Evaluation of comparative effect of pre‐ and posttreatment of selenium on mercury‐induced oxidative stress,histological alterations,and metallothionein mRNA expression in rats

To evaluate the effect of pre‐ or posttreatment of selenium (6 μmol/kg b.w., single intraperitoneal injection) in mercury intoxication, rats were exposed to mercury (12 μmol/kg b.w., single intraperitoneal injection). Exposure to mercury resulted in induced oxidative stress in liver, kidney, and brain tissues. Marked changes in serum biochemical parameters together with alterations in histopathology and an induction in metallothionein‐I and metallothionein‐II mRNA expression in the liver and kidney were observed. Pretreatment with selenium to mercury‐exposed animals had protective effect on the liver, whereas posttreatment had partial protection on restoration of altered oxidative stress parameters. In the kidney, pretreatment with selenium showed partial protection on restoration of altered biochemical parameters, whereas no protection was observed in posttreatment. The pretreatment with selenium resulted in restoration of mercury‐induced metallothionein‐I and metallothionein‐II mRNA expression, which was completely restored in the liver whereas partial restoration was observed in the kidney. Posttreatment with selenium resulted in further induction in metallothionein‐I and metallothionein‐II mRNA expression in the liver and kidney. In the brain, selenium showed partial protection on alerted biochemical parameters. Results indicate that pretreatment with selenium is beneficial in comparison to posttreatment in mercury intoxication. Thus, dietary intake of selenium within safe limit may, therefore, enable us in combating any foreseen effects due to mercury exposure. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:123–135, 2010; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20320     

In vivo and ex vivo displacement of zinc from metallothionein by cadmium and by mercury

Divalent cadmium and mercury ions are capable in vitro of displacement of zinc from metallothionein. This process has now been studied in vivo and ex vivo, using the isolated perfused rat liver system, in order to determine if this process can occur in the intact cell. Rats with normal and elevated (via preinduction with zinc) levels of hepatic zinc thionein were studied. Cd(II) completely displaces zinc from normal levels of metallothionein and on a one-to-one basis from elevated levels of metallothionein, both in vivo and ex vivo. Hg(II) displaces zinc from metallothionein (normal or elevated) rather poorly, as compared with Cd(II), in vivo, probably due to the kidneys preference for absorbing this metal. Ex vivo Hg(II) displaces zinc from metallothionein (normal or elevated) on a one-to-one basis, with considerably more mercury being incorporated into the protein than in vivo. The results of double-label ex vivo experiments using metal and [35S]cysteine (+/- cycloheximide) were consistent with the above experiments, indicating that de novo thionein synthesis was not required for short term incorporation of cadmium and mercury into metallothionein. These data are supportive of the hypothesis that cadmium and mercury incorporation into rat hepatic metallothionein during the first few hours after exposure to these metals can occur primarily by displacement of zinc from preexisting zinc thionein by a process which does not require new protein synthesis.     

The localization of mercury and metallothionein in the cerebellum of rats experimentally exposed to methylmercury

Summary Rats were dosed with methylmercuric chloride, either by gastric gavage (5 × 10 mg kg^-1 body weight over a 15-day period), or in their drinking water (20 mg methylmercuric chloride l^–1 for 14 or 42 days). Localization of mercury within the cerebellum was performed with a silver physical development technique, and metallothionein with dinitrophenyl hapten-sandwich immunohistochemistry. Mercury was detected in structurally undamaged Purkinje neurones and adjacent Bergmann glial cells; no mercury was detected in granule cells even though these small cells nearest the Purkinje layer had a high incidence of pyknotic nuclei. In general, metallothionein was detected mainly in Bergmann glial cells, Purkinje cells, astrocytes and glial cells of white matter; no metallothionein was detected in granule cells. We hypothesized that the resistance of Purkinje cells to methylmercuric chloride reflects their ability to transform organic mercurials to inorganic mercury that, in turn, induces the synthesis of radical-scavenging metallothionein molecules.     

Variation of the level of mercury and metallothionein in the kidneys and liver of rats with time of exposure to sodium selenite

Sodium selenite was administered to rats before, after, and simultaneously with mercuric chloride. In all animal groups, mercury was administered intravenously in doses of 0.5 mg/kg every other day for two weeks. Selenium was given intragastrically either in a single dose of 7.0 mg Se/kg or in repeated doses of 0.1 mg Se/kg every day for weeks. It was demonstrated that, depending on the administration schedule, selenium induced significant changes in the binding of mercury by soluble fraction proteins both in the kidneys and in the liver. In every exposure, the mercury content decreased mainly in the low-molecular weight proteins, and the level of metallothionein-like proteins was diminished in the both organs. In the kidneys, the mercury content showed a correlation with the level of metallothionein (r=0.78). Amounts of mercury below 10 μg/g kidney do not stimulate metallothionein biosynthesis in this organ. A distinct interaction effect was observed in the case of a simultaneous administration of equimolar amounts of both the metals in question.     

Reactivity of propylene oxides towards deoxycytidine and identification of reaction products

In order to elucidate the mechanism of the stimulative effect of molybdenum on mercury-mediated renal metallothionein induction, the levels of translatable metallothionein mRNA (MT mRNA) in the kidneys of rats treated with saline or Na2MoO4 or HgCl2 or Na2MoO4 and HgCl2 were measured by translation experiments in cell-free protein synthesizing systems. The time course of accumulation of mercury in renal nuclei of rats given HgCl2 with or without Na2MoO4-pretreatment was also investigated. Molybdenum, itself, did not elevate levels of MT mRNA compared to saline controls at all time points (0, 6 and 14 h after exposure to HgCl2) but rapidly elevated the levels of the mRNA more than Hg-dosed rats when HgCl2 was also administered. On the other hand, the time course study in renal nuclei showed that the mercury content of nuclei was consistently lower in Mo-Hg-dosed rats than in Hg-dosed rats at all time points (4, 8 and 24 h after exposure to HgCl2). These results suggest that the stimulative effect of molybdenum on mercury-mediated metallothionein induction is coupled with an increase of the mRNA coding for the low molecular weight protein and that such an increase in the levels of translatable MT mRNA is not due to the difference in uptake of mercury into renal nuclei.     

Involvement of metallothionein and copper in cell proliferation

Metallothionein is a low-molecular weight, cysteine-rich, metal-binding protein which has been implicated in the detoxification of toxic metals (cadmium, mercury), metabolism of zinc and copper, as well as in the scavenging of free radicals. Recent evidence suggests that the protein may also be involved in cell proliferation. Based on the experiments carried out so far, it is assumed that the fundamental role of metallothionein in cell proliferation may be to detoxify and/or transfer copper ions from the cytoplasm to the nucleus at the G₁/S phase, which in turn participate in some way in nuclear DNA synthesis.     

Multi-biomarker responses in the freshwater mussel Dreissena polymorpha exposed to polychlorobiphenyls and metals

Contaminant related changes in behavioral, phase I and II metabolizing enzymes and pro-oxidant/antioxidant processes in the freshwater mussels Dreissena polymorpha exposed to metals and PCBs were assessed. Behavioral and biochemical responses including filtering rates, key phase I, II and antioxidant enzymes and levels of metallothioneins, glutathione, lipid peroxidation and DNA strand breaks were determined in digestive glands of mussels after being exposed to sublethal levels of mercury chloride, methyl mercury, cadmium and Aroclor 1260 during 5 days. In 7 out of 12 responses analyzed, mussels showed significant differences across treatments. Unusual properties of measured ethoxyresorufin-O-deethylase (EROD) activities indicated that mussels lack an inducible CYP1A enzymatic activity. Despite of using similar exposure levels, inorganic and organic mercury showed different biomarker patterns of response with methyl mercury being more bio-available and unable to induce metallothionein proteins. Mussels exposed to Cd presented higher levels of metallothioneins and an enhanced metabolism of glutathione, whereas those exposed to Aroclor showed their antioxidant glutathione peroxidase related enzyme activities inhibited. Although there was evidence for increased lipid peroxidation under exposure to inorganic and organic mercury, only mussels exposed to Aroclor had significant greater levels than those in controls.     

Low-molecular weight metalloproteins in tissues of the narwhal (Monodon monoceros)

Narwhal (Monodon monoceros) liver and kidney cytosol were fractionated by gel chromatography, anion-exchange chromatography and electrophoresis. Cadmium was associated largely with low molecular weight proteins, while mercury was associated also with high molecular weight proteins, but apparently not because of saturation of the metallothionein mechanism. Eight different electrophoretic bands, four of which were metalloproteins, were found under the "metallothionein" peak. Anion-exchange chromatography yielded five metal peaks while further fractionation on G-50 gave two peaks, one containing almost pure metallothionein (Mt-1) and the other a metalloprotein having twice the molecular weight of metallothionein. Mt-2 was observed, at a much lower concentration than Mt-1, in liver but not kidney.     

Comparative distributions of zinc, cadmium and mercury in the tissues of experimental mice

Different groups of mice were injected with cadmium, zinc and mercury. Zinc injections had no effect on zinc tissue levels while both mercury and cadmium accumulated in various tissues. Cadmium persisted in the tissues much longer than mercury, and while the mercury concentrations began to decline as soon as dosing ceased, cadmium concentrations in kidney and intestine increased even after dosing ceased. There appeared to be an interrelationship between cadmium concentrations in spleen and intestine which warrants some further investigations. There was a linear, but discontinuous, effect of cadmium on zinc concentrations in liver, kidney and pancreas which may depend on metallothionein biochemistry. Mercury injections had no effect on zinc metabolism. It is proposed that differences in the rate of excretion of cadmium and mercury from the kidney could explain the differential accumulation of cadmium and mercury in animals.     

Yeast metallothionein function in metal ion detoxification

A genetic approach was taken to test the function of yeast metallothionein in metal ion detoxification. A yeast strain was constructed in which the metallothionein locus was deleted (cup1 delta). The cup1 delta strain was complemented with normal or mutant metallothionein genes under normal or constitutive regulatory control on high copy episomal plasmids. Metal resistance of the cup1 delta strain with and without the metallothionein-expressing vectors was analyzed. The normally regulated metallothionein gene conferred resistance only to copper (1000-fold); constitutively expressed metallothionein conferred resistance to both copper (500-fold) and cadmium (1000-fold), but not to mercury, zinc, silver, cobalt, nickel, gold, platinum, lanthanum, uranium, or tin. Two mutant versions of the metallothionein gene were constructed and tested for their ability to confer metal resistance in the cup1 delta background. The first had a deletion of a highly conserved amino acid sequence (Lys-Lys-Ser-Cys-Cys-Ser). The second was a hybrid gene consisting of the sequences coding for the first 20 amino acids of the yeast protein fused to the monkey metallothionein gene. Expression of these genes under the CUP1 promoter provided significant protection from copper, but none of the other metals tested. These results demonstrate that there is significant flexibility in the structural requirements for metallothionein to function in copper detoxification and that yeast metallothionein is also capable of detoxifying cadmium under conditions of constitutive expression.     

Bioaccumulation of mercury from wastewater by genetically engineered Escherichia coli

Genetically engineered E. coli, which express both a Hg2+ transport system and metallothionein, were tested for their ability to remove mercury from wastewater. The wastewater contained more than ten different ions, including 2.58 mg/l mercury, and its pH was 9.6. Mercury uptake was faster from the wastewater than from distilled water, probably because of the higher ionic strength, as the high pH had little effect on mercury accumulation. EDTA also stimulated mercury uptake rather than inhibiting it. A hollow-fiber bioreactor was used to retain induced cells for continuous mercury uptake. The cells removed more than 99% of the mercury in the wastewater and the final amount of mercury accumulated was 26.8 mg/g cell dry weight, while none of the other ions were removed from the water. These results indicated that the induced cells had a high affinity and specificity for mercury.     

Spectroscopic characterization of rat kidney Hg,Cu-metallothionein

Absorption, circular dichroism (CD), magnetic circular dichroism (MCD) and emission spectra are reported for rat kidney Hg,Cu-metallothionein isoform 3 isolated following induction of the metallothionein with HgCl2. While the absorption spectrum is featureless, both the CD and MCD spectra show resolved bands that arise from the Cu-thiolate and Hg-thiolate groups. The emission spectrum at 77 K is much more complicated than would be expected for a copper (I)-containing metallothionein. It is suggested the emission only arises from the copper-thiolate groups but that the presence of the mercury results in copper ions in several different environments depending on the nature of the nearest neighbour.     

Expression of the mouse metallothionein-I gene in Escherichia coli: increased tolerance to heavy metals

The cDNA of mouse metallothionein, a small metal-binding protein rich in cysteine, has been cloned downstream from a bacterial inducible promoter and expressed in Escherichia coli. Upon induction, E. coli harboring this cDNA clone contained a protein species readily labelled by [35S]cysteine in vivo and incorporated 10-times as much 109Cd from the medium than would otherwise be the case. We show that expression of metallothionein endows resistance in E. coli to heavy metal ions such as mercury, silver, copper, cadmium and zinc by sequestering rather than exclusion or conversion, common mechanisms of metal resistance in bacteria.     

Protective effect of sodium molybdate on the acute toxicity of mercuric chloride. V. Enhancement of renal regeneration after exposure to HgCl2

Pretreatment with Na2MoO4 protected rats from HgCl2-induced decreases in the renal concentration of amino acids, RNA, DNA, ATP and dry matter. It also reduced the mercury-induced increases in renal water, Ca and serum creatinine. Ma2MoO4 considerably elevated the RNA/DNA ratio in the renal cortex after treatment with HgCl2. In addition, subcellular distribution of mercury was markedly altered by pretreatment with Na2MoO4, specifically Na2MoO4 pretreatment decreased the mercury content in the particulate fractions such as the nuclei and mitochondria while increasing the mercury content of the cytosol. Sephadex G-75 gel filtration showed that the increase in mercury content in the cytosol of Na2MoO4-pretreated rats is due to an increase in the metal content of a metallothionein-like fraction. These results suggest that Na2MoO4-pretreatment protects against HgCl2 renal toxicity by stimulating mercury-mediated metallothionein induction in the renal cortex and renal regenerative processes.     

Urinary excretion of mercury, copper and zinc in subjects exposed to mercury vapour

The excretion of mercury, copper and zinc in urine, and mercury in whole blood andplasma, was determined in 40 chloralkali workers exposed to mercury vapour and 40age-matched referents. The Hg concentrations in whole blood, plasma and urine werehigher in the exposed group (35 nmol l, 30 nmol l,and 11.5 nmol mmol creatinine, respectively) in comparison with thereference group (15 nmol l, 6.3 nmol l, and 1.8nmol mmol creatinine, respectively). The urinary copper excretionwas similar in the two groups, while U-Zn excretion was significantly higher (P = 0.04)in the exposed group, median 0.83 mmol mmol creatinine versus 0.76mnmol mmol creatinine in the reference group. In a subgroup of exposedworkers with current U-Hg above 11.5 nmol lmmolcreatinine (20 mg g creatinine) the medianU-Zn was 1.1 mmol mmol creatinine. In both groups smokers had highU-Zn levels than non smokers. When both U-Hg and smoking were taken into account in alinear regression model, there was a significant association between U-Hg and U-Zn inthe combined group of exposed and referents (P = 0.002). This study indicates thatmercury exposure in humans, as in animals, causes increased urinary excretion of zinc.The mechanisms may be induced synthesis of metallothionein in the kidneys, displacementof Zn from preexisting metallothionein by Hg, or a decreased reabsorption of zinc in thekidneys owing to a slight tubular dysfunction.     

Mercury distribution and renal metallothionein induction after subchronic oral exposure in rats

The effects of long-term daily intake of low and high levels of mercury on its organ distribution and binding to renal metallothionein (MT) in male rats were studied. The animals were exposed to mercuric chloride labelled with²⁰³Hg via drinking water for 8 weeks (5, 50 and 500 m Hg). The greatest concentration of mercury was found in the kidneys. Similar levels of radioactivity in the buccal cavity and oesophagus were also observed by whole-body autoradiography. In the kidneys, the mercury was accumulated in the outer stripe of the outer zone of the medulla and, to a minor degree, in the renal cortex. Almost 50% the total renal mercury was associated to MT. The binding capacity of the renal MT for mercury tends to saturate with increasing doses, thus this means that the capacity of the kidneys to accumulate mercury is limited.     

Biochemical changes in rat kidney on exposure to elemental mercury vapor: effect on biosynthesis of metallothionein.

Evidence is presented that exposure of rats to elemental mercury vapor results in increased amounts of a metallothionein-like protein in kidney tissue but not in liver. After three or more daily exposures, each of 2 h duration, to elemental mercury vapor, more than 50% of the mercury in kidney tissue is bound to a protein having a molecular weight (mol. wt.) of about 10 000 as determined by Sephadex G-75 gel filtration chromatography. Cystine is incorporated into a 10 000 mol. wt. protein fraction from kidneys of rats which were injected with [U-14C] cystine after five daily 2-h exposures to mercury vapor. In contrast, no significant incorporation of [U-14C] cystine into this protein fraction was observed in kidneys of control rats or in livers of both control and mercury vapor-exposed rats. The in vivo incorporation of 109Cd into the fraction followed the same pattern as that of [14C] cystine in rats injected with tracer doses of CdCl2 labeled with radioactive 109Cd isotope. This 10 000 mol. wt. protein, newly synthesized in response to repeated exposures to mercury vapor, exhibited identical properties to metallothionein, namely in its subcellular localization, molecular weight, heat stability and isoelectric points. A significant incorporation of [U-14C]-cystine into this protein in rat kidney alone on exposure to mercury vapor confirms its induced biosynthesis in the kidney.