, haptoglobin and leptin mRNA levels by 24 h, but adiponectin and haptoglobin mRNA was initially increased. In contrast, TNF-alpha induced rapid and substantial increases in expression of the genes encoding IL-6, MCP-1, NGF and TNF-alpha itself; IL-6 and TNF-alpha mRNA levels peaked at 2 h with 75-fold and 600-fold increases, respectively. The elevated MCP-1, NGF and VEGF mRNA levels were sustained between 4 and 24 h. The adipokine secretion pattern largely paralleled cellular mRNA levels; IL-6 (transiently), MCP-1, NGF and VEGF release were stimulated by TNF-alpha, with an accelerating rate of MCP-1 secretion over 24 h. TNF-alpha has rapid and substantial effects on the synthesis of key inflammation-related adipokines in human adipocytes, with highly gene-specific responses.

Changes of Metallothionein 1 and 3 mRNA Levels with Age in Brain of Senescence-Accelerated Mice and The Effects of Acupuncture

The effects of aging and acupuncture on brain MT1 and MT3 mRNA levels in senescence-accelerated mice (SAMP10) and accelerated senescence resistant mice (SAMR1) were analyzed by Northern blot analysis. Both MT1 and MT3 mRNA levels in SAMR1 were increased significantly from birth to month 4 and decreased gradually thereafter. In SAMP10, the MT3 mRNA level followed the same pattern as in SAMR1 before month 4, then decreased from month 4 to 6, but was over-expressed and exceeded the previous level at month 8. The MT1 mRNA expression in SAMP10 showed a zigzag pattern. Of two groups of SAMP10 mice treated with acupuncture, the xingnao group (PC6 and Du26 as acupoints) and the zibuganshen group (BL18 and BL23 as acupoints), both showed a higher MT1 mRNA level and a lower MT3 mRNA level than the age-matched control group. Meanwhile, in both of the acupuncture groups, the ratios of MT3 to MT1 were down-regulated to the normal range. Overall, these results suggested that over-expression of MT3 mRNA and the increase in MT3 to MT1 ratios in SAMP10 were correlated with aging, and could be an important physiological and pathological event in the aging process. Acupuncture altered the expression levels of MT1 and MT3 mRNA and differences between the effects of the two stimulated acupoints were seen. Therefore, maintenance of the balance between MTs mRNA expression and correct MTs concentrations is crucial for brain-endocrine-immune response and normal aging. Acupuncture could improve zinc ion bioavailability, by maintaining the balance between MT1 and MT3 mRNA expression levels and might explain one of the mechanisms by which acupuncture treatments defer aging and treat some age-related neurodegenerative diseases.

Tilapia metallothionein genes: PCR-cloning and gene expression studies

Genomic PCR reactions were performed to isolate gene sequences of tilapia metallothionein (tiMT) from Oreochromis mossambicus and Oreochromis aureus. Two AP1 binding sites, four metal responsive elements, and a TATA box are the major cis-acting elements identified in the 800-bp 5′ flanking region of the tiMTs obtained in this study. The tiMT gene promoter cloned from O. aureus was characterized in vitro using PLHC-1 cell-line, a hepatocellular carcinoma of a desert topminnow ( Poecciliopsis lucida), following the administrations of Cd 2+, Co 2+, Cu 2+, Ni 2+, Pb 2+ and Zn 2+. Only Cd 2+, Pb 2+ and Zn 2+ were able to induce the transcription of tiMT gene promoter in PLHC-1 cells in a dose-dependent manner. Zn 2+ had the highest fold induction of tiMT gene promoter activity. Deletion mutants were tested for their abilities to drive the transcription of reporter gene following Cd 2+ and Zn 2+ administrations. However, Cu 2+ and Ni 2+ also induced the production of hepatic MT mRNA in vivo. Northern blot analysis showed that liver gave the highest fold induction of MT gene expression following the administration of heavy metal ions. These data indicated that hepatic MT mRNA level in tilapia is a potential sensitive biomarker of exposure to various metal ions including Cu 2+, Cd 2+, Ni 2+, Pb 2+, Hg 2+ and Zn 2+ ions.

Effect of quercetin on metallothionein, nitric oxide synthases and cyclooxygenase-2 expression on experimental chronic cadmium nephrotoxicity in rats

Inflammation can play a key role in Cd-induced dysfunctions. Quercetin is a potent oxygen free radical scavenger and a metal chelator. Our aim was to study the effect of quercetin on Cd-induced kidney damage and metallothionein expression. The study was performed in Wistar rats that were administered during 9 weeks with either cadmium (1.2 mg Cd/kg/day, s.c.), quercetin (50 mg/kg/day, i.p.) or cadmium + quercetin. Renal toxicity was evaluated by measuring blood urea nitrogen concentration and urinary excretion of enzymes marker of tubular damage. Endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) renal expression were assessed by Western blot. Renal expression of metallothionein 1 and 2 (MT-1, MT-2) and eNOS mRNA was assessed by Northern blot. Our data demonstrated that Cd-induced renal toxicity was markedly reduced in rats that also received quercetin. MT-1 and MT-2 mRNA levels in kidney were substantially increased during treatment with Cd, being even higher when the animals received Cd and quercetin. Renal eNOS expression was significantly higher in rats receiving Cd and quercetin than in animals receiving Cd alone or in control rats. In the group that received Cd, COX-2 and iNOS expression was markedly higher than in control rats. In the group Cd+quercetin, no changes in COX-2 and iNOS expression were observed compared with the control group. Our results demonstrate that quercetin treatment prevents Cd-induced overexpression of iNOS and COX-2, and increases MT expression. These effects can explain the protection by quercetin of Cd-induced nephrotoxicity.

Regulation of mitochondrial gene expression by energy demand in neural cells

Mitochondrial DNA (mtDNA) encodes critical subunit proteins of the oxidative phosphorylation (OXPHOS) complex that generates ATP. This study tested the hypothesis that mitochondrial gene expression in neural cells is regulated by energy demand, as modified via stimulation of cellular sodium transport. Exposure of PC12S cells to the sodium ionophore monensin (250 nm) for 1-6 h caused a 13-60% decrease in cellular ATP (from 15 to 5 nmol per mg protein at 6 h). Levels of mitochondrial DNA-encoded mRNAs (mt-mRNAs) increased significantly (150%) within the first hour of exposure to monensin, and then decreased significantly (50%) at 3-4 h. Levels of mtDNA-encoded 12S rRNA and nuclear DNA-encoded OXPHOS subunit mRNAs were not significantly affected. Exposure of primary cerebellar neuronal cultures to the excitatory amino acid glutamate caused a similar rapid and significant increase followed by a significant decrease in cell mt-mRNA levels. The monensin-induced initial increase in mt-mRNA levels was abolished by pretreatment with actinomycin D or by reducing extracellular sodium ion concentration. The monensin-induced delayed reduction in mt-mRNA levels was accelerated in the presence of actinomycin D, and was accompanied by a 67% reduction in the half-life (from 3.6 to 1.2 h). Exposure of PC12S cells to 2-deoxy-d-glucose significantly decreased cellular ATP levels (from 14.2 to 7.1 nmol per mg protein at 8 h), and increased mt-mRNA levels. These results suggest a physiological transcriptional mechanism of regulation of mitochondrial gene expression by energy demand and a post-transcriptional regulation that is independent of energy status of the cell.

Interferon alpha induction of metallothionein in rat liver is not linked to interleukin-1, interleukin-6, or tumor necrosis factor alpha

Synthesis of metallothionein (MT) is induced by interferon-alpha (IFN-α) in vitro and in vivo. In addition, IFN-α promotes redistribution of zinc (Zn) from the plasma to the liver in mice. However, it is not clear if IFN-α induces hepatic MT synthesis directly or indirectly via liberation of other cytokines. In order to address this issue, we determined hepatic MT levels, Zn concentration in plasma, liver, and urine, and plasma levels interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNFα) in rats following intramuscular injection of human IFN-α (1.5 × 10 6 UI/m 2). Animals were housed in metabolic cages and sacrificed at various times after IFN-α administration. Zn concentrations in serum, urine, and hepatic tissue were determined by atomic absorption spectrophotometry. MT protein was measured using the MT silver saturation method and expression of MT-1 and MT-2 mRNA was measured by RT-PCR. Plasma levels of rat IL-1, IL-6, and TNFα were determined using an ELISA method. Hepatic MT levels began to increase at 2 h following IFN-α administration and reached maximum levels at 12 h post-treatment. Induction of MT gene expression was confirmed by increases in MT-1 and MT-2 mRNA levels 6, 12, and 18 h after IFN-α administration. IFN-α treatment also resulted in biphasic increases in hepatic Zn, with levels peaking at 2 h, the time-point when MT levels are first increased, and again at 18 h. Concurrently, there were decreases in serum Zn levels at these time points, suggesting IFN-α induced movement of Zn from the blood to hepatic tissue. The decrease in serum Zn was not due to increased excretion since urinary Zn levels were unaffected following IFN-α treatment. IFN-α administration had no effect on plasma IL-1, IL-6, and TNFα levels. These results show that IFN-α promotes the increase of hepatic MT levels and plasma/liver redistribution directly, without IL-1, IL-6, or TNFα participation.

Metallothionein gene expression in peripheral lymphocytes and renal dysfunction in a population environmentally exposed to cadmium

In order to study the validity of metallothionein (MT) gene expression in peripheral blood lymphocytes (PBLs) as a biomarker of cadmium exposure and susceptibility to renal dysfunction, MT mRNA levels were measured using reverse transcription polymerase chain reaction (RT-PCR) in PBLs from residents living in a cadmium-contaminated area. MT mRNA levels were found to increase with the increase of blood cadmium (BCd) and urinary cadmium (UCd) levels. Basal MT mRNA levels were significantly correlated with the logarithm of BCd levels and the logarithm of UCd levels confirming that MT expression in PBLs is a biomarker of cadmium exposure and internal dose. An inverse relationship was observed between in vitro induced MT-mRNA level in PBLs and urinary N-acetyl-β- d-glucosaminidase (UNAG) suggesting that MT gene expression in PBLs may be used as a biomarker of susceptibility to renal toxicity of cadmium.

Protective role of metallothionein in stress-induced gastric ulcer in rats

To illustrate the pathophysiological role of metallothionein (MT) in gastric ulcer induced by stress. Wistar rats underwent water-immersion-restraint (WIR) stress, ZnSO(4) (an MT inducer) treatment, WIR+ZnSO(4) or WIR+MT, and the ulcer index (UI) was estimated in excised stomach and liver tissues. The mRNA level of gastric MT was determined by semi-quantitative RT-PCR. The MT content in gastric and hepatic tissues was determined by Cd/hemoglobin affinity assay. The lipid peroxidation products malondialdehyde (MDA) and conjugated dienes (CD) were estimated by use of thiobarbituric acid reactive species and ultraviolet spectrophotometry. WIR stress induced severe gastric mucosal lesions in rats. Compared with control rats, stressed rats had increased lipid peroxide content in serum and stomach and liver tissues. MDA content was increased by 34%, 21% and 29% and CD level by 270%, 83% and 28%, respectively. MT content in the stomach and liver was increased by 0.74- and 1.8-fold, and the MT-mRNA level in the stomach was increased by 26%. Pretreatment with ZnSO(4) prevented gastric lesion development (the UI was 87% lower than that without pretreatment), and the MDA and CD content in serum and tissues was lower. The MT content in the liver was double in rats that were not pretreated, and the MT mRNA level in the stomach was 35% higher. MT administration 1 h before the WIR stress prevented gastric lesion development (the UI decreased by 47% compared with that in rats not pretreated), and the MDA and CD content in serum and tissues was significantly lower. In WIR-stressed rats, the MT level was increased in serum and in stomach and liver tissues. Pre-administration of exogenous MT or pre-induction of endogenous MT can protect the gastric mucosa against stress-induced ulcers and inhibits the formation of stress-induced lipid peroxide. MT could have a gastroprotective effect and might be a new interventive and therapeutic target in stress-induced gastric ulcers.

Metallothionein gene expression under different time in testicular Sertoli and spermatogenic cells of rats treated with cadmium

The rodent testes are generally more susceptible to cadmium (Cd)-induced toxicity than the liver. To clarify the molecular mechanism underlying tissue and cell differences in Cd sensitivity, we compared metallothionein (MT) gene expression, MT protein accumulation, and Cd retention under different times in freshly isolated testicular Sertoli and spermatogenic cells and liver of rats treated with Cd. Adult male Sprague–Dawley rats received a s.c. injection of 4.0 μmol Cd/kg and 1, 3, 6, or 24 h later and untreated animals (0 h) tissue were sampled and testicular Sertoli and spermatogenic cells isolated. MT1 and MT2 mRNA levels were determined by semi-quantitative RT-PCR analysis followed by densitometry scanning, and MT was estimated by the enzyme-linked immunosorbent assay (ELISA) method. Cadmium content was determined by atomic absorption spectrophotometry. Testicular lesions were not grossly or histologically observed in rats treated with 4.0 μmol Cd/kg. In the present study, we demonstrated that the rat testis indeed expressed MT1 and MT2, the major isoforms. We also found that untreated animals contained relatively high basal levels of both isoform mRNA, which were increased after Cd treatment in liver and peaked at 3 h, followed by a decline, in contrast, the mRNA levels in Sertoli cells peaked at 6 h. Interestingly, the induction of MT1 mRNA was lower than MT2 mRNA in Sertoli cells and liver of rats treated with Cd. However, the MT1 mRNA levels of spermatogenic cells decreased 0–3 h after Cd treatment, followed by an increase; in contrast, MT2 mRNA levels increased 0–3 h after Cd treatment, followed by a reduction, but induced extents of them are lower than those of Sertoli cells and liver. Cd exposure substantially increased hepatic MT, but did not increase MT translation in Sertoli and spermatogenic cells. These results indicate: (1) that Cd-induced MT mRNA expression is cell- and time-dependent; (2) that the inability to induce the metal-detoxicating MT protein in response to Cd, might account for higher susceptibility of testes to Cd toxicity and carcinogenesis relative to liver.