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Using a newly identified organomercury lyase gene (merB3) expression system from Tn MERI1, the mercury resistance transposon first found in Gram-positive bacteria, a dual-purpose system to detect and remove organomercurial contamination was developed. A plasmid was constructed by fusing the promoterless luxAB genes as bioluminescence reporter genes downstream of the merB3 gene and its operator/promoter region. Another plasmid, encoding mer operon genes from merR1 to merA, was also constructed to generate an expression regulatory protein, MerR1, and a mercury reductase enzyme, MerA. These two plasmids were transformed into Escherichia coli cells to produce a biological system that can detect and remove environmental organomercury contamination. Organomercurial compounds, such as neurotoxic methylmercury at nanomolar levels, were detected using the biomonitoring system within a few minutes and were removed during the next few hours.
When pretreated with the organomercurial compound that reversibly blocks the adhesion of pyocin R1 to sensitive cells, this bacteriocin became susceptible to trypsin. Trypsinization resulted in the exclusive fragmentation of subunit protein no. 2 among 20 different subunit components and the disappearance of distal knobs from the fibers as well as in irreversible inactivation.
Mercury (Hg), especially in organic form, is a highly toxic pollutant affecting plants, animals, and man. In plants, the primary target of Hg damage is the chloroplast; Hg inhibits electron transport and photosynthesis. In the present study, chloroplast genetic engineering is used for the first time to our knowledge to enhance the capacity of plants for phytoremediation. This was achieved by integrating a native operon containing the merA and merB genes (without any codon modification), which code for mercuric ion reductase (merA) and organomercurial lyase (merB), respectively, into the chloroplast genome in a single transformation event. Stable integration of the merAB operon into the chloroplast genome resulted in high levels of tolerance to the organomercurial compound, phenylmercuric acetate (PMA) when grown in soil containing up to 400 micro M PMA; plant dry weights of the chloroplast transformed lines were significantly higher than those of wild type at 100, 200, and 400 micro M PMA. That the merAB operon was stably integrated into the chloroplast genome was confirmed by polymerase chain reaction and Southern-blot analyses. Northern-blot analyses revealed stable transcripts that were independent of the presence or absence of a 3'-untranslated region downstream of the coding sequence. The merAB dicistron was the more abundant transcript, but less abundant monocistrons were also observed, showing that specific processing occurs between transgenes. The use of chloroplast transformation to enhance Hg phytoremediation is particularly beneficial because it prevents the escape of transgenes via pollen to related weeds or crops and there is no need for codon optimization to improve transgene expression. Chloroplast transformation may also have application to other metals that affect chloroplast function.
The method of matrix isolation has been used to investigate mechanisms of gas-phase chemical reactions, in particular the pyrolysis of some organomercury compounds. A molecular beam of pyrolysis products was condensed simultaneously with a large excess of rare gas at temperatures from 5 to 15 degrees K to form a matrix that was subsequently studied by infrared spectroscopy. In the case of C(6)H(5)HgCCl(3), we found that pyrolysis in the temperature range 220-400 degrees C produced mainly dichlorocarbene. In addition, some trichloromethyl radical was observed and increased in relative importance at increased temperatures. Another identified product of pyrolysis was C(6)H(5)HgCl. In general, the same reactive intermediates, CCl(2) and CCl(3), were found from pyrolysis of Hg(CCl(3))(2) in the temperature range 250-500 degrees C, along with CCl(3)HgCl and HgCl(2). The identity of CCl(2) and CCl(2) was demonstrated by measurement of the relative intensities and isotopic splittings of stretching vibrations due to the chlorine isotopes. Isotopic patterns found for CCl(2) are: v(3) (745.8, 744.0, 741.8 cm(-1)), v(1) (719.5, 717.0, 714.9 cm(-1)) and for CCl(3) are: v(3) (897.8, 896.4, 895.2, 893.9 cm(-1)). Less dilution with the rare gas or warming of the matrix produced a decrease of CCl(2) and CCl(3) spectral bands and an increase of bands due to C(2)Cl(4), C(2)Cl(6), and other products. These results show the usefulness of matrix isolation in the study of such reactive species as CCl(2) produced by pyrolysis in the gas phase.
A former mercury plant, where mercury salts and organomercurials for pesticide use were produced, caused soil contamination in high concentrations. Typical organomercurial products included ethylmercury, phenylmercury, methoxyethylmercury and ethoxyethylmercury compounds. Risk assessment of these sites must be carried out before any major clean-up processes can be planned. A sensitive speciation technique for the various organomercury species in environmental matrices is a prerequisite for toxicity investigations. In this connection, a high-performance liquid chromatography-atomic fluorescence spectrometry (HPLC-AFS) technique has been developed to differentiate between and determine the presence of eight organomercury compounds in environmental samples. Using this technique, methylmercury, ethylmercury and phenylmercury and some unknown organomercury species were found in soil samples collected from the sites of an old mercury products producing plant. With regard to risk assessment, it is necessary to assess the toxicity of the organomercurials. As different microbial metabolic pathways react differently to mercury and its compounds, batteries of bioassays are, therefore, useful to evaluate the toxicity of pollutants. To describe the toxicity and genotoxicity of MeHg+, MeOEtHg+, EtHg+, EtOEtHg+ and PhHg+, p-tolymercury chloride, nitromersol and Hg2+ six bioassays were used: resazurin reduction method, Spirillum volutans test, nematode toxicity assay Panagrellus redivivus, Toxi-Chromotest and SOS-Chromotest. A ranking of the toxicity of the organomercurial is shown. The SOS-Chromotest indicated genotoxicity for 5-7 organomercurials.
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The hepatobiliary excretion of the anionic organic mercury compound (mersalyl) was studied in the isolated perfused rat liver and in isolated rat liver plasma membrane vesicles. In the isolated perfused liver, mersalyl is immediately taken up from the perfusion medium and concentratively excreted into bile. Uptake is characterized by saturation kinetics (S)0.5 = 20 microM, Vmax = 117 nmoles/min/g liver, cooperatively of mersalyl binding sites, stimulation by extracellular sodium and temperature dependence. Uptake of mersalyl into basolateral membrane vesicles also exhibits characteristics of a carrier mediated transport: saturation kinetics (S)0.5 = 28 microM, Vmax = 1.6 nmoles/min/mg protein, dependence on extravesicular sodium, cooperativity of mersalyl binding sites, temperature dependence and transstimulation by intravesicular non-radioactive mersalyl. Uptake was inhibited by alpha-naphthylacetic acid and mercapto group reagents, indicating involvement of mercapto groups on the carrier and a binding site for carboxylic anions. Data from the isolated perfused liver and from isolated basolateral vesicles indicate that mersalyl uptake into the liver is carrier mediated. Uptake mechanism and driving forces appear analogous to those for the uptake of chemically related compounds such as taurocholic acid. Therefore it is speculated that mersalyl may be transported by carrier molecules which apparently accept numerous chemically unrelated compounds.
Trace metals such as mercury, especially its organic compounds, are an important risk to the environment and to man due to their accumulation in the food chain. For this reason, the routine determination of the very toxic methylmercury, and of other organic and inorganic mercury compounds in marine and land animals, vegetables, fruits and fresh water is of increasing importance in health and environmental control programmes throughout the world. The majority of speciation methods for organomercurials involve a series of fundamental steps for the identification and quantification of samples of biological origin: extraction or isolation from the matrix; derivatisation and concentration; detection; separation of different species of interest and of interference. Each of these steps, as part of the chromatographic analysis of MeHg and of other organomercurials is revised in this study using food samples.
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The complexing properties of conjugates between 8-hydroxyquinoline and bovine serum albumin (Ox-BSA) towards inorganic and organic mercury were studied. Two Ox-BSA conjugates (different substitution ratio) were prepared and their complexing properties were studied. Through the use of titration curves with mercury (II), methylmercury and ethylmercury an evaluation of the complex stoichiometry and stability was obtained, showing that Ox-BSA has good affinity for all investigated mercuric compounds and that the stability increases in the order: Hg (II) < CH3Hg+ < C2H5Hg+, whatever conjugate is considered. Complexes show a stoichiometry of 1:1 between mercury and 8-hydroxyquinoline residues, except with the high substituted conjugate and Hg2+ ion. The skill of the high substituted conjugate to bind inorganic and organic mercury in the presence of NaCl was also studied. Organic mercuric complexes do not show significant modification due to NaCl. Nevertheless, considering inorganic mercury, the number of retained metal ions per protein molecule increases if the NaCl concentration becomes higher than 0.1 M, probably because at high NaCl concentrations 1:1 complexes between mercury and 8-hydroxyquinoline are preferred to 1:2 complexes.
Acidithiobacillus ferrooxidans MON-1 which is highly resistant to Hg2+ could grow in a ferrous sulfate medium (pH 2.5) with 0.1 microM p-chloromercuribenzoic acid (PCMB) with a lag time of 2 d. In contrast, A. ferrooxidans AP19-3 which is sensitive to Hg2+ did not grow in the medium. Nine strains of A. ferrooxidans, including seven strains of the American Type Culture Collection grew in the medium with a lag time ranging from 5 to 12 d. The resting cells of MON-1, which has NADPH-dependent mercuric reductase activity, could volatilize Hg0 when incubated in acidic water (pH 3.0) containing 0.1 microM PCMB. However, the resting cells of AP19-3, which has a similar level of NADPH-dependent mercuric reductase activity compared with MON-1, did not volatilize Hg0 from the reaction mixture with 0.1 microM PCMB. The activity level of the 11 strains of A. ferrooxidans to volatilize Hg0 from PCMB corresponded well with the level of growth inhibition by PCMB observed in the growth experiments. The resting cells of MON-1 volatilized Hg0 from phenylmercury acetate (PMA) and methylmercury chloride (MMC) as well as PCMB. The cytosol prepared from MON-1 could volatilize Hg0 from PCMB (0.015 nmol mg(-1) h(-1)), PMA (0.33 nmol mg(-1) h(-1)) and MMC (0.005 nmol mg(-1) h(-1)) in the presence of NADPH and beta-mercaptoethanol.
Mercury is a ubiquitous contaminant, and a range of chemical species is generated by human activity and natural environmental change. Elemental mercury and its inorganic and organic compounds have different toxic properties, but all them are considered hazardous in human exposure. In an equimolecular exposure basis, organomercurials with a short aliphatic chain are the most harmful compounds and they may cause irreversible damage to the nervous system. Methylmercury (CH(3)Hg(+)) is the most studied following the neurotoxic outbreaks identified as Minamata disease and the Iraq poisoning. The first description of the CNS pathology dates from 1954. Since then, the clinical neurology, the neuropathology and the mechanisms of neurotoxicity of organomercurials have been widely studied. The high thiol reactivity of CH(3)Hg(+), as well as all mercury compounds, has been suggested to be the basis of their harmful biological effects. However, there is clear selectivity of CH(3)Hg(+) for specific cell types and brain structures, which is not yet fully understood. The main mechanisms involved are inhibition of protein synthesis, microtubule disruption, increase of intracellular Ca(2+) with disturbance of neurotransmitter function, oxidative stress and triggering of excitotoxicity mechanisms. The effects are more damaging during CNS development, leading to alterations of the structure and functionality of the nervous system. The major source of CH(3)Hg(+) exposure is the consumption of fish and, therefore, its intake is practically unavoidable. The present concern is on the study of the effects of low level exposure to CH(3)Hg(+) on human neurodevelopment, with a view to establishing a safe daily intake. Recommendations are 0.4 micro g/kg body weight/day by the WHO and US FDA and, recently, 0.1 micro g/kg body weight/day by the US EPA. Unfortunately, these levels are easily attained with few meals of fish per week, depending on the source of the fish and its position in the food chain.
Averaged ERGs of rats treated with phenylmercury borate and methylmercury chloride were recorded. Amplitude differences were seen only with the latter treatment. A high Hg tot. level was found in eyes of animals treated with methylmercury.