Maleate induced change in the kidney binding of mercury in rats pretreated with cadmium

The kidney uptake of Hg2+ was increased by Cd2+-pretreatment when Hg2+ was given intraperitoneally but not subcutaneously. Subsequent s.c. administration of maleate increased Hg2+ release from the kidneys only if Hg2+ was given subcutaneously. Neither the effect of Cd2+, nor that of maleate, on the distribution of Hg2+ among the renal soluble protein fractions was affected by the route of Hg2+ administration. The protective effect of Cd2+-pretreatment against the nephrotoxic effect of Hg2+ was also independent of the route of Hg2+ administration. Maleate given in nephrotoxic doses removed Hg2+ and copper, but not Cd2+ from the renal metallothionein fraction. Mercury in the urine, however, was not complexed by proteins with the molecular weight of thionein, but was bound to high molecular weight proteins and diffusible molecules. These findings are discussed in relation to the role of metallothionein in the interaction between Cd2+ and Hg2+.     

Role of cell wall in Saccharomyces cerevisiae mutants resistant to Hg2+.

Hg2+-resistant mutants were isolated from Saccharomyces cerevisiae. Although they were very much like the parental strains in terms of colony-forming ability, they grew faster than the parental strains in the presence of sublethal doses of Hg2+. The Hg2+-resistant mutations were dominant. They were centromere linked and were divided into two groups by means of recombination; one of the mutations, designated HGR1-1, was mapped on chromosome IV because of its linkage to the TRP1 locus. The Hg2+-resistant mutants took up Hg2+ as much as, or slightly more than, the parental strains did. The mutants and parental strains retained only about 5 and 15%, respectively, of the cell-associated Hg2+ after removal of the cell wall; therefore, the mutants had less spheroplast-associated Hg2+ than did the parental strains. These results indicate that the cell wall plays an important role in protection against Hg2+ by acting as an adsorption filter and that the mutations described confer Hg2+ resistance by increasing the Hg2+-binding capacity of the cell wall.     

Environmental significance of the potential for mer(Tn21)-mediated reduction of Hg2+ to Hg0 in natural waters

The role of mer(Tn21) in the adaptation of aquatic microbial communities to Hg2+ was investigated. Elemental mercury was the sole product of Hg2+ volatilization by freshwater and saline water microbial communities. Bacterial activity was responsible for biotransformation because most microeucaryotes did not survive the exposure conditions, and removal of larger microbes (greater than 1 micromole) from adapted communities did not significantly (P greater than 0.01) reduce Hg2+ volatilization rates. DNA sequences homologous to mer(Tn21) were found in 50% of Hg2+-resistant bacterial strains representing two freshwater communities, but in only 12% of strains representing two saline communities (the difference was highly significant; P less than 0.001). Thus, mer(Tn21) played a significant role in Hg2+ resistance among strains isolated from fresh waters, in which microbial activity had a limited role in Hg2+ volatilization. In saline water environments in which microbially mediated volatilization was the major mechanism of Hg2+ loss, other bacterial genes coded for this biotransformation.     

Environmental significance of the potential for mer(Tn21)-mediated reduction of Hg2+ to Hg0 in natural waters.

The role of mer(Tn21) in the adaptation of aquatic microbial communities to Hg2+ was investigated. Elemental mercury was the sole product of Hg2+ volatilization by freshwater and saline water microbial communities. Bacterial activity was responsible for biotransformation because most microeucaryotes did not survive the exposure conditions, and removal of larger microbes (greater than 1 micromole) from adapted communities did not significantly (P greater than 0.01) reduce Hg2+ volatilization rates. DNA sequences homologous to mer(Tn21) were found in 50% of Hg2+-resistant bacterial strains representing two freshwater communities, but in only 12% of strains representing two saline communities (the difference was highly significant; P less than 0.001). Thus, mer(Tn21) played a significant role in Hg2+ resistance among strains isolated from fresh waters, in which microbial activity had a limited role in Hg2+ volatilization. In saline water environments in which microbially mediated volatilization was the major mechanism of Hg2+ loss, other bacterial genes coded for this biotransformation.     

Application of a modified 203Hg binding assay for metallothionein

A sensitive and rapid method to estimate concentrations of functional metallothionein in small biological samples, based upon the acid stability of ²⁰³Hg binding and solubility of this protein in trichloroacetic acid is described. Sephadex G-10 minicolumns supported in centrifuge tubes afforded separation and quantitation of isotope bound metallothionein from unbound metal. Elution of metallothionein bound ²⁰³Hg was achieved by short term-low speed centrifugation that segregated chelator-ligand complex into the eluate while unbound ligand remained in the gel. A well characterized standard of pure metallothionein protein was utilized to verify the specificity and sensitivity of the modified assay. Metallothionein levels were estimated by ²⁰³Hg binding in extracts of wild type and cadmium resistant Chinese hamster ovary cells treated with maximum tolerable concentrations of CdCl₂. Similar separation methods demonstrated [³⁵S]-cysteine incorporation into induced metallothionein. Additionally, induction of metallothionein was observed after treatment with particulate CdS but not crystalline NiS particles. These results demonstrate that the modified assay system is easily applied to serial measurement of metallothionein levels in multiple small biological samples.     

金属硫蛋白突变体的植物高效表达载体的构建及其在烟草中的表达

金属硫蛋白有α、β两个结构域（ｄｏｍ ａｉｎ）,其中α结构域优先结合Ｃｄ２＋ 和Ｈｇ２＋ ．小鼠αα突变体在大肠杆菌中已经构建并得到表达,其转基因植株已得到,可在Ｃｄ３００（３００ μｍ ｏｌ／Ｌ）中生长．为了进一步提高外源基因在烟草中的表达量,首先用ＰＣＲ 的方法设计引物,在基因翻译起始密码子ＡＴＧ 附近加入植物偏爱的碱基组合ＡＡＣＡＡＴＧ．另外,将该突变体基因插入具有双３５ Ｓ（ＣａＭＶ３５Ｓ）强启动子的植物双元表达载体ｐＧＰＴＶｄ３５Ｓ－ＢＡＲ中,获得了带有αα突变体的植物双元表达载体．通过农杆菌介导的叶盘转化法转化烟草ＮＣ８９,获得了抗除草剂的转基因植株．经ＰＣＲ－Ｓｏｕｔｈｅｒｎ 和蛋白Ｄｏｔ－ｂｌｏｔｔｉｎｇ 检测,证明了αα突变体在烟草中的嵌合与表达．抗重金属实验证明转基因烟草可以在Ｃｄ４００（４００ μｍ ｏｌ／Ｌ）中生长．     

Mercuric reductase enzymes from Streptomyces species and group B Streptococcus

Mercury volatilization (Hg2+ reductase) activity has been found with Hg2+-resistant isolates of three Streptomyces species and with three Hg2+-resistant strains of group B Streptococcus from clinical sources in Japan. Hg2+ reductase activities in crude cell extracts showed the temperature sensitivity, the requirement for an added thiol compound and the characteristic dependence on NAD(P)H cofactors of similar enzymes isolated from other bacteria.     

Occurrence of antibiotic and metal resistance and plasmids in Bacillus strains isolated from marine sediment

Eleven hundred Bacillus strains isolated from marine sediment from the Minas Basin, Nova Scotia, Canada, were purified on LB agar supplemented with ampicillin, chloramphenicol, erythromycin, streptomycin, tetracycline, or mercuric chloride. Seventy-seven isolates were examined for plasmid DNA, and for resistance to 11 antibiotics, HgCl2, and phenylmercuric acetate. Minimum inhibitory concentrations of Ag, Cd, Co, Cu, and Zn were also determined. Forty-three percent of antibiotic- and mercury-resistant strains contained one or more plasmids ranging from 1.9 to 210 MDa. Fifty-four percent carried plasmids greater than 20 MDa, and 97% were resistant to two or more metals. There was no correlation between plasmid content and resistance either to antibiotics or to mercurial compounds in these strains. Mercury-resistant isolates were unable to transform Hg2+ to volatile Hg0 by virtue of a mercuric reductase enzyme system (mer). Strains resistant to Hg2+ were investigated for their ability to produce H2S and intracellular acid-labile sulfide when grown in the absence and presence of HgCl2. Lower levels of H2S and intracellular sulfide were detected only in metal-resistant strains grown in the presence of HgCl2, suggesting that cellular sulfides complexed with Hg2+ in these strains.     

Genes encoding mercuric reductases from selected gram-negative aquatic bacteria have a low degree of homology with merA of transposon Tn501

An investigation of the Hg2+ resistance mechanism of four freshwater and four coastal marine bacteria that did not hybridize with a mer operonic probe was conducted (T. Barkay, C. Liebert, and M. Gillman, Appl. Environ. Microbiol. 55:1196-1202, 1989). Hybridization with a merA probe, the gene encoding the mercuric reductase polypeptide, at a stringency of hybridization permitting hybrid formation between evolutionarily distant merA genes (as exists between gram-positive and -negative bacteria), detected merA sequences in the genomes of all tested strains. Inducible Hg2+ volatilization was demonstrated for all eight organisms, and NADPH-dependent mercuric reductase activities were detected in crude cell extracts of six of the strains. Because these strains represented random selections of bacteria from three aquatic environments, it is concluded that merA encodes a common molecular mechanism for Hg2+ resistance and volatilization in aerobic heterotrophic aquatic communities.     

Genes encoding mercuric reductases from selected gram-negative aquatic bacteria have a low degree of homology with merA of transposon Tn501.

An investigation of the Hg2+ resistance mechanism of four freshwater and four coastal marine bacteria that did not hybridize with a mer operonic probe was conducted (T. Barkay, C. Liebert, and M. Gillman, Appl. Environ. Microbiol. 55:1196-1202, 1989). Hybridization with a merA probe, the gene encoding the mercuric reductase polypeptide, at a stringency of hybridization permitting hybrid formation between evolutionarily distant merA genes (as exists between gram-positive and -negative bacteria), detected merA sequences in the genomes of all tested strains. Inducible Hg2+ volatilization was demonstrated for all eight organisms, and NADPH-dependent mercuric reductase activities were detected in crude cell extracts of six of the strains. Because these strains represented random selections of bacteria from three aquatic environments, it is concluded that merA encodes a common molecular mechanism for Hg2+ resistance and volatilization in aerobic heterotrophic aquatic communities.     

Hypersensitivity to Hg2+ and hyperbinding activity associated with cloned fragments of the mercurial resistance operon of plasmid NR1.

The region of plasmid NR1 concerned with resistance to Hg2+ and organomercurials consists of sequences found on restriction endonuclease fragments EcoRI-H and EcoRI-I. When both fragments were cloned together into a derivative of plasmid ColE1, the hybrid plasmid conferred properties indistinguishable from those of the parental plasmid, NR1: resistance to Hg2+ and to the organomercurials merbromin and fluoresceinmercuric acetate and the inducible synthesis of the enzyme mercuric reductase. When fragment EcoRI-I was cloned into plasmid ColE1, cells containing the plasmid was as sensitive to Hg2+ and organomercurials as plasmidless strains. When fragment EcoRI-H was cloned into ColE1, cells with the hybrid plasmid were hypersensitive to Hg2+ and organomercurials. This hypersensitivity was inducible by prior exposure to low, subtoxic Hg2+ or merbromin levels. It was associated with an inducible hyperbinding activity attributed to a gene governing Hg2+ uptake and found on fragment EcoRI-H (which contains the proximal portion of a mercuric resistance [mer] operon).     

Increased urinary excretion of zinc and copper by mercuric chloride injection in rats

The effects of HgCl₂ on urinary excretion of Zn, Cu and metallothionein at different time intervals were observed in male Wistar rats. The rats were given a daily intraperitoneal injection of²⁰³HgCl₂ (0.5 or 1.0 mg Hg kg^–1) for 2 days.²⁰³Hg, Zn, Cu and metallothionein in urine, kidney and liver were analyzed. Significant increases in urinary Zn and Cu concentrations were found in HgCl₂-dosed groups. Elevated urinary Zn and Cu concentrations were accompanied by an increased metallothionein excretion in urine at different time periods. Zn concentration in urine remained elevated during the entire observation period of 7 days. There were also increased concentrations of Cu and Zn in the renal cortex in one of the two exposed groups. The results indicate that urinary Cu and Zn are related to the manifestation of renal toxicity and/or the synthesis of metallothionein in kidney induced by mercury.     

Uptake and binding of cadmium and mercury to metallothionein in rat hepatocyte primary cultures

The administration of inorganic Cd and Hg in vivo has been shown to result in markedly different metal concentrations in rat liver. Primary cultures of rat hepatocytes were utilized to gain insight into the dispositional differences between these chemically similar metals. Hepatocyte monolayer cultures were exposed to several concentrations of Cd or Hg (3, 10 and 30μm) in serum-containing medium for 30min. The cells were then washed and incubated in fresh medium for the remainder of the experiment. Hepatocytes exposed to Cd accumulated significantly more metal than hepatocytes exposed to equimolar concentrations of Hg. In cells exposed to 3μm-Cd there was an initial loss of Cd from the hepatocytes when placed in fresh medium, followed by a gradual re-uptake of metal, concomitant with increased binding to metallothionein. In hepatocytes exposed to 3 and 10μm-Cd, 87 and 77% of the intracellular Cd was bound to metallothionein within 24h. Loss of Hg from hepatocytes pulsed with 30μm-Hg was also observed upon the addition of fresh medium and continued for the duration of the experiment. No time-dependent increase in Hg binding to metallothionein was observed. A maximum of about 10% of the intracellular Hg was found associated with metallothionein in hepatocytes exposed to 30μm-Hg. Studies utilizing [³⁵S]cysteine incorporation indicated significant increases in the amount of metallothionein synthesized in hepatocytes exposed to 3 and 10μm-Cd (300% of control value) and 30μm-Hg (150% of control value) 24h after metal pulsing. Time-course studies revealed a 6–12h lag in metallothionein synthesis, followed by a significant elevation in [³⁵S]cysteine incorporation into metallothionein between 12 and 24h. These studies suggest that (a) isolated hepatocytes differentiate between Cd and Hg and preferentially accumulate the former, and (b) Cd strongly stimulates the induction of, and preferentially binds to, metallothionein, whereas Hg induces weakly, and does not preferentially bind to, metallothionein.     

Induction of metallothionein mRNA in rat liver and kidney after copper chloride injection.

The kinetics of the increase of metallothionein mRNA in rat liver and kidney after CuCl2 injection was determined by cell-free translation and dot-blot hybridization of total RNA isolated at various times after the injection. Both assay procedures gave essentially the same result: a 16-fold increase in hepatic metallothionein mRNA was observed 7h after CuCl2 injection, with a decline to basal values by 15 h. The response in the kidney was less dramatic, with a 6-fold increase in metallothionein mRNA 5 h after injection, and basal values were attained by 12h. The rise in Cu2+ concentration in both organs was closely correlated with the increase in metallothionein mRNA; hepatic Cu2+ was increased 5.9-fold by 5h after injection and renal Cu2+ was increased 4.3-fold 5h after injection. The Zn2+ concentration in the liver had not risen significantly within 5h of Cu2+ injection. Renal Zn2+ concentrations did not alter appreciably in the Cu2+-treated animals. These results support the conclusion that Cu2+ is acting as a primary inducer of metallothionein mRNA in the rat.     

Bacterium-based heavy metal biosorbents: enhanced uptake of cadmium by E. coli expressing a metallothionein fused to beta-galactosidase

We investigated the potential utility of a recombinant E. coli that expresses the human metallothionein II gene as a fusion protein with beta-galactosidase as a heavy metal biosorbent. E. coli cells expressing the metallothionein fusion demonstrated enhanced binding of Cd2+ compared to cells that lack the metallothionein. It was shown that the metallothionein fusion was capable of efficiently removing Cd2+ from solutions. Approximately 40% of the Cd2+ accumulated by the recombinant cells free in suspension was associated with the outer cell membrane, and 60% of that was present in the cytoplasm.     

Formation of Methyl Mercury by Bacteria

Twenty-three Hg2+-resistant cultures were isolated from sediment of the Savannah River in Georgia; of these, 14 were gram-negative short rods belonging to the genera Escherichia and Enterobacter, six were gram-positive cocci (three Staphylococcus sp. and three Streptococcus sp.) and three were Bacillus sp. All the Escherichia, Enterobacter, and the Bacillus strain were more resistant to Hg2+ than the strains of staphylococci and streptococci. Adaptation using serial dilutions and concentration gradient agar plate techniques showed that it was possible to select a Hg2+-resistant strain from a parent culture identified as Enterobacter aerogenes. This culture resisted 1,200 mug of Hg2+ per ml of medium and produced methyl mercury from HgCl2, but was unable to convert Hg2+ to volatile elemental mercury (Hg0). Under constant aeration (i.e., submerged culture), slightly more methyl mercury was formed than in the absence of aeration. Production of methyl mercury was cyclic in nature and slightly decreased if DL-homocysteine was present in media, but increased with methylcobalamine. It is concluded that the bacterial production of methyl mercury may be a means of resistance and detoxification against mercurials in which inorganic Hg2+ is converted to organic form and secreted into the environment.     

Volatilization of mercury by an iron oxidation enzyme system in a highly mercury-resistant Acidithiobacillus ferrooxidans strain MON-1

A highly mercury-resistant strain Acidithiobacillus ferrooxidans MON-1, was isolated from a culture of a moderately mercury-resistant strain, A. ferrooxidans SUG 2-2 (previously described as Thiobacillus ferrooxidans SUG 2-2), by successive cultivation and isolation of the latter strain in a Fe2+ medium with increased amounts of Hg2+ from 6 microM to 20 microM. The original stain SUG 2-2 grew in a Fe2+ medium containing 6 microM Hg2+ with a lag time of 22 days, but could not grow in a Fe2+ medium containing 10 microM Hg2+. In contrast, strain MON-1 could grow in a Fe2+ medium containing 20 microM Hg2+ with a lag time of 2 days and the ability of strain MON-1 to grow rapidly in a Fe2+ medium containing 20 microM Hg2+ was maintained stably after the strain was cultured many times in a Fe2+ medium without Hg2+. A similar level of NADPH-dependent mercury reductase activity was observed in cell extracts from strains SUG 2-2 and MON-1. By contrast, the amounts of mercury volatilized for 3 h from the reaction mixture containing 7 microM Hg2+ using a Fe(2+)-dependent mercury volatilization enzyme system were 5.6 nmol for SUG 2-2 and 67.5 nmol for MON-1, respectively, indicating that a marked increase of Fe(2+)-dependent mercury volatilization activity conferred on strain MON-1 the ability to grow rapidly in a Fe2+ medium containing 20 microM Hg2+. Iron oxidizing activities, 2,3,5,6-tetramethyl-p-phenylenediamine (TMPD) oxidizing activities and cytochrome c oxidase activities of strains SUG 2-2 and MON-1 were 26.3 and 41.9 microl O2 uptake/mg/min, 15.6 and 25.0 microl O2 uptake/mg/min, and 2.1 and 6.1 mU/mg, respectively. These results indicate that among components of the iron oxidation enzyme system, especially cytochrome c oxidase activity, increased by the acquisition of further mercury resistance in strain MON-1. Mercury volatilized by the Fe(2+)-dependent mercury volatilization enzyme system of strain MON-1 was strongly inhibited by 1.0 mM sodium cyanide, but was not by 50 nM rotenone, 5 microM 2-n-heptyl-4-hydroxy-quinoline-N-oxide (HQNO), 0.5 microM antimycin A, or 0.5 microM myxothiazol, indicating that cytochrome c oxidase plays a crucial role in mercury volatilization of strain MON-1 in the presence of Fe2+.     

One-step purification of metallothionein extracted from two different sources

We describe a one-step purification of hepatic metallothionein from the Amazon fish Colossoma macropomum injected with cadmium and from the copper-loaded metallothionein from the yeast Saccharomyces cerevisiae, performed by affinity chromatography through metal-chelating columns. Yeast metallothionein was purified from Cu2+-loaded resin and eluted by a continuous EDTA gradient whereas hepatic metallothionein extracted from fishes was purified by Ni2+-loaded resin and eluted by a continuous imidazol gradient. Purified metallothioneins were evaluated by SDS-PAGE and characterized by UV spectra of the apo- and Cd2+-loaded protein. This method allowed high purity and yield as well as rapid one-step extraction of both metal-loaded and apoprotein.