Comparison of mercury sulfides with mercury chloride and methylmercury on hepatic P450, phase-2 and transporter gene expression in mice

Zuotai (mainly β-HgS) and Zhusha (also called as cinnabar, mainly α-HgS) are used in traditional medicines in combination with herbs or even drugs in the treatment of various disorders, while mercury chloride (HgCl₂) and methylmercury (MeHg) do not have known medical values but are highly toxic. This study aimed to compare the effects of mercury sulfides with HgCl₂ and MeHg on hepatic drug processing gene expression. Mice were orally administrated with Zuotai (β-HgS, 30 mg/kg), α-HgS (HgS, 30 mg/kg), HgCl₂ (33.6 mg/kg), or MeHg (3.1 mg/kg) for 7 days, and the expression of genes related to phase-1 drug metabolism (P450), phase-2 conjugation, and phase-3 (transporters) genes were examined. The mercurials at the dose and duration used in the study did not have significant effects on the expression of cytochrome P450 1–4 family genes and the corresponding nuclear receptors, except for a slight increase in PPARα and Cyp4a10 by HgCl₂. The expressions of UDP-glucuronosyltransferase and sulfotransferase were increased by HgCl₂ and MeHg, but not by Zuotai and HgS. HgCl₂ decreased the expression of organic anion transporter (Oatp1a1), but increased Oatp1a4. Both HgCl₂ and MeHg increased the expression of multidrug resistance-associated protein genes (Mrp1, Mrp2, Mrp3, and Mrp4). Zuotai and HgS had little effects on these transporter genes. In conclusion, Zuotai and HgS are different from HgCl₂ and MeHg in hepatic drug processing gene expression; suggesting that chemical forms of mercury not only affect their disposition and toxicity, but also affect their effects on the expression of hepatic drug processing genes.     

Inhibition of the human thioredoxin system. A molecular mechanism of mercury toxicity

Mercury toxicity mediated by different forms of mercury is a major health problem; however, the molecular mechanisms underlying toxicity remain elusive. We analyzed the effects of mercuric chloride (HgCl(2)) and monomethylmercury (MeHg) on the proteins of the mammalian thioredoxin system, thioredoxin reductase (TrxR) and thioredoxin (Trx), and of the glutaredoxin system, glutathione reductase (GR) and glutaredoxin (Grx). HgCl(2) and MeHg inhibited recombinant rat TrxR with IC(50) values of 7.2 and 19.7 nm, respectively. Fully reduced human Trx1 bound mercury and lost all five free thiols and activity after incubation with HgCl(2) or MeHg, but only HgCl(2) generated dimers. Mass spectra analysis demonstrated binding of 2.5 mol of Hg(2+) and 5 mol of MeHg(+)/mol of Trx1 with the very strong Hg(2+) complexes involving active site and structural disulfides. Inhibition of both TrxR and Trx activity was observed in HeLa and HEK 293 cells treated with HgCl(2) or MeHg. GR was inhibited by HgCl(2) and MeHg in vitro, but no decrease in GR activity was detected in cell extracts treated with mercurials. Human Grx1 showed similar reactivity as Trx1 with both mercurial compounds, with the loss of all free thiols and Grx dimerization in the presence of HgCl(2), but no inhibition of Grx activity was observed in lysates of HeLa cells exposed to mercury. Overall, mercury inhibition was selective toward the thioredoxin system. In particular, the remarkable potency of the mercury compounds to bind to the selenol-thiol in the active site of TrxR should be a major molecular mechanism of mercury toxicity.     

Change in permeability of liposomes caused by methylmercury and inorganic mercury

The effects of two mercurial compounds, methylmercury and inorganic mercury, on lipids were examined by measuring permeability change of lipid bilayer, liposome. Both decrease in the cholesterol content and increase in the content of unsaturated fatty acid moieties in the lipid bilayers, augmented to susceptibility of the liposomes to the mercurial compounds. Inorganic mercury and methylmercury disrupted the lipid membrane to essentially the same extent. The influence on the permeability seems to be specific for mercury compounds. The significant increase in the permeability of some liposomal preparation noted even at the mercurial concentration of 10(-7) M strongly suggests that lipid in biomembrane could be one of the primary targets of these toxic substances.     

Human serum albumin-mercurial species interactions

Binding of metal ions to the heteroatomic sites of proteins is undoubtedly fundamental to their observed physiological effects. In this paper, the interactions of inorganic mercury (Hg2+), methylmercury (MeHg+), ethylmercury (EtHg+), and phenylmercury (PhHg+) with human serum albumin (HSA) were studied from the electrophoretic behaviors, stoichiometry, thermodynamics, and kinetics by using a new hybrid technique, capillary electrophoresis on-line coupled with electrothermal atomic absorption spectrometry (CE-ETAAS), together with the consequent structural information from circular dichroism and Raman spectroscopy. The stoichiometry (mercurial species to HSA) for the interactions of Hg2+, MeHg+, EtHg+, and PhHg+ with HSA was found to be 6:1, 4:1, 4:1, and 3:1, respectively. Two types of binding sites in HSA were observed for the binding of mercurial species with the orders of magnitude of binding constants of 10(7) and 10(6) L mol-1, respectively, showing strong affinity of mercurial species for HSA. The interactions of mercurial species with both types of binding sites in HSA are exothermic and thermodynamically favorable and are both enthalpically and entropically driven. The binding of mercurial species to HSA follows the first-order kinetics for mercurial species and zero-order kinetics for HSA with the apparent activation energy of 57-59 kJ mol-1. Among the four mercurial species examined, only Hg2+ induces the secondary structure transition of HSA. Mercury-HSA adducts are formed mainly through metal-sulfur binding with participation of C=O and/or C-N groups of amino acid residues in HSA molecules. The present work represents the most comprehensive study on the interactions between various mercurial species with HSA and provides new evidence for and insights into the interactions of mercurial species with HSA for further understanding of the toxicological effects of mercurial species.     

Aspects of bioavailability of mercury for methylation in pure cultures of Desulfobulbus propionicus (1pr3)

We have previously hypothesized that sulfide inhibits Hg methylation by decreasing its bioavailability to sulfate-reducing bacteria (SRB), the important methylators of Hg in natural sediments. With a view to designing a bioassay to test this hypothesis, we investigated a number of aspects of Hg methylation by the SRB Desulfobulbus propionicus, including (i) the relationship between cell density and methylmercury (MeHg) production, (ii) the time course of Hg methylation relative to growth stage, (iii) changes in the bioavailability of an added inorganic Hg (Hg(I)) spike over time, and (iv) the dependence of methylation on the concentration of dissolved Hg(I) present in the culture. We then tested the effect of sulfide on MeHg production by this microorganism. These experiments demonstrated that under conditions of equal bioavailability, per-cell MeHg production was constant through log-phase culture growth. However, the methylation rate of a new Hg spike dramatically decreased after the first 5 h. This result was seen whether methylation rate was expressed as a fraction of the total added Hg or the filtered Hg(I) concentration, which suggests that Hg bioavailability decreased through both changes in Hg complexation and formation of solid phases. At low sulfide concentration, MeHg production was linearly related to the concentration of filtered Hg(I). The methylation of filtered Hg(I) decreased about fourfold as sulfide concentration was increased from 10(-6) to 10(-3) M. This decline is consistent with a decrease in the bioavailability of Hg(I), possibly due to a decline in the dissolved neutral complex, HgS(0).     

Total mercury and methylmercury in garfish (Belone belone) of different body weights,sizes, ages,and sexes

BackgroundGarfish, (Belone belone) is a migratory pelagic fish that inhabits the waters of coastal Europe, North Africa, the North Sea, the Mediterranean Sea. Little information about garfish has been disseminated mainly because of its low abundance and its brief occurrence in various water bodies. Data is lacking on mercury compounds, particularly dangerous the toxic organic form of methylmercury (MeHg), which endangers the health of fish and their consumers.MethodsThe research material was garfish caught off the southern Baltic Sea coast in Puck Bay during the spawning period. Total mercury (THg) content was assayed with the cold vapour atomic absorption method in an AMA 254 mercury analyser. The MeHg extraction procedure was based on three-step sequential extraction method: hydrolysis using of hydrochloric acid, extract by toluene, bind the MeHg by L-cysteine.ResultsThe concentrations of THg and MeHg was determined in the muscle of garfish. The highest concentrations of THg (0.210 mg kg-1) and MeHg (0154 mg kg-1) were detected in the longest specimens (80 cm). The THg and MeHg concentrations in garfish muscles increased with specimens length, weight and age, which was confirmed by positive correlations. Differences were also noted depending on sex. Males accumulated more THg and MeHg than did females. The mercury in garfish from the southern Baltic Sea occurred mainly in its organic form MeHg and accounted for 84.7% of the THg.ConclusionSignificant differences were noted in mercury concentrations depends on length, weight, age and sex. Concentration of MeHg in garfish must be done by length class, and fish sex when selecting this fish for contamination studies and risk assessment. The toxic MeHg in garfish tissues did not pose a threat to the health of consumers, as indicated by the low values of EDI, TWI and THQ indices.     

Functional Significance of the Effects of Neurotransmitters on the Na+,K+-ATPase System

The aim of the present experiments was to study the effects of the neurotransmitters acetylcholine, noradrenaline, 5-hydroxytryptamine, and dopamine on the Na+,K+-ATPase of rat brain synaptosomal fractions. It is shown that dopamine at low concentrations specifically inhibits the Na+,K+-ATPase of synaptic membranes from the brain regions rich in dopaminergic endings, but has no effect on the synaptosomal Na+,K+-ATPase from the other parts of brain. Acetylcholine and noradrenaline have similar specific effects on Na+,K+-ATPase from cholinergic and adrenergic synaptosomes. The Na+,K+-ATPase of synaptic membranes from the different brain regions, characterised by different distributions of cholinergic, adrenergic, and 5-hydroxytryptaminergic endings, show different reactions with neurotransmitters. These data indicate a functional significance of the effects of the neurotransmitters on the synaptosomal Na+,K+-ATPase.     

Natural incorporation of mercury in bone

Mercury (Hg) is a highly toxic element that causes bone defects and malformations. Structure and surface analyses using quantitative x-ray diffraction using the Rietveld method, High-Resolution Transmission Electron Microscopy and nanodiffraction analyses, and Fourier-Transformed Infrared spectroscopy showed that bone enriched naturally with Hg (≤ 2.3 %) contained Hg₃PO₄ [(Hg₂)₃(PO₄)₂] and HgO. Bone [mostly as apatite, verified as carboxyapatite Ca₁₀(PO₄)₄(CO₃)₃(OH)₂(s)] and cinnabar (HgS) dissolved releasing Hg⁺ (existing as dimer Hg₂²⁺) and PO₄³⁻, both of which became immobilized as (Hg₂)₃(PO₄)₂. Besides, released Hg²⁺ became oxidized to form HgO. The outcome of this work is novel, provided that only a handful of stable compounds of Hg₂²⁺ are found in nature.     

Experimental Methyl Mercury Neurotoxicity: Locus of Mercurial Inhibition of Brain Protein Synthesis In Vivo and In Vitro

Brain cell-free protein synthesis is inhibited by methyl mercury chloride (MeHg) following in vivo or in vitro administration. In this report, we have identified the locus of mercurial inhibition of translation. Intraperitoneal injection of MeHg (40 nmol/g body wt) induced variable inhibition of amino acid incorporation into the post-mitochondrial supernatant (PMS) harvested from the brain of young (10-20-day-old) rats. No mercurial-induced disaggregation of brain polyribosomes nor change in the proportion of 80S monoribosomes was detected on sucrose density gradients. No difference in total RNA was found in the PMS. Initiation complex formation was stimulated by MeHg, as detected by radiolabelled methionine binding to 80S monoribosomes following continuous sucrose density gradient centrifugation. After micrococcal nuclease digestion of endogenous mRNA, both in vivo and in vitro MeHg inhibited polyuridylic acid-directed incorporation of [3H]phenylalanine. However, the in vivo inhibition was no longer observed when [3H]phenylalanyl-tRNAPhe replaced free [3H]phenylalanine in the incorporation assay. The formation of peptidyl[3H]puromycin revealed no difference from controls. There was significant mercurial inhibition of phenylalanyl-tRNA Phe synthetase activity in pH 5 enzyme fractions derived from brain PMS of MeHg-poisoned rats. These experiments revealed that the apparent MeHg inhibition of brain translation in vivo and in vitro is due primarily to perturbation in the aminoacylation of tRNA and is not associated with defective initiation, elongation, or ribosomal function.     

The transfer of cadmium, mercury, methylmercury, and zinc in an intertidal rocky shore food chain

We examined the transfer of cadmium (Cd), inorganic mercury [Hg(II)], methylmercury (MeHg), and zinc (Zn) in an intertidal rocky shore food chain, namely from marine phytoplankton to suspension-feeding rock oysters (Saccostrea cucullata) and finally to predatory whelks Thais clavigera. The uptake of metals from the dissolved phase was also concurrently quantified in the oysters and the whelks. Metal uptake by the oysters was not directly proportional, whereas metal uptake by the whelks was directly proportional to metal concentration in the water. The order of uptake was MeHg>Hg(II)>Zn>Cd, and was much higher in the oysters than in the whelks. The relative uptake of Zn and Cd was comparable between oysters and whelks, whereas MeHg and Hg(II) showed disproportionally higher uptake in oysters than in whelks as compared to Zn and Cd. The assimilation efficiencies (AEs) were in the order of MeHg>Zn>Cd=Hg(II) in oysters, whereas the AEs were highest for MeHg and comparable for Zn, Cd, and Hg(II) in the whelks. Pre-exposure of the oysters to different dissolved concentrations of Cd significantly elevated the AEs of Cd and Hg(II) but not of Zn, in association with the induction of metallothioneins in the oysters. The whelks significantly assimilated Cd and Zn from various prey (barnacles, oysters, mussels, and snails) with contrasting strageties of metal sequestration and storage. There was no significant relationship between the metal AE and the metal partitioning in the soluble fraction (including metallothionein-like proteins, heat stable protein, and organelles). The insoluble fraction of metals was also available for metal assimilation. Our calculations show that the dietary uptake of metals can be dominant in the overall bioaccumulation in the oysters and whelks, and the trophic transfer factor was >1 for all metals. Thus, the four metals have a high potential of being biomagnified in the intertidal rocky shore food chain. MeHg possessed the highest and Hg(II) and Cd the lowest potential of trophic transfer among the four metals considered.     

Enhancement of learning and memory in mice by a benzodiazepine antagonist

Benzodiazepines, a class of drugs widely employed as anxiolytics and anticonvulsants, can induce impairments of learning and memory. The purpose of the present investigation was to determine if a benzodiazepine receptor antagonist, flumazenil (Ro 15-1788), could enhance learning and memory. Pretraining injection of flumazenil (2.5 to 40.0 mg/kg) was found to enhance both learning and memory in a test requiring young mice to discriminate the correct arm of a T-maze to escape mild electric shock. In a second test, which required mice to passively avoid a dark chamber after shock, flumazenil pretreatment prevented the occurrence of amnesia induced by the cholinergic receptor antagonist scopolamine. It is hypothesized that flumazenil may facilitate learning or memory processes by reversing a negative modulatory influence of endogenous diazepam-like ligands for benzodiazepine receptors.     

Selenium and mercury levels in rat liver slices co-treated with diphenyl diselenide and methylmercury

Organoseleno-compounds have been investigated for its beneficial effects against methylmercury toxicity. In this way, diphenyl diselenide (PhSe)₂ was demonstrated to decrease Hg accumulation in mice, protect against MeHg-induced mitochondrial dysfunction, and protect against the overall toxicity of this metal. In the present study we aimed to investigate if co-treatment with (PhSe)₂ and MeHg could decrease accumulation of Hg in liver slices of rats. Rat liver slices were co-treated with (PhSe)₂ (0.5; 5 µM) and/or MeHg (25 µM) for 30 min at 37 °C and Se and Hg levels were measured by inductively coupled plasma mass spectrometry (ICP-MS) in the slices homogenate, P1 fraction, mitochondria and incubation medium. Co-treatment with (PhSe)₂ and MeHg did not significantly alter Se levels in any of the samples when compared with compounds alone. In addition, co-treatment with (PhSe)₂ and MeHg did not decrease Hg levels in any of the samples tested, although, co-incubation significantly increased Hg levels in homogenate. We suggest here that (PhSe)₂ could exert its previously demonstrated protective effects not by reducing MeHg levels, but forming a complex with MeHg avoiding it to bind to critical molecules in cell.     

Metallothioneins are required for formation of cross-adaptation response to neurobehavioral toxicity from lead and mercury exposure in nematodes

Metallothioneins (MTs) are small, cysteine-rich polypeptides, but the role of MTs in inducing the formation of adaptive response is still largely unknown. We investigated the roles of metallothionein genes (mtl-1 and mtl-2) in the formation of cross-adaptation response to neurobehavioral toxicity from metal exposure in Caenorhabditis elegans. Pre-treatment with mild heat-shock at L2-larva stage effectively prevented the formation of the neurobehavioral defects and the activation of severe stress response in metal exposed nematodes at concentrations of 50 and 100 μM, but pre-treatment with mild heat-shock did not prevent the formation of neurobehavioral defects in 200 μM of metal exposed nematodes. During the formation of cross-adaptation response, the induction of mtl-1 and mtl-2 promoter activity and subsequent GFP gene expression were sharply increased in 50 μM or 100 μM of metal exposed Pmtl-1::GFP and Pmtl-2::GFP transgenic adult animals after mild heat-shock treatment compared with those treated with mild heat-shock or metal exposure alone. Moreover, after pre-treatment with mild heat-shock, no noticeable increase of locomotion behaviors could be observed in metal exposed mtl-1 or mtl-2 mutant nematodes compared to those without mild heat-shock pre-treatment. The defects of adaptive response to neurobehavioral toxicity induced by metal exposure formed in mtl-1 and mtl-2 mutants could be completely rescued by the expression of mtl-1 and mtl-2 with the aid of their native promoters. Furthermore, over-expression of MTL-1 and MTL-2 at the L2-larval stage significantly suppressed the toxicity on locomotion behaviors from metal exposure at all examined concentrations. Therefore, the normal formation of cross-adaptation response to neurobehavioral toxicity induced by metal exposure may need the enough accumulation of MTs protein in animal tissues.     

Cinnabar mineralization in fossil small mammal remains as a consequence of diagenetic processes

The peculiar red colour of fossil small mammal remains from a late Miocene section in southern Spain suggests an unusual diagenetic alteration. These remains have been studied by means of environmental scanning electron microscope equipped with different detectors, energy dispersive X‐ray spectroscopy and an inductively coupled plasma optical emission spectrometer. The red colour is caused by the presence of cinnabar (HgS) in the pores of the fossil bones and teeth, filling the dentinal and bone tubules and other cavities that at life were filled by organic matter. A nearby cinnabar outcrop in the metamorphic materials of Sierra Nevada is interpreted as the source of mercury. This element was mobilized by meteoric diagenesis and incorporated as cinnabar in a palustrine environment, occupying pores and cavities of mammal remains shortly after the degradation of the organic matter (post‐mortem enrichment), during the early diagenesis.     

Elimination of the vesicular acetylcholine transporter in the striatum reveals regulation of behaviour by cholinergic-glutamatergic co-transmission

Cholinergic neurons in the striatum are thought to play major regulatory functions in motor behaviour and reward. These neurons express two vesicular transporters that can load either acetylcholine or glutamate into synaptic vesicles. Consequently cholinergic neurons can release both neurotransmitters, making it difficult to discern their individual contributions for the regulation of striatal functions. Here we have dissected the specific roles of acetylcholine release for striatal-dependent behaviour in mice by selective elimination of the vesicular acetylcholine transporter (VAChT) from striatal cholinergic neurons. Analysis of several behavioural parameters indicates that elimination of VAChT had only marginal consequences in striatum-related tasks and did not affect spontaneous locomotion, cocaine-induced hyperactivity, or its reward properties. However, dopaminergic sensitivity of medium spiny neurons (MSN) and the behavioural outputs in response to direct dopaminergic agonists were enhanced, likely due to increased expression/function of dopamine receptors in the striatum. These observations indicate that previous functions attributed to striatal cholinergic neurons in spontaneous locomotor activity and in the rewarding responses to cocaine are mediated by glutamate and not by acetylcholine release. Our experiments demonstrate how one population of neurons can use two distinct neurotransmitters to differentially regulate a given circuitry. The data also raise the possibility of using VAChT as a target to boost dopaminergic function and decrease high striatal cholinergic activity, common neurochemical alterations in individuals affected with Parkinson's disease.     

Spatial and temporal dynamics of mercury in Precambrian Shield upland runoff

Methylated and total Hg, and TOC concentrations were measured in precipitation and runoff in a first order Precambrian Shield watershed, and in precipitation, throughfall, shallow groundwater and runoff in a zero Precambrian Shield watershed. Plots dominated by open lichen-covered bedrock and another containing small patches of conifer forest and thin discontinuous surficial deposits were monitored within the zero order catchment. Methyl (3–10 fold) and non-methyl (1.4–2.8 fold) Hg concentrations changed irregularly during rainfall and snowmelt runoff events in all catchments. Temporal patterns of Hg concentration in runoff included flushing and subsequent dilution as well as peak concentrations coinciding with peak or recession flow. Mercury export was highest from lichen-covered bedrock surfaces as a result of high runoff yields and minimal opportunity for physical retention and in the case of MeHg demethylation. Forest canopy and lichen/bedrock surfaces were often net sources for Hg while forest soils were mostly sinks. However, upland soils undergoing periodic reducing conditions appear to be sites for the in situ production of MeHg.     

A possible mechanism of participation of dopaminergic cells and striatal cholinergic interneurons in the conditioned selection of motor activity

A possible mechanism of participation of cholinergic striatal interneurons and dopaminergic cells in conditioned selection of a certain types of motor activity is proposed. This selection is triggered by simultaneous increase in the activity of dopaminergic cells and a pause in the activity of cholinergic interneurons in response to a conditioned stimulus. This pause is promoted by activation of striatal inhibitory interneurons and action of dopamine at D2 receptors on cholinergic cells. Opposite changes in dopamine and acetylcholine concentration synergistically modulate the efficacy of corticostriatal inputs, modulation rules for the "strong" and "weak" corticostriatal inputs are opposite. Subsequent reorganization of neuronal firing in the loop cortex--basal ganglia--thalamus--cortex results in amplification of activity of the group of cortical neurons that strongly activate striatal cells, and simultaneous suppression of activity of another group of cortical neurons that weakly activate striatal cells. These changes can underlie a conditioned selection of motor activity performed with involvement of the motor cortex. As follows from the proposed model, if the time delay between conditioned and unconditioned stimuli does not exceed the latency of responses of dopaminergic and cholinergic cells (about 100 ms), conditioned selection of motor activity and learning is problematic.     

Dynamics of mercury and methylmercury in forest floor and runoff of a forested watershed in Central Europe

Forested watersheds are an important part of the terrestrialmercury and methylmercury cycle, and a link between theatmospheric and aquatic environment. This study was conducted todetermine the contribution of the forest floor to the pools andfluxes of total Hg (Hg_total) and methylmercury (MeHg) in aforested catchment, and to identify factors influencing themobility of both compounds. Throughfall deposition, litterfall,runoff and fluxes with forest floor percolate of Hg_total and MeHgwere sampled during one year in a coniferous catchment inGermany. Total deposition of Hg_total was 552 mg ha_–1 a_–1 withlitterfall contributing one third. Nearly 60% of the total inputof Hg_total reached the mineral soil with the forest floorpercolate, but less than half of this fraction was found in therunoff of the catchment. Total deposition of MeHg was 2.6 mg ha_–1a_–1, with litterfall as the dominating pathway. Only 19% of theMeHg deposition was discharged from the forest floor, but theflux of MeHg with runoff was nearly twice as high. Only fewcorrelations with other solution parameters were found. Fluxes ofboth compounds with forest floor percolates depended mainly onwater fluxes, which was not true for the runoff. The forest floorof the upland soil is an effective sink for MeHg, but not forHg_total. Differences in the mobility of both compounds in theforest floor disappeared at the catchment scale, probably becauseother processes (i.e. Hg_total immobilization and MeHg formation)dominated.     

Mercury methylation by Desulfovibrio desulfuricans ND132 in the presence of polysulfides

The extracellular speciation of mercury may control bacterial uptake and methylation. Mercury-polysulfide complexes have recently been shown to be prevalent in sulfidic waters containing zero-valent sulfur. Despite substantial increases in total dissolved mercury concentration, methylation rates in cultures of Desulfovibrio desulfuricans ND132 equilibrated with cinnabar did not increase in the presence of polysulfides, as expected due to the large size and charged nature of most of the complexes. In natural waters not at saturation with cinnabar, mercury-polysulfide complexes would be expected to shift the speciation of mercury from HgS(0)((aq)) toward charged complexes, thereby decreasing methylation rates.