metallothionein and methylmercuric-chloride

Methyl mercury chloride "MMC" (CH

Topics: Animals; Antioxidants; Cichlids; Diet; Dietary Exposure; Dietary Supplements; Hematology; Liver; Metallothionein; Methylmercury Compounds; Oxidative Stress; RNA, Messenger

Methylmercury (MeHg) is a potent neurotoxin, and human beings are mainly exposed to this pollutant through fish consumption. We addressed the question of whether a diet mimicking the fish consumption of Wayanas Amerindians from French Guiana could result in observable adverse effects in mice. Wayanas adult men are subjected to a mean mercurial dose of 7 g Hg/week/kg of body weight. We decided to supplement a vegetarian-based mice diet with 0.1% of lyophilized Hoplias aimara fish, which Wayanas are fond of and equivalent to the same dose as that afflicting the Wayanas Amerindians. Total mercury contents were 1.4 ± 0.2 and 5.4 ± 0.5 ng Hg/g of food pellets for the control and aimara diets, respectively. After 14 months of exposure, the body parts and tissues displaying the highest mercury concentration on a dry weight (dw) basis were hair (733 ng/g) and kidney (511 ng/g), followed by the liver (77 ng/g). Surprisingly, despite the fact that MeHg is a neurotoxic compound, the brain accumulated low levels of mercury (35 ng/g in the cortex). The metallothionein (MT) protein concentration only increased in those tissues (kidney, muscles) in which MeHg demethylation had occurred. This can be taken as a molecular sign of divalent mercurial contamination since only Hg(2+) has been reported yet to induce MT accumulation in contaminated tissues. The suppression of the synthesis of the chemokine CCL2 in the corresponding knockout (KO) mice resulted in important changes in gene expression patterns in the liver and brain. After three months of exposure to an aimara-containing diet, eight of 10 genes selected (Sdhb, Cytb, Cox1, Sod1, Sod2, Mt2, Mdr1a and Bax) were repressed in wild-type mice liver whereas none presented a differential expression in KO Ccl2(-/-) mice. In the wild-type mice brain, six of 12 genes selected (Cytb, Cox1, Sod1, Sod2, Mdr1a and Bax) presented a stimulated expression, whereas all remained at the basal level of expression in KO Ccl2(-/-) mice. In the liver of aimara-fed mice, histological alterations were observed for an accumulated mercury concentration as low as 32 ng/g, dw, and metal deposits were observed within the cytoplasm of hepatic cells.

Topics: Adult; Animals; Chemokine CCL2; Fish Products; Food Contamination; French Guiana; Gene Expression Regulation; Humans; Male; Metallothionein; Methylmercury Compounds; Mice; Mice, Knockout; Organ Specificity

Among the naturally occurring three mercury species, metallic mercury (Hg(0)), inorganic mercury (Hg(II)) and methylmercury (MeHg), Hg(II) is well documented to induce metallothionein (MT) in tissues of injected animals. Although Hg(0) and MeHg are considered to be inert in terms of directly inducing MT, MT can be induced by them after in vivo conversion to Hg(II) in an animal body. In the present study we examined accumulations of inorganic mercury and MT inductions in mouse tissues (brain, liver and kidney) up to 72 hr after treatment by one of three mercury compounds of sub-lethal doses. Exposure to mercury compounds caused significant mercury accumulations in mouse tissues examined, except for the Hg(II)-treated mouse brain. Although MeHg caused the highest total mercury accumulation in all tissues among mercury compounds, the rates of inorganic mercury were less than 10% through the experimental period. MT inductions that depended on the inorganic mercury accumulation were observed in kidney and brain. However, MT induction in the liver could not be accounted for by the inorganic mercury accumulation, but by plasma IL6 levels, marked elevation of which was observed in Hg(II) or MeHg-treated mouse. The present study demonstrated that MT was induced in mouse tissues after each of three mercury compounds, Hg(0), Hg(II) and MeHg, but the induction processes were different among tissues. The induction would occur directly through accumulation of inorganic mercury in brain and kidney, whereas the hepatic MT might be induced secondarily through mercury-induced elevation in the plasma cytokines, rather than through mercury accumulation in the tissue.

Topics: Administration, Inhalation; Animals; Cerebellum; Cerebrum; Environmental Pollutants; Gene Expression; Interleukin-6; Kidney; Male; Mercuric Chloride; Mercury; Mercury Compounds; Metallothionein; Methylmercury Compounds; Mice; Mice, Inbred C57BL; RNA, Messenger

Mangiferin (MGN), a C-glucosylxanthone was investigated for its ability to protect against methylmercury (MeHg) induced neurotoxicity by employing IMR-32 (human neuroblastoma) cell line. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and clonogenic cell survival assays confirmed the efficacy of MGN supplementation in attenuating MeHg-induced cytotoxicity. Pre-treatment with MGN significantly (p<0.01) inhibited MeHg-induced DNA damage (micronuclei, olive tail moment and % tail DNA) thereby demonstrating MGN's antigenotoxic potential. Also, pre-treatment with MGN significantly reduced MeHg-induced oxidative stress, intra-cellular Ca(2+) influx and inhibited depolarization of mitochondrial membrane. MGN pre-treated cells demonstrated a significant (p<0.05) increase in the GSH and GST levels followed by a significant (p<0.05) decrease in malondialdehyde (MDA) formation. In addition, inhibition of MeHg induced apoptotic cell death by MGN was demonstrated by microscopic, Annexin-V FITC and DNA fragmentation assays and further confirmed by western blot analysis. The present findings indicated the protective effect of MGN against MeHg induced toxicity, which may be attributed to its anti-genotoxic, anti-apoptotic and anti-lipid peroxidative potential plausibly because of its free radical scavenging ability, which reduced the oxidative stress and in turn facilitated the down-regulation of mitochondrial apoptotic signalling pathways.

Topics: Annexin A5; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Calcium; Caspase 3; Cell Line, Tumor; Cell Nucleus; Cell Survival; Comet Assay; Cytochromes c; Cytokinesis; Cytoprotection; DNA Damage; DNA Fragmentation; Dose-Response Relationship, Drug; Drug Interactions; Free Radical Scavengers; Humans; Inhibitory Concentration 50; Intracellular Space; Membrane Potential, Mitochondrial; Metallothionein; Methylmercury Compounds; Micronucleus Tests; Necrosis; Neuroblastoma; NF-E2-Related Factor 2; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Tubulin; Tubulin Modulators; Tumor Stem Cell Assay; Xanthones

A neuron spinal chord x hybrid (NSC-34) cell culture derived from neonatal mouse was characterized for studies on mercury toxicity. Exposure of NSC-34 cells to methyl mercury chloride (MeHgCl) (0-16 microM) resulted in significant dose-dependent cell damage and death (P < 0.05). MeHgCl was more toxic than inorganic mercury (Hg2+) for both the NSC-34 cells and its parent neuroblastoma cell line N18TG-2 (P < 0.05). Hg2+, but not ZnCl2 or MeHg exposure induced metallothionein (MT) (P < 0.05). To mimic the increase in Hg2+ in the mammalian brain with long term MeHg exposure, the cells were treated with 1 microM mercuric chloride (HgCl2) for five passages before exposure to MeHgCl (1-16 microM) for 48 h. MeHgCl toxicity was measured by trypan blue exclusion, reduction of resazurin dye and acid phosphatase activity. Pre-exposure to HgCl2 lessened the toxicity as shown by trypan blue exclusion (P = 0.0559) and reduction of resazurin (P = 0.0001). Pre-exposure to HgCl2 also resulted in induction of MT (P = 0.0066) and lessened the decrease of reduced glutathione (GSH) (P = 0.0013). These results suggest that MT and GSH may play a protective role in methyl mercury induced neurotoxicity of neuron spinal chord cells. The NSC-34 hybrid cell line can be a useful model for the study of MeHg neurotoxicity.

Topics: Acid Phosphatase; Animals; Cells, Cultured; Glutathione; Hybrid Cells; Mercury; Metallothionein; Methylmercury Compounds; Mice; Neurons; Spinal Cord; Zinc

Metallothionein (MT) is one of the stress proteins which can easily be induced by various kind of heavy metals. However, MT in the brain is difficult to induce because of blood-brain barrier impermeability to most heavy metals. In this paper, we have attempted to induce brain MT in rats by exposure to methylmercury (MeHg) or metallic mercury vapor, both of which are known to penetrate the blood-brain barrier and cause neurological damage. Rats treated with MeHg (40 micromol/kg per day x 5 days, p.o.) showed brain Hg levels as high as 18 microg/g with slight neurological signs 10 days after final administration, but brain MT levels remained unchanged. However, rats exposed to Hg vapor for 7 days showed 7-8 microg Hg/g brain tissue 24 h after cessation of exposure. At that time brain MT levels were about twice the control levels. Although brain Hg levels fell gradually with a half-life of 26 days, MT levels induced by Hg exposure remained unchanged for > 2 weeks. Gel fractionation revealed that most Hg was in the brain cytosol fraction and thus bound to MT. Hybridization analysis showed that, despite a significant increase in MT-I and -II mRNA in brain, MT-III mRNA was less affected. Although significant Hg accumulation and MT induction were observed also in kidney and liver of Hg vapor-exposed rats, these decreased more quickly than in brain. The long-lived MT in brain might at least partly be accounted for by longer half-life of Hg accumulated there. The present results showed that exposure to Hg vapor might be a suitable procedure to provide an in vivo model with enhanced brain MT.

Topics: Animals; Brain; Kidney; Liver; Male; Mercury; Metallothionein; Methylmercury Compounds; Rats; Rats, Wistar; Time Factors

Mercuric chloride (MC, Hg2+) and methylmercury (MeHg, CH3Hg+) significantly inhibited the initial rates of uptake of 86Rb (a tracer for K+), as well as the Na(+)-dependent uptake of [3H]-L-glutamate. Both mercury species were also found to increase [3H]-D-aspartate and 86Rb release from cultured astrocytes. Astrocytes were more sensitive to the effects of MC with IC50's for glutamate and Rb uptake an order of magnitude lower than those noted for the organic species (MeHg). Increased potency, and irreversibility relative to MeHg, were also noted for MC induced astrocytic D-aspartate and Rb release. These observations support the hypothesis that the astrocyte plasma membrane is an important target for mercurials and specifically that low concentrations of MC and MeHg inhibit the ability of astrocytes to maintain transmembrane ion gradients. The propensity of MC to interfere with astrocytic functions, relative to MeHg, was also corroborated by measurements on the inducibility of the astrocytic metalloprotein, metallothionein (MT). Whilst a dose-dependent increase in MT protein synthesis occurred upon exposure to either MC or MeHg, MC was shown to be the more potent of the mercurials. The greater susceptibility of astrocytes to MC compared with MeHg lends support, at the cellular level, to the hypothesis that accumulation of inorganic mercury (MC) at an order of magnitude lower concentration than MeHg, may be equally neurotoxic.

Topics: Animals; Aspartic Acid; Astrocytes; Dose-Response Relationship, Drug; Glutamic Acid; Mercuric Chloride; Metallothionein; Methylmercury Compounds; Potassium; Rats; Rats, Sprague-Dawley

Rats were dosed with methylmercuric chloride, either by gastric gavage (5 x 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.

Topics: Administration, Oral; Animals; Cerebellar Cortex; Cerebellum; Frozen Sections; Immunoenzyme Techniques; Male; Mercury; Metallothionein; Methylmercury Compounds; Neuroglia; Neurons; Purkinje Cells; Rats; Rats, Wistar; Silver Staining

Simultaneous treatment of rats with ethanol (EtOH) and methylmercury (MeHg) increases the frequency of lesions in the rat kidney. Therefore, it was of interest to us to study the effects of simultaneous treatment of rats with MeHg and EtOH on kidney metallothionein (MT) and mercury residues levels in kidneys of rats maintained on 70% of ad libitum diet. Treatment with MeHg alone induced kidney MT the most (twice) compared to its pair-fed control. Simultaneous treatment with MeHg and EtOH also induced kidney MT but to a lesser degree than treatment with MeHg alone (by about 30%). Ethanol by itself caused a slight increase in kidney MT although starvation resulting from pair-feeding with mercury-treated animals may have contributed to this observation. Simultaneous treatment with MeHg and 2 g/kg EtOH caused a significant reduction in inorganic mercury levels in the kidney (P less than 0.05) compared to treatment with MeHg alone or in combination with 1 g/kg EtOH. Corresponding with the decrease in kidney inorganic mercury levels was a significant increase in urine inorganic mercury levels in this group compared to treatment with 1 g/kg ethanol + MeHg.

Topics: Animals; Diet; Drug Synergism; Ethanol; Kidney; Male; Mercury; Metallothionein; Methylmercury Compounds; Rats; Rats, Inbred Strains

Cockroach ileum has a high capability to concentrate mercury compared with other tissues. Part of the mercury contained in the soluble phase of this organ is bound to metallothionein. It is suggested that mercury of the insoluble phase is stored in lysosomes under a polymerized metallothionein form.

Topics: Animals; Cockroaches; Female; Ileum; Liver; Male; Mercury; Metallothionein; Methylmercury Compounds; Protein Binding; Spectrophotometry, Ultraviolet

The biotransformation efficiency of alkylmercurial compounds was studied in rat liver, kidneys, blood, and brain after 2-week administration of methylmercuric chloride (MeHg) and ethylmercuric chloride (EtHg) at doses of 0.25 or 2.5 mg Hg/kg, alone or in combination with sodium selenite (Se) at a level of 0.5 mg Se/kg. Simultaneously, the level of metallothioneinlike proteins (MTP) and endogenous copper (Cu) was monitored in tissues of control rats and intoxicated rats. Regardless of the dose, the highest concentrations of inorganic mercury from both the alkylmercurials was found in the rat kidneys. Sodium selenite had a variable effect on the amount of inorganic mercury liberated, depending on the organ and the molar ratio of Hg:Se administered. A statistically significant increase in the levels of MTP and endogenous Cu, compared with control group, was found only in the kidneys of intoxicated rats. This increase was dependent on the concentration of inorganic mercury liberated by biotransformation of alkylmercurials. The observed changes appeared when the level of inorganic mercury exceeded 10 micrograms Hg/g tissue and reached a plateau at about 40 micrograms Hg/g tissue. In the presence of selenium the plateau of MTP and Cu levels were no observed in the kidneys, regardless of the amount of inorganic mercury liberated.

Topics: Animals; Biotransformation; Brain; Copper; Dose-Response Relationship, Drug; Drug Interactions; Ethylmercuric Chloride; Ethylmercury Compounds; Female; Kidney; Liver; Metallothionein; Methylmercury Compounds; Rats; Rats, Inbred Strains; Selenious Acid; Selenium

The combined use of ultrastructural morphometry and X-ray microanalysis in conjunction with biochemical analysis is one approach to elucidating mechanisms of metal nephrotoxicity at the cellular level. Ultrastructural morphometry conducted on proximal tubule cells of rats exposed to low levels of methyl mercury for prolonged periods of time showed statistically significant increases in the volume densities of the lysosomal and mitochondrial compartments. These findings were associated with marked changes in lysosomal marker enzymes and mitochondrial heme biosynthesis enzymes leading to the detection of a renal porphyrinuria that occurred before changes in standard tests of renal function. Ultrastructural morphometry, X-ray microanalysis, and biochemical studies of the low-molecular-weight tubular proteinuria produced by injection of cadmium metallothionein (CdMT) showed a rapid proximal tubule cell lysosome uptake and degradation of the CdMT complex, which led to a subsequent decrease in the numerical density (Nv) and average diameter of lysosomes and to an increase in the Nv of apical pinocytolic vesicles with time. The data indicate disruption of the normal primary lysosome-pinocytolic vesicle fusion process and related development of tubular proteinuria. Ultrastructural techniques may provide information useful in elucidating mechanisms of ongoing metal-induced nephrotoxic processes when consideration is given to sampling strategies for morphometric analysis and the inherent detection limits, elemental volatility, translocation effects, and limitations of quantification for X-ray microanalysis in soft biological tissues.

Topics: Animals; Dose-Response Relationship, Drug; Electron Probe Microanalysis; Kidney; Kidney Tubules, Proximal; Metallothionein; Metals; Methylmercury Compounds; Organoids; Rats