Reduction of Hg(II) to Hg(0) by Biogenic Magnetite from two Magnetotactic Bacteria

Understanding the biogeochemical cycle of the highly toxic element mercury (Hg) is necessary to predict its fate and transport. In this study, we determined that biogenic magnetite isolated from Magnetospirillum gryphiswaldense MSR-1 and Magnetospirillum magnetotacticum MS-1 was capable of reducing inorganic mercury [Hg(II)] to elemental mercury [Hg(0)]. These two magnetotactic bacteria (MTB) lacked mercuric resistance operons in the genomes. However, they revealed high resistance to Hg(II) under atmospheric conditions and an even higher resistance under microaerobic conditions (1% O₂ and 99% N₂). Neither strain reduced Hg(II) to Hg(0) under atmospheric conditions. However, a slow rate (0.05–0.21 µM·d^?1) of Hg(II) loss occurred from late log phase to stationary phase in two MTBs' culture media under microaerobic conditions. Increased Hg(II) entered both cells under microaerobic conditions relative to atmospheric conditions. The majority of Hg(II) was still blocked by the cell membrane. Hg(II) reduction was more effective when biogenic magnetite was extracted out, with or without the magnetosome membrane envelope. When magnetosome membrane was present, 8.55–13.53% of 250 nM Hg(II) was reduced to Hg(0) by 250 mg/L biogenic magnetite suspension within 2 hours. This ratio increased to 55.07–64.70% while magnetosome membrane was removed. We concluded that two MTBs contributed to the reduction of Hg(II) to Hg(0) at a slow rate in vivo. Such reduction was more favorable to occur when biogenic magnetite is released from dead cells. It proposed a new biotic pathway for the formation of Hg(0) in aquatic systems.     

Mercury(II) complex formation with glutathione in alkaline aqueous solution

The structure and speciation of the complexes formed between mercury(II) ions and glutathione (GSH = L-glutamyl-L-cysteinyl-glycine) have been studied for a series of alkaline aqueous solutions (\( C_{{{\text{Hg}}^{{2 + }}}}\,{\sim18\,{\rm{mmol}}\,{\rm{{dm^{-3}}}}}\) and C _GSH = 40–200 mmol dm^?3 at pH ～10.5) by means of extended X-ray absorption fine structure (EXAFS) and ¹⁹⁹Hg NMR spectroscopy at ambient temperature. The dominant complexes are [Hg(GS)₂]^4? and [Hg(GS)₃]^7?, with mean Hg–S bond distances of 2.32(1) and 2.42(2) Å observed in digonal and trigonal Hg–S coordination, respectively. The proportions of the Hg²⁺–glutathione complexes were evaluated by fitting linear combinations of model EXAFS oscillations representing each species to the experimental EXAFS spectra. The [Hg(GS)₄]^10? complex, with four sulfur atoms coordinated at a mean Hg–S bond distance of 2.52(2) Å, is present in minor amounts (<30%) in solutions containing a large excess of glutathione (C _GSH ≥ 160 mmol dm^?3). Comparable alkaline mercury(II) cysteine (H₂Cys) solutions were also investigated and a reduced tendency to form higher complexes was observed, because the deprotonated amino group of Cys^2? allows the stable [Hg(S,N-Cys)₂]^2? chelate to form. The effect of temperature on the distribution of the Hg²⁺–glutathione complexes was studied by comparing the EXAFS spectra at ambient temperature and at 25 K of a series of glycerol/water (33/67, v/v) frozen glasses with \( C_{{{\text{Hg}}^{{2 + }} }} \,{\sim7\,{\rm{mmol}}\,{\rm{{dm^{-3}}}}} \) and C _GSH = 16–81 mmol dm^?3. Complexes with high Hg–S coordination numbers, [Hg(GS)₃]^7? and [Hg(GS)₄]^10?, became strongly favored when just a moderate excess of glutathione (C _GSH ≥28 mmol dm^?3) was used in the glassy samples, as expected for a stepwise exothermic bond formation. Addition of glycerol had no effect on the Hg(II)–glutathione speciation, as shown by the similarity of the EXAFS spectra obtained at room temperature for two parallel series of Hg(II)-glutathione solutions with \( C_{{{\text{Hg}}^{{2 + }} }} \,{\sim7\,{\rm{mmol}}\,{\rm{{dm^{-3}}}}},\) with and without 33% glycerol. Also, the ¹⁹⁹Hg NMR chemical shifts of a series of ～18 mmol dm^?3 mercury(II) glutathione solutions with 33% glycerol were not significantly different from those of the corresponding series in aqueous solution.     

Effect of NAD+-dependent formate dehydrogenase on anaerobic respiration of Shewanella oneidensis MR-1

An expression plasmid was constructed in order to carry out heterologous expression of the gene of the NAD⁺-dependent formate dehydrogenase (FDH) from methylotrophic bacterium Moraxella sp. in the cells of Shewanella oneidensis MR-1 under aerobic and anaerobic conditions. In both modes of cell cultivation, recombinant FDH activity was revealed in the cell lysate of the transformants. In the medium with la? tate as a carbon source, the rate of anaerobic respiration determined as the rate of conversion of fumarate (the electron acceptor) to succinate was higher in the transformant with recombinant FDH. Anaerobic cultivation of the FDH-containing transformant of S. oneidensis MR-1 in a microbial fuel cell (MFC) revealed increased current density.     

Antioxidant system activation by mercury in Pfaffia glomerata plantlets

Oxidative stress caused by mercury (Hg) was investigated in Pfaffia glomerata plantlets grown in nutrient solution using sand as substrate. Thirty-day-old acclimated plants were treated for 9 days with four Hg levels (0, 1, 25 and 50 μM) in the substrate. Parameters such as growth, tissue Hg concentration, toxicity indicators (δ-aminolevulinic acid dehidratase, δ-ALA-D, activity), oxidative damage markers (TBARS, lipid peroxidation, and H₂O₂ concentration) and enzymatic (superoxide dismutase, SOD, catalase, CAT, and ascorbate peroxidase, APX) and non-enzymatic (non-protein thiols, NPSH, ascorbic acid, AsA, and proline concentration) antioxidants were investigated. Tissue Hg concentration increased with Hg levels. Root and shoot fresh weight and δ-ALA-D activity were significantly decreased at 50 μM Hg, and chlorophyll and carotenoid concentration were not affected. Shoot H₂O₂ concentration increased curvilinearly with Hg levels, whereas lipid peroxidation increased at 25 and 50 μM Hg, respectively, in roots and shoots. SOD activity showed a straight correlation with H₂O₂ concentration, whereas CAT activity increased only in shoots at 1 and 50 μM Hg. Shoot APX activity was either decreased at 1 μM Hg or increased at 50 μM Hg. Conversely, root APX activity was only increased at 1 μM Hg. In general, AsA, NPSH and proline concentrations increased upon addition of Hg, with the exception of proline in roots, which decreased. These changes in enzymatic and non-enzymatic antioxidants had a significant protective effect on P. glomerata plantlets under mild Hg-stressed conditions.     

The thioredoxin system as a target for mercury compounds

BackgroundMercury interaction with selenium in vivo has been recognized for >50 years. Several researchers attempted to use selenium to mitigate the detrimental effects of mercurial compounds but the results were controversial. Selenium pools in living organisms are quite low and the high affinity of mercury to bind selenols pointed out selenoproteins as possible targets of toxicity. Such was the case of the selenoenzyme thioredoxin reductase (TrxR) which is an integrant part of the thioredoxin system. Given the important role of this redox system for cellular functioning and the high affinity of mercury for TrxR's active site, this interaction can be key to understand the mechanism by which Hg causes cell death.Scope of the reviewThis review discusses the current state of knowledge concerning the interaction between mercury compounds and the thioredoxin system, its implications for the development of toxicity and the effects of selenium co-exposure.Major conclusionsThe mechanism of toxicity of mercurials is a complex chain of events starting with inhibition of the selenoenzyme, TrxR. Selenium supplementation protects TrxR from the toxicity of inorganic forms of mercury (i.e., Hg(II)) to a certain extent, but not from methylmercury.When TrxR is inhibited, thioredoxin is reduced by alternative mechanisms involving glutathione and glutaredoxin and only when this pathway is hampered does cell death occur.General significanceUnderstanding the molecular mechanism of mercury toxicity and the mechanisms of enzymatic compensation allows the design of mitigation strategies and, since TxrR and Trx exist in the plasma, puts forward the possibility for future use of changes in activity/expression of these enzymes as biomarkers of mercury toxicity, thus refining the risk assessment process.     

Chromate/nitrite interactions in Shewanella oneidensis MR-1: evidence for multiple hexavalent chromium [Cr(VI)] reduction mechanisms dependent on physiological growth conditions

Inhibition of hexavalent chromium [Cr(VI)] reduction due to nitrate and nitrite was observed during tests with Shewanella oneidensis MR-1 (previously named Shewanella putrefaciens MR-1 and henceforth referred to as MR-1). Initial Cr(VI) reduction rates were measured at various nitrite concentrations, and a mixed inhibition kinetic model was used to determine the kinetic parameters-maximum Cr(VI) reduction rate and inhibition constant [V(max,Cr(VI)) and K(i,Cr(VI))]. Values of V(max,Cr(VI)) and K(i,Cr(VI)) obtained with MR-1 cultures grown under denitrifying conditions were observed to be significantly different from the values obtained when the cultures were grown with fumarate as the terminal electron acceptor. It was also observed that a single V(max,Cr(VI)) and K(i,Cr(VI)) did not adequately describe the inhibition kinetics of either nitrate-grown or fumarate-grown cultures. The inhibition patterns indicate that Cr(VI) reduction in MR-1 is likely not limited to a single pathway, but occurs via different mechanisms some of which are dependent on growth conditions. Inhibition of nitrite reduction due to the presence of Cr(VI) was also studied, and the kinetic parameters V(max,NO2) and K(i,NO2) were determined. It was observed that these coefficients also differed significantly between MR-1 grown under denitrifying conditions and fumarate reducing conditions. The inhibition studies suggest the involvement of nitrite reductase in Cr(VI) reduction. Because nitrite reduction is part of the anaerobic respiration process, inhibition due to Cr(VI) might be a result of interaction with the components of the anaerobic respiration pathway such as nitrite reductase. Also, differences in the degree of inhibition of nitrite reduction activity by chromate at different growth conditions suggest that the toxicity mechanism of Cr(VI) might also be dependent on the conditions of growth. Cr(VI) reduction has been shown to occur via different pathways, but to our knowledge, multiple pathways within a single organism leading to Cr(VI) reduction has not been reported previously.     

Trace elements in tissues of white-chinned petrels (Procellaria aequinoctialis) from Kerguelen waters,Southern Indian Ocean

The use of seabirds to assess marine contamination by trace elements in areas remote from pollutant emission points has already been done at various latitudes. Nevertheless, little information is available concerning the Southern Indian Ocean. Determining the contaminants levels, there appears necessary not only due to several deleterious effects reported in literature, but also as previous studies have highlighted elevated concentrations of cadmium (Cd) and mercury (Hg) in mollusks, crustaceans and fish. Within this context, the white-chinned petrel appears as a key species due to its lifespan, diet and trophic position. Thirty-three accidentally killed (collision with lights/bycatch in longline vessels) individuals collected in Kerguelen waters were analysed for Cd, copper (Cu), Hg, selenium (Se) and zinc (Zn) in liver, kidney, pectoral muscle, feathers and for mature males, testis. Elevated Hg concentrations (average 58.4 μg g^?1 dw in liver) are likely due to the presence of mesopelagic prey in the diet of Procellaria aequinoctialis. Cd concentrations (average of 65.7 μg g^?1 dw in kidney) can be attributed to a high level of fisheries offal consumption, as well as crustacean and squid ingestion. Correlation of Hg with Se indicates its detoxification by co-precipitation, and correlation of Cd with Zn suggests its displacement by Cd on metallothioneins binding sites. This work also indirectly confirms ecological data (range and diet composition) from the wintering period of the species, which is rather scarce. Seasonal diet change and moulting accounted more for the obtained results than sex of the birds.     

Biotransformation of Hg(II) by Cyanobacteria

The biotransformation of Hg(II) by cyanobacteria was investigated under aerobic and pH-controlled culture conditions. Mercury was supplied as HgCl₂ in amounts emulating those found under heavily impacted environmental conditions where bioremediation would be appropriate. The analytical procedures used to measure mercury within the culture solution, including that in the cyanobacterial cells, used reduction under both acid and alkaline conditions in the presence of SnCl₂. Acid reduction detected free Hg(II) ions and its complexes, whereas alkaline reduction revealed that meta-cinnabar (β-HgS) constituted the major biotransformed and cellularly associated mercury pool. This was true for all investigated species of cyanobacteria: Limnothrix planctonica (Lemm.), Synechococcus leopoldiensis (Racib.) Komarek, and Phormidium limnetica (Lemm.). From the outset of mercury exposure, there was rapid synthesis of β-HgS and Hg(0); however, the production rate for the latter decreased quickly. Inhibitory studies using dimethylfumarate and iodoacetamide to modify intra- and extracellular thiols, respectively, revealed that the former thiol pool was required for the conversion of Hg(II) into β-HgS. In addition, increasing the temperature enhanced the amount of β-HgS produced, with a concomitant decrease in Hg(0) volatilization. These findings suggest that in the environment, cyanobacteria at the air-water interface could act to convert substantial amounts of Hg(II) into β-HgS. Furthermore, the efficiency of conversion into β-HgS by cyanobacteria may lead to the development of applications in the bioremediation of mercury.     

Higher sensitivity of pad2-1 and vtc2-1 mutants to cadmium is related to lower subcellular glutathione rather than ascorbate contents

Cadmium (Cd) interferes with ascorbate and glutathione metabolism as it induces the production of reactive oxygen species (ROS), binds to glutathione due to its high affinity to thiol groups, and induces the production of phytochelatins (PCs) which use glutathione as a precursor. In this study, changes in the compartment specific distribution of ascorbate and glutathione were monitored over a time period of 14 days in Cd-treated (50 and 100 μM) Arabidopsis Col-0 plants, and two mutant lines deficient in glutathione (pad2-1) and ascorbate (vtc2-1). Both mutants showed higher sensitivity to Cd than Col-0 plants. Strongly reduced compartment specific glutathione, rather than decreased ascorbate contents, could be correlated with the development of symptoms in these mutants suggesting that higher sensitivity to Cd is related to low glutathione contents rather than low ascorbate contents. On the subcellular level it became obvious that long-term treatment of wildtype plants with Cd induced the depletion of glutathione and ascorbate contents in all cell compartments except chloroplasts indicating an important protective role for antioxidants in chloroplasts against Cd. Additionally, we could observe an immediate decrease of glutathione and ascorbate in all cell compartments 12 h after Cd treatment indicating that glutathione and ascorbate are either withdrawn from or not redistributed into other organelles after their production in chloroplasts, cytosol (production centers for glutathione) and mitochondria (production center for ascorbate). The obtained data is discussed in respect to recently proposed stress models involving antioxidants in the protection of plants against environmental stress conditions.     

Construction and elementary mode analysis of a metabolic model for Shewanella oneidensis MR-1

A stoichiometric model describing the central metabolism of Shewanella oneidensis MR-1 wild-type and derivative strains was developed and used in elementary mode analysis (EMA). Shewanella oneidensis MR-1 can anaerobically respire a diverse pool of electron acceptors, and may be applied in several biotechnology settings, including bioremediation of toxic metals, electricity generation in microbial fuel cells, and whole-cell biocatalysis. The metabolic model presented here was adapted and verified by comparing the growth phenotypes of 13 single- and 1 double-knockout strains, while considering respiration via aerobic, anaerobic fumarate, and anaerobic metal reduction (Mtr) pathways, and utilizing acetate, n-acetylglucosamine (NAG), or lactate as carbon sources. The gene ppc, which encodes phosphoenolpyruvate carboxylase (Ppc), was determined to be necessary for aerobic growth on NAG and lactate, while not essential for growth on acetate. This suggests that Ppc is the only active anaplerotic enzyme when cultivated on lactate and NAG. The application of regulatory and substrate limitations to EMA has enabled creation of metabolic models that better reflect biological conditions, and significantly reduce the solution space for each condition, facilitating rapid strain optimization. This wild-type model can be easily adapted to include utilization of different carbon sources or secretion of different metabolic products, and allows the prediction of single- and multiple-knockout strains that are expected to operate under defined conditions with increased efficiency when compared to wild type cells.     

Methods for imaging Shewanella oneidensis MR-1 nanofilaments

Nanofilament production by Shewanella oneidensis MR-1 was evaluated as a function of lifestyle (planktonic vs. sessile) under aerobic and anaerobic conditions using different sample preparation techniques prior to imaging with scanning electron microscopy. Nanofilaments could be imaged on MR-1 cells grown in biofilms or planktonically under both aerobic and anaerobic batch culture conditions after fixation, critical point drying and coating with a conductive metal. Critical point drying was a requirement for imaging nanofilaments attached to planktonically grown MR-1 cells, but not for cells grown in a biofilm. Techniques described in this paper cannot be used to differentiate nanowires from pili or flagella.     

Impact of mercury on denitrification and denitrifying microbial communities in nitrate enrichments of subsurface sediments

The contamination of groundwater with mercury (Hg) is an increasing problem worldwide. Yet, little is known about the interactions of Hg with microorganisms and their processes in subsurface environments. We tested the impact of Hg on denitrification in nitrate reducing enrichment cultures derived from subsurface sediments from the Oak Ridge Integrated Field Research Challenge site, where nitrate is a major contaminant and where bioremediation efforts are in progress. We observed an inverse relationship between Hg concentrations and onset and rates of denitrification in nitrate enrichment cultures containing between 53 and 1.1 μM of inorganic Hg; higher Hg concentrations increasingly extended the time to onset of denitrification and inhibited denitrification rates. Microbial community complexity, as indicated by terminal restriction fragment length polymorphism (tRFLP) analysis of the 16S rRNA genes, declined with increasing Hg concentrations; at the 312 nM Hg treatment, a single tRFLP peak was detected representing a culture of Bradyrhizobium sp. that possessed the merA gene indicating a potential for Hg reduction. A culture identified as Bradyrhizobium sp. strain FRC01 with an identical 16S rRNA sequence to that of the enriched peak in the tRFLP patterns, reduced Hg(II) to Hg(0) and carried merA whose amino acid sequence has 97 % identity to merA from the Proteobacteria and Firmicutes. This study demonstrates that in subsurface sediment incubations, Hg may inhibit denitrification and that inhibition may be alleviated when Hg resistant denitrifying Bradyrhizobium spp. detoxify Hg by its reduction to the volatile elemental form.     

Effect of pH on Intracellular Accumulation of Trace Concentrations of Hg(II) in Escherichia coli under Anaerobic Conditions, as Measured Using a mer-lux Bioreporter

The effects of pH on the uptake and accumulation of Hg(II) by Escherichia coli were determined at trace, environmentally relevant, concentrations of Hg and under anaerobic conditions. Hg(II) accumulation was measured using inducible light production from E. coli HMS174 harboring a mer-lux bioreporter plasmid (pRB28). The effect of pH on the toxicity of higher concentrations of Hg(II) was measured using a constitutive lux plasmid (pRB27) in the same bacterial host. In this study, intracellular accumulation and toxicity of Hg(II) under anaerobic conditions were both significantly enhanced with decreasing pH over the pH range of 8 to 5. The pH effect on Hg(II) accumulation was most pronounced at pHs of <6, which substantially enhanced the Hg(II)-dependent light response. This enhanced response did not appear to be due to pH stress, as similar results were obtained whether cells were grown at the same pH as the assay or at a different pH. The enhanced accumulation of Hg(II) was also not related to differences in the chemical speciation of Hg(II) in the external medium resulting from the changes in pH. Experiments with Cd(II), also detectable by the mer-lux bioreporter system, showed that Cd(II) accumulation responded differently to pH changes than the net accumulation of Hg(II). Potential implications of these findings for our understanding of bacterial accumulation of Hg(II) under anaerobic conditions and for bacteria-mediated cycling of Hg(II) in aquatic ecosystems are discussed. Arguments are provided suggesting that this differential accumulation is due to changes in uptake of mercury.     

A glutathione reductase mutant of yeast accumulates high levels of oxidized glutathione and requires thioredoxin for growth.

A glutathione reductase null mutant of Saccharomyces cerevisiae was isolated in a synthetic lethal genetic screen for mutations which confer a requirement for thioredoxin. Yeast mutants that lack glutathione reductase (glr1 delta) accumulate high levels of oxidized glutathione and have a twofold increase in total glutathione. The disulfide form of glutathione increases 200-fold and represents 63% of the total glutathione in a glr1 delta mutant compared with only 6% in wild type. High levels of oxidized glutathione are also observed in a trx1 delta, trx2 delta double mutant (22% of total), in a glr1 delta, trx1 delta double mutant (71% of total), and in a glr1 delta, trx2 delta double mutant (69% of total). Despite the exceptionally high ratio of oxidized/reduced glutathione, the glr1 delta mutant grows with a normal cell cycle. However, either one of the two thioredoxins is essential for growth. Cells lacking both thioredoxins and glutathione reductase are not viable under aerobic conditions and grow poorly anaerobically. In addition, the glr1 delta mutant shows increased sensitivity to the thiol oxidant diamide. The sensitivity to diamide was suppressed by deletion of the TRX2 gene. The genetic analysis of thioredoxin and glutathione reductase in yeast runs counter to previous studies in Escherichia coli and for the first time links thioredoxin with the redox state of glutathione in vivo.     

Toxic effects of chromium(VI) on anaerobic and aerobic growth of Shewanella oneidensis MR-1

Cr(VI) was added to early- and mid-log-phase Shewanella oneidensis (S. oneidensis) MR-1 cultures to study the physiological state-dependent toxicity of Cr(VI). Cr(VI) reduction and culture growth were measured during and after Cr(VI) reduction. Inhibition of growth was observed when Cr(VI) was added to cultures of MR-1 growing aerobically or anaerobically with fumarate as the terminal electron acceptor. Under anaerobic conditions, there was immediate cessation of growth upon addition of Cr(VI) in early- and mid-log-phase cultures. However, once Cr(VI) was reduced below detection limits (0.002 mM), the cultures resumed growth with normal cell yield values observed. In contrast to anaerobic MR-1 cultures, addition of Cr(VI) to aerobically growing cultures resulted in a gradual decrease of the growth rate. In addition, under aerobic conditions, lower cell yields were also observed with Cr(VI)-treated cultures when compared to cultures that were not exposed to Cr(VI). Differences in response to Cr(VI) between aerobically and anaerobically growing cultures indicate that Cr(VI) toxicity in MR-1 is dependent on the physiological growth condition of the culture. Cr(VI) reduction has been previously studied in Shewanella spp., and it has been proposed that Shewanella spp. may be used in Cr(VI) bioremediation systems. Studies of Shewanella spp. provide valuable information on the microbial physiology of dissimilatory metal reducing bacteria; however, our study indicates that S. oneidensis MR-1 is highly susceptible to growth inhibition by Cr(VI) toxicity, even at low concentrations [0.015 mM Cr(VI)].     

Mercury (Hg) Exposure in Breast-Fed Infants and Their Mothers and the Evidence of Oxidative Stress

The objective of this work was to assess exposure to mercury (Hg) and its induction of oxidative stress in 155 healthy lactating Saudi mothers and their infants. Samples of breast milk and blood were collected from the mothers, while urine was taken from both infants and mothers. Both urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) and malondialdehyde (MDA) were measured in mothers and infants as biomarkers of oxidative stress. The mean concentration of Hg in breast milk was 1.19 μg/L (range 0.012–6.44 μg/L) with only one mother having Hg >4 μg/L, the upper limit established by the US Agency for Toxic Substance and Disease Registry. However, 57.4 % had Hg ≥1 μg/L, the background level for Hg in human milk. The mean urinary Hg corrected for creatinine (Hg-C) in mothers and infants was 1.47 and 7.90 μg/g creatinine, respectively, with a significant correlation between the two (p?<?0.001). Urinary Hg levels over 5 μg/g creatinine (the background level in an unexposed population) were found in 3.3 % of mothers and 50.1 % of infants. None of the mothers had total blood Hg above the US Environmental Protection Agency’s maximum reference dose of 5.8 μg/L. No correlation was noted between urinary Hg in infants and Hg in breast milk (p?>?0.05). Hg in breast milk, though, was associated with Hg in blood (p?<?0.001), suggesting the efficient transfer of Hg from blood to milk. Hg in the breast milk of mothers and in the urine of infants affected the excretion of urinary MDA and 8-OHdG, respectively, in a dose-related manner. These findings reveal for the first time lactational exposure to Hg-induced oxidative stress in breast-fed infants, which may play a role in pathogenesis, particularly during neurodevelopment. This will also contribute to the debate over the benefits of breast milk versus the adverse effects of exposure to pollutants. Nevertheless, breastfeeding should not be discouraged, but efforts should be made to identify and eliminate the source of Hg exposure in the population.     

Mercury analysis of acid- and alkaline-reduced biological samples: identification of meta-cinnabar as the major biotransformed compound in algae

The biotransformation of Hg(II) in pH-controlled and aerated algal cultures was investigated. Previous researchers have observed losses in Hg detection in vitro with the addition of cysteine under acid reduction conditions in the presence of SnCl2. They proposed that this was the effect of Hg-thiol complexing. The present study found that cysteine-Hg, protein and nonprotein thiol chelates, and nucleoside chelates of Hg were all fully detectable under acid reduction conditions without previous digestion. Furthermore, organic (R-Hg) mercury compounds could not be detected under either the acid or alkaline reduction conditions, and only beta-HgS was detected under alkaline and not under acid SnCl2 reduction conditions. The blue-green alga Limnothrix planctonica biotransformed the bulk of Hg(II) applied as HgCl2 into a form with the analytical properties of beta-HgS. Similar results were obtained for the eukaryotic alga Selenastrum minutum. No evidence for the synthesis of organomercurials such as CH3Hg+ was obtained from analysis of either airstream or biomass samples under the aerobic conditions of the study. An analytical procedure that involved both acid and alkaline reduction was developed. It provides the first selective method for the determination of beta-HgS in biological samples. Under aerobic conditions, Hg(II) is biotransformed mainly into beta-HgS (meta-cinnabar), and this occurs in both prokaryotic and eukaryotic algae. This has important implications with respect to identification of mercury species and cycling in aquatic habitats.     

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.     

Evidence of elevated mercury levels in carnivorous and omnivorous fishes downstream from an Amazon reservoir

Hydroelectric reservoirs can stratify, producing favorable conditions for mercury methylation in the hypolimnion. The methylmercury (MeHg) can be exported downstream, increasing its bioavailability below the dam. Our objective was to assess the mercury levels in plankton, suspended particulate matter (SPM) and fish collected upstream (UP) and downstream (DW) from the Reservatório de Samuel dam, an Amazonian reservoir that stratifies during half of the year. Mercury concentrations in both SPM and plankton were similar between the two sites, which could indicate there are no conditions favoring methylation at the moment of sampling (absence of stratification). Almost all mercury found in the muscle of fishes was in organic form, and differences of mercury levels between sites were dependent on the fishes trophic level. Herbivores showed similar mean organic mercury levels (UP = 117 μg g^?1; DW = 120 μg g^?1; n = 12), whereas omnivores (UP = 142 μg g^?1; DW = 534 μg g^?1; n = 27) and carnivores (UP = 545 μg g^?1; DW = 1,366 μg g^?1; n = 69) showed significantly higher values below the dam. The absence of a reservoir effect in herbivores is expected, since they feed on grassy vegetation, near the riverbanks, which is not much influenced by mercury in aquatic systems. On the other hand, the higher mercury levels below the dam observed for omnivores and carnivores suggest a possible influence of the reservoir since they feed on items that could be contaminated by MeHg exported from upstream. The results highlight the necessity of assessing areas downstream of reservoirs.