Metallothionein-like proteins and zinc--copper interaction in the hindgut of Porcellio scaber (Crustacea: Isopoda) exposed to zinc.

Metallothioneins (MTs) are ubiquitous low-molecular-weight metal-binding proteins, with a variety of functions in metal metabolism ascribed to them. Among terrestrial invertebrates, MTs have been studied in nematodes, insects, snails, and earthworms. The aim of this study was the characterization of MT-like proteins in the terrestrial isopod crustacean Porcellio scaber in order to analyze their probable role in the metabolism of copper (Cu) and zinc (Zn). Dietary Zn supplementation (793 microg Zn/g dry food, 6 d) was applied to stimulate MT synthesis. After separation of the hindgut post-microsomic supernatant (cytosol) of Zn-exposed animals by gel filtration on a Sephadex G-75 column, a Cu- and Zn-containing peak was detected in the position of Ve/Vo approximately 2, where MTs are expected to elute. Rechromatography of these fractions by size-exclusion chromatography-high-performance liquid chromatography revealed that the 215-nm absorbance peak coincided with the absorbance peak of the rabbit MT II standard. These low-molecular-weight Cu- and Zn-binding compounds, detected in the cytosol of the hindgut cells in Zn-exposed P. scaber, are considered to be Cu, Zn-MT-like proteins. To our knowledge, this is the first report on the characterization of MT-like proteins in isopod crustaceans. These results also indicate that both Zn and Cu dynamics in P. scaber hindgut are affected at the given dietary Zn supplementation and that MT-like proteins are involved in this Zn-Cu interaction.

A study of low level selenium determination by hydride generation atomic fluorescence spectrometry in water soluble protein and peptide fractions.

Development of a method for very low level selenium determination in water soluble protein and peptide fractions, obtained after various separation procedures, is presented. A hydride generation atomic fluorescence spectrometry (HG-AFS) detection system was optimised and the influence of Cu(II), Sb(V), As(III) and HNO3 interferences in the measurement of Se by HG-AFS was investigated. A destruction procedure using HNO3 and H2O2 was also optimised and the average recovery of the digestion of a solution of selenomethioneine was 92 +/- 4% (n=14). Combination of this digestion with the detection system gave reliable results. Accuracy was tested by comparison with two independent methods. A very low detection limit (DL) of 0.2 ng/g of measuring solution was achieved. The whole procedure from weighing to measuring was performed in the same Teflon tube. The addition of HNO3 to the fractions before long term storage at -20 degrees C was necessary to prevent adsorption on the test tubes. Selenium was measured in water soluble protein and peptide fractions obtained after extraction, and Sephadex G-75 chromatography performed on liver samples from: i) hens exposed to As2O3, ii) hens fed with a high fat feed and iii) the certified reference material dogfish liver (CRM DOLT-2). Because of the very low DL we were able to observe the Se distribution in chromatographic fractions of samples of organisms which were not exposed to excess amounts of Se. The presence of selenium associated with metallothioneins was observed.

Hepatic metallothioneins in two neotenic salamanders, Proteus anguinus and Necturus maculosus (Amphibia, Caudata).

The presence of metallothionein (MT) and the subcellular distribution of copper, zinc and cadmium were investigated in livers of two neotenic salamanders, Proteus anguinus and Necturus maculosus. In P. anguinus, caught in the wild, hepatic MTs were present as a single isoform of (Zn, Cu, Cd)-thioneins, whose molecular weight was estimated to be approximately 12000 by size exclusion chromatography. The percentage of zinc and cadmium was higher in the cytosol and of copper in the pellet. Cytosolic cadmium was almost exclusively associated with MTs (80%), while zinc and copper were also present in the regions of higher-molecular weight proteins. In laboratory bred N. maculosus, MTs were isolated from the liver cytosol and extract of the pellet as (Cu, Zn)- and (Zn, Cu)-thioneins, respectively. According to the low amount of copper extracting from liver pellets of N. maculosus, the presence of water insoluble aggregated forms of Cu-thioneins should be checked in further investigations.

DNA damage and metallothionein synthesis in human hepatoma cells (HepG2) exposed to cadmium.

Cadmium is an important heavy metal environmental toxicant, which is classified as a human carcinogen. The comet assay was used to evaluate the levels of DNA damage in a metabolically competent HepG2 cell line after treatment with low, non-cytotoxic and physiologically relevant concentrations of cadmium, alone and in combination with the dietary mutagen 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) and with the environmental mutagen benzo[a]pyrene (B(a)P). After exposure of the cells to 10, 100 and 1000 nM CdCl(2), a dose- and time-dependent increase of DNA damage was detected. Maximal damage was found after 12 h of treatment, but declined with further incubation with CdCl(2). The increased synthesis of metallothioneins on exposure to CdCl(2) up to 12 h suggests that they are responsible for the adaptation of HepG2 cells to the DNA damaging effects of CdCl(2). Co-treatment of the cells with CdCl(2) (10-1000 nM) and IQ (300 microM) induced a dose-dependent increase of DNA damage compared to cells treated with IQ alone. Co-genotoxic activity was also observed by increased formation of micronuclei in cells exposed to IQ and 1000 nM CdCl(2); at this concentration, CdCl(2) alone also induced micronuclei in HepG2 cells. Our results support the hypothesis that direct and indirect mechanisms are involved in cadmium-induced DNA damage.

The action of mercury on cell membranes.

The action of mercuric chloride and methyl mercuric chloride on the membrane lateral domain organization of bovine, equine, and canine erythrocytes was studied. Electron paramagnetic resonance (EPR) spectra of spin-labeled erythrocytes were analyzed with respect to their lateral domain structure. Continuous alteration of the membrane domain populations revealed that mercuric compounds affect the membrane via the evolution of toxic events in the cells.

Mercury, selenium, and cadmium in human autopsy samples from Idrija residents and mercury mine workers.

Total Hg and Se concentrations were determined in autopsy samples of retired Idrija mercury mine workers, Idrija residents living in a Hg-contaminated environment, and a control group with no known Hg exposure from the environment. In selected samples we also checked the presence of MeHg. The highest Hg concentrations were found in endocrine glands and kidney cortex, regardless of the group. MeHg contributed only to a negligible degree to the total mercury concentrations in all analyzed samples. In the Hg-exposed groups the coaccumulation and retention of mercury and selenium was confirmed. Selenium coaccumulation with a Hg/Se molar ratio near 1 or higher was notable only in those tissue samples (thyroid, pituitary, kidney cortex, nucleus dentatus) where the mercury concentrations were >1 microg/g. After tissue separation of such samples the majority of these elements were found in the cell pellet. Because the general population is continuously exposed to Cd and possibly also to Pb from water, food, and/or air, in some samples the levels of these elements were also followed. In all examined control tissue samples the average values of Cd (kidney cortex, thyroid, hippocampus, cortex cerebellum, nucleus dentatus) and Pb (thyroid, hippocampus) exceeded the average values of Hg. Cd concentrations were the highest, particularly in kidney cortex and thyroids (microg/g), but no relationship between Cd and Se concentration was evident at the tissue level. Regarding the results in the control group, it is debatable which element is the more hazardous for the general population as concerns neurotoxicity.

Preliminary study on the determination of selenium compounds in some selenium-accumulating mushrooms.

Using various chromatographic techniques (size exclusion, anion exchange, and cation exchange) combined with several detectors (neutron activation analysis and atomic fluorescence spectrometry), an attempt was made to characterize selenium compounds in some edible, selenium-accumulating mushrooms (Albatrellus pes-caprae and Boletus edulis). The mushrooms contained mostly low-molecular-weight (6 kDa) selenium compounds. After proteolysis, only a small fraction of the extractable selenium could be identified as selenite (3.0-9.2%, Albatrellus pes-caprae), selenocystine (minor, Albatrellus pes-caprae; 7.5%, Boletus edulis), or selenomethionine (1.0%, Boletus edulis), leaving the form of the bulk still to be elucidated.

Use of nitric acid in sample pretreatment for determination of trace elements in various biological samples by ETAAS.

Trace elements in liquid biological samples may be determined by direct electrothermal atomic absorption spectrometry (ETAAS). In our previous work it was found that samples containing proteins or DNA may leak out of the graphite tube before the drying step, despite the addition of various modifiers. In order to keep the sample to the graphite tube, samples were diluted before analysis 1 + 1 with 32% v/v nitric acid, or 5 microl of 32% v/v nitric acid was added to the graphite tube before ETAAS determination. Applying the proposed procedure, the concentrations of lead in eluted fractions after gel chromatographic separation of human cerebellar nucleus dentatus supernatant and platinum in isolated DNA samples were determined. The use of nitric acid in sample pretreatment prevent sample leakage out of the graphite tube, provided for even drying and considerably reduced nonspecific absorption in lead determination. The repeatability of measurements was better than + 6%. The accuracy of the procedure was checked by spiking samples. The recoveries for both elements lay between 93--104%. Nitric acid was found to be a better modifier than TRITON X-100.

A methodological study of mercury speciation using dogfish liver CRM (DOLT-2).

The purpose of the study was to optimise analytical methods for determination of the chemical speciation of mercury in studies of protective mechanisms of selenium. Optimisation of the methods was performed using CRM DOLT-2 (Dogfish liver), both in its original form and after separation of various fractions. The sample was homogenised with 10 mM Tris-HCl buffer (pH 7.6) and ultracentrifuged. The soluble phase obtained was applied to a size exclusion chromatography column (Sephadex G-75 column) for separation of various protein fractions. Total mercury (total Hg), monomethyl mercury (MeHg) and selenium (Se) were determined in whole dogfish liver tissue and its soluble and insoluble phases (pellet). Different approaches for determination of total Hg and MeHg were compared. Simultaneous determination of MeHg and inorganic mercury (Hg2+) was based on alkaline dissolution and/or acid leaching, followed by ethylation, room temperature precollection, isothermal gas chromatography (GC), pyrolysis and detection with cold vapour atomic fluorescence spectrometry (CVAFS). The sum of MeHg and Hg2+ was compared to total Hg results obtained by acid digestion and CVAAS detection. The accuracy of MeHg determination was checked by its determination using acid leaching at room temperature, solvent extraction, back extraction into Milli-Q water, ethylation, GC and CVAFS detection. For the insoluble phase it is recommended to use solvent extraction for MeHg and acid digestion CVAAS for total Hg. For determination of MeHg and Hg2+ in the lyophilised sample and water soluble fractions containing low concentrations of mercury species, the simultaneous measurement of MeHg and Hg2+ after alkaline dissolution is the most appropriate method.

Effect of arsenic trioxide on metallothionein and its conversion to different arsenic metabolites in hen liver.

The metabolism of arsenic, its affinity to metallothionein (MT), its influence on selenium levels, and its biotransformation to different metabolites in the liver tissue of laying hens exposed to arsenic trioxide (As2O3) was investigated. The experiment was performed with two groups of hens fed for 19 d with either a standard diet or with the same diet enriched in arsenic (30 microg/g). The major findings were as follows: 1. After 19 d exposure, about 65% of the total liver As was found in the water-soluble phase (100,000g centrifuged supernatant). In liver supernatant, As binding was found mostly in the range of very low-molecular-weight proteins (Mr < 10,000). Although after exposure the amount of MT-like proteins increased, the As bound to it was only in trace amounts. The protein was identified by convential procedures as Zn,Cu-thionein with traces of selenium and arsenic. 2. Arsenic exposure resulted in almost unchanged Se levels regarding its tissue concentrations and distribution between supernatant and pellet, where about 10% of total Se was found in the supernatant. On the contrary, As exposure did affect Cd levels. Tissue Cd concentration was slightly diminished, but the percentage of tissue Cd found in the water-soluble phase was increased from 20% to 40%. 3. In methanol extracts of tissue and supernatant of the As-exposed group, only two arsenic compounds were detected, As(III) and dimethylarsinic acid (DMA), the latter prevailing.

Mercury toxicokinetics in Wistar rats exposed to elemental mercury vapour: modeling and computer simulation.

The kinetics of total mercury (Hg) absorption, distribution and elimination in Wistar rats exposed for long periods to elemental mercury vapour (Hg zero) in the Idrija mercury mine were studied. From the experimental data base a compartmental model was built as a framework for experimental data interpretation and prediction of organ mercury levels under different conditions. Using the model the exposures of rats under conditions comparable to those of professionally exposed workers (mercury miners, workers in the chloralkali industry) and individuals with amalgam fillings were simulated.

Interactions of mercury in rat brain.

In order to study the metabolism of mercury (Hg), its affinity to metallothionein (MT), and its influence on levels of the essential metals copper and zinc in the brain tissue of rats exposed to elemental mercury (HgO) vapor was investigated. The major findings were: 1. After long-term exposure, about 40% of mercury was found in the brain water-soluble phase (supernatant); 2. In brain supernatant, about 80% of Hg was found in the range of low-molecular-weight proteins; the MT-like protein Hg-Cu-Zn-thionein was isolated and partially characterized; 3. HgO vapor exposure resulted in increased tissue levels of essential Cu and Zn in addition to exogenous Hg; and 4. Experiments showed that HgO vapor exposure can induce the stimulation of rat brain MT synthesis.