Liver Metallothionein Research Articles

A competitive ELISA for detection and quantitation of oxidative and non‐oxidative polymeric forms of metallothionein 1 and 2 in normal and Ames dwarf mice

The physiological functions assigned to the metallothionein (MT) protein family (MT1, 2, 3 and 4), while not unambiguous, include heavy metal detoxification (A domain), oxidative stress reduction via zinc and cadmium-associated redox activity to protect against reactive oxygen species (B domain) and inflammation signal transduction (B domain). While these oxidoreductive mechanisms may require MT polymeric participation, the biological significance of monomeric and multimeric cellular MT remains unknown. We employed ELISA, SDS-PAGE and Western blot analysis to assess the immunoreactivity of purified rabbit MT1 and 2 using monoclonal mouse anti-horse MT (E9), which is specifically and equally neutralized by glutaraldehyde-polymerized MT1 and 2. MT was analyzed +/− glutaraldehyde treatment, and +/− added 2-mercaptoethanol. Our data indicated that, without glutaraldehyde, E9 recognizes non-oxidative (metal-bridged) polymeric MT species but not monomers or oxidative (cysteine-bridged) polymers. Glutaraldehyde polymerization of MT, however, imparts immunoreactivity to the oxidized species but not to monomers. Moreover, this signal is lost if MT is reduced by 2-mercaptoethanol prior to glutaraldehyde polymerization. Importantly, the non-oxidized signal is retained. We predicted that E9 could be used to generate a sensitive and reliable method for accurate quantitation, not only of total polymerized MT1+2, but also of oxidized and non-oxidized species. Data are presented for mouse heart and liver MT levels that validate this prediction.

Gill damage, metallothionein gene expression and metal accumulation in Tilapia sparrmanii from selected field sites at Rustenburg and Potchefstroom, South Africa

Fish were collected from field sites in the mining and agricultural areas of Potchefstroom and Rustenburg, North-West Province, South Africa. Water and sediment samples from each site, together with fish muscle and gills, were analysed by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) for Li, Na, Mg, Al, Ca, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Cd, Hg, Pb and U. All metal levels were within the levels set by the South African Department of Water Affairs and Forestry, except for copper, which was still within the levels set by other guidelines. Water hardness, pH, dissolved oxygen concentration and conductivity were measured at each site. Fish gills were analysed morphometrically for arithmetic mean epithelial thickness (Har) and with Reverse Transcriptase PCR for metallothionein (MT) gene expression levels. Fish liver was also analysed for MT expression. Har was found to increase with changing copper levels in gills of fish, while gill and liver MT levels corresponded to changes in sediment and gill cadmium levels, respectively. There were highly significant differences in Har between different sampling sites, while no significant differences in gill and liver MT expression were found.

Interaction between copper and zinc in metal accumulation in rats with particular reference to the synthesis of induced-metallothionein

The effectiveness of Zn at moderating the pro-oxidant effects of Cu was evaluated in two rat models that differed in the route and mode of administration. The endpoints investigated included measurement of the concentrations of Cu, Zn, metallothionein and glutathione concentrations, as well as SOD and catalase activity, in liver, kidneys and intestine. In a sub-chronic animal model, the hepatic accumulation of Cu was achieved by administration of dietary Cu (1.8 g/kg solid diet) for 30 days after which oral Zn (6 g/kg solid diet) was given. Cu treatment induced an increase in the hepatic and intestinal concentration of Cu of 66 and 455%, respectively, that was not associated with synthesis of metallothionein synthesis, but rather appeared to be related to the higher activity of SOD. Subsequent administration with Zn after dietary Cu induced an increase in the hepatic and intestinal metallothionein content of more twice and reduced the Cu content to control values. Thus, Zn could act as both a competitor for absorption on the luminal side of the intestinal epithelium inducing the synthesis of metallothionein. In the second animal model, we studied the effects of interaction between Cu and Zn administered by i.p. injection at the dose of 3 and 10 mg/kg, respectively; Zn was administered subsequent to Cu overload. In this case, when Zn was administered, Cu was already deposited in tissues and thus there is no competition between two metals at the level of membrane transport. In this experimental model treatment with Cu alone induced liver metallothionein synthesis, and the subsequent treatment with Zn did not decrease the hepatic content of Cu. One explanation for these observations is that Zn induces the synthesis of metallothionein, which binds Cu for which it has a higher affinity. Moreover, after treatment with Zn, SOD activity in the liver decreases of almost 30% with respect to treatment with alone Cu, suggesting that Zn has a protective effect.

An alternative interface for CE–ICP–MS cadmium speciation in metallothioneins based on volatile species generation

An alternative CE–ICP–MS interface based on volatile species generation (VSG) is here developed, evaluated and compared to the conventional sample introduction systems via nebulisation. For this purpose, the speciation of Cd–metallothioneins (MTs) in rabbit liver is taken as a model. Cd, bound to the different MT isoforms previously separated by CE, is transformed into volatile species at the exit of the capillary and on-line detected by ICP–MS. Optimum conditions for Cd VSG have been investigated in a flow injection device, using NaBH 4 as hydrogenation reagent in a HCl medium containing cobalt and thiourea as catalysts. Sample volume injected, CE separation voltage and reagents flows have been optimised. Analytical performance characteristics of the CE–VSG–ICP–(Q)MS coupling developed were evaluated, in terms of repeatability and linearity of response, using standard rabbit liver metallothionein isoforms (MT1 and MT2). Detection limits for Cd–MTs turned out to be almost one order of magnitude better than those derived from using a conventional Babington nebuliser-based interface. Compared to a MicroMist-based interface detection limits resulted to be similar, but the observed peak height was eight times higher using the VSG interface, indicating the enhanced analyte transport efficiency derived from VSG sample introduction systems.

XAFS Spectral Analysis of the Cadmium Coordination Geometry in Cadmium Thiolate Clusters in Metallothionein

We report the combination of measurement and prediction of X-ray absorption fine structure (XAFS) data, where the term XAFS refers to the overall spectrum that encompasses both the X-ray Absorption Near Edge Structure (XANES) region as well as the Extended X-ray Absorption Fine Structure (EXAFS) region, to evaluate the cadmium thiolate cluster structures in the metalloprotein metallothionein. XAFS spectra were simulated using coordinates from molecular models of the protein calculated by molecular mechanics/molecular dynamics (MM3/MD), from NMR analyses, and from analysis of X-ray diffraction data. XAFS spectra were also simulated using the coordinates from X-ray crystallographic data for [Cd(SPh)4]2-, CdS, [Cd2(mu-SPh)2(SPh)4]2-, and [Cd4(mu-SPh)6(SPh)4]2-. The simulated XAFS data that were calculated using the FEFF8 program closely resemble the experimental data reported for [Cd(SPh)4]2-, CdS, [Cd2(mu-SPh)2(SPh)4]2-, [Cd4(mu-SPh)6(SPh)4]2-, rabbit liver metallothionein cadmium alpha-domain (Cd4-alpha MT), and cadmium rabbit liver betaalpha metallothionein (Cd7-betaalpha MT). MM3 force field parameters were modified to include cadmium-sulfur bonding and were initially set to values derived from published X-ray diffraction and EXAFS experimental data. The force field was further calibrated and adjusted through comparison between experimental spectra taken from the literature and simulated XAFS spectra calculated using the FEFF8 program in combination with atomic coordinates from MM3/MD energy minimization. MM3/MD techniques were used with the calibrated force field to predict the high-resolution structure of the metal clusters in rabbit liver Cd7-MT. Structures for Cd3S9 (beta) MT and Cd4S11 (alpha) MT domains from MM3/MD calculations and those previously reported for Cd7-MT on the basis of 1H and 113Cd NMR data were compared. Structural differences between the different models for these cadmium thiolate clusters were evident. Combining the measurement and simulation of XAFS data provides an excellent method of assessing, modeling, and predicting metal-binding sites in metalloproteins when X-ray absorption spectroscopy (XAS) data are available.

Interferon alpha induction of metallothionein in rat liver is not linked to interleukin-1, interleukin-6, or tumor necrosis factor alpha

Synthesis of metallothionein (MT) is induced by interferon-alpha (IFN-α) in vitro and in vivo. In addition, IFN-α promotes redistribution of zinc (Zn) from the plasma to the liver in mice. However, it is not clear if IFN-α induces hepatic MT synthesis directly or indirectly via liberation of other cytokines. In order to address this issue, we determined hepatic MT levels, Zn concentration in plasma, liver, and urine, and plasma levels interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNFα) in rats following intramuscular injection of human IFN-α (1.5 × 10 6 UI/m 2). Animals were housed in metabolic cages and sacrificed at various times after IFN-α administration. Zn concentrations in serum, urine, and hepatic tissue were determined by atomic absorption spectrophotometry. MT protein was measured using the MT silver saturation method and expression of MT-1 and MT-2 mRNA was measured by RT-PCR. Plasma levels of rat IL-1, IL-6, and TNFα were determined using an ELISA method. Hepatic MT levels began to increase at 2 h following IFN-α administration and reached maximum levels at 12 h post-treatment. Induction of MT gene expression was confirmed by increases in MT-1 and MT-2 mRNA levels 6, 12, and 18 h after IFN-α administration. IFN-α treatment also resulted in biphasic increases in hepatic Zn, with levels peaking at 2 h, the time-point when MT levels are first increased, and again at 18 h. Concurrently, there were decreases in serum Zn levels at these time points, suggesting IFN-α induced movement of Zn from the blood to hepatic tissue. The decrease in serum Zn was not due to increased excretion since urinary Zn levels were unaffected following IFN-α treatment. IFN-α administration had no effect on plasma IL-1, IL-6, and TNFα levels. These results show that IFN-α promotes the increase of hepatic MT levels and plasma/liver redistribution directly, without IL-1, IL-6, or TNFα participation.

Voltammetric Methods in Metallothionein Research

The application of voltammetric methods using different rates of polarisation on HMDE reveal inert or labile behaviour of Cd- or Zn- complexes in the presence of excessive cadmium or zinc ions in solution. This phenomenon was demonstrated first on the simplest phytochelatin – complex of peptide (γ-Glu-Cys)2 Gly with cadmium, later on rabbit liver metallothioneins – Cd7 MT in the presence of cadmium and Cd5 Zn2 MT in the presence of zinc. Voltammetric methods can distinguish between labile and inert complexes present simultaneously and therefore could elucidate their role in reactions of metal ion transfer. Another method using different rates of polarisation – elimination voltammetry with linear scan – proved that S-tetracoordinated complexes of Cd(II) or Zn(II) in the above-mentioned metallothioneins on HMDE are reduced in the adsorbed state. This implies the possibility of increasing the sensitivity of identification or determination of the above complexes. On carbon composite electrode, similar behaviour of Cd-complexes as on HMDE was observed using differential pulse voltammetry.

The roles of metallothionein on cadmium-induced testes damages in Sprague–Dawley rats

The present study was to investigate whether metallothionein (MT) was involved in sensitivity of testis to cadmium (Cd) and protection of rats from Cd-induced testis damages. The rats were treated by intraperitoneal injection with 0.2, 0.4, and 0.8 mg Cd/kg BW for 7 days. The atomic absorption spectrophotometry and cadmium hemoglobin affinity assay were applied to evaluate the contents of Cd and MT in testis and liver. The testis glutathione (GSH), malondialdehyde (MDA) and daily sperm production were measured. There were substantial increases of both Cd and MT in the liver after Cd exposure. The testis Cd and MT contents were lower than those in the corresponding liver in Cd-exposed rats. Low doses of Cd (0.2 and 0.4 mg/kg BW) induced MT in testis, while a significant decline of MT was found in rats treated with 0.8 mg Cd/kg BW. By a concomitant decrease of MT, there was an obvious increase of MDA and marked decreases of GSH, daily sperm production in rats treated with 0.8 mg Cd/kg BW. These findings suggested MT was more difficult to be induced in the testis than in the liver by Cd, which might account for the high susceptibility of testis to Cd. MT, increased by a low dose of Cd, played an important role in protecting testis against Cd toxicity by sequestering and antioxidating.

Metal release in metallothioneins induced by nitric oxide: X-ray absorption spectroscopy study.

Metallothioneins (MTs) are low molecular weight proteins that include metal ions in thiolate clusters. The capability of metallothioneins to bind different metals has suggested their use as biosensors for different elements. We study here the interaction of nitric oxide with rat liver MTs by using in situ X-ray absorption spectroscopy techniques. We univocally show that the presence of NO induces the release of Zn atoms from the MT structure to the solution. Zn ions transform in the presence of NO from a tetrahedral four-fold coordinated environment in the MT into a regular octahedral six-fold coordinated state, with interatomic distances compatible with those of Zn solvated in water.

Maximum limits of organic and inorganic mercury in fish feed

The relatively high levels of mercury found in fish feeds might form a fish health and food safety risk. The present study aims to establish sublethal toxic threshold levels in fish and assess feed-fillet transfer of dietary mercury. Atlantic salmon (Salmo salar L.) parr were fed for 4 months on fish meal-based diets supplemented with mercuric chloride (0, 0.1, 1, 10 or 100 mg Hg kg−1 dry weight (DW)) or methylmercuric chloride (0, 0.1, 0.5, 5 or 10 mg MeHg kg−1 DW). At the end of the experiment, dietary inorganic mercury mainly accumulated in intestine (80% of body burden) and assimilation was low (6%). In contrast, methylmercury readily accumulated in internal organs and muscle (80% of body burden) and had a relatively high assimilation (23%). Highest accumulation of dietary inorganic mercury was observed in the gut and kidney. Fish fed 10 mg Hg kg−1 had an early (after 2 months) significant increase in renal metallothionein (MT) level and intestinal cell proliferation, followed by intestinal pathological conditions after 4 months of exposure. At 100 mg Hg kg−1, intestinal and renal function were reduced as seen from the significantly reduced protein and glycogen digestibility and increased plasma creatinine levels. For dietary methylmercury (MeHg), highest accumulation was found in blood and muscle. Intestinal cell proliferation and liver MT significantly increased at 5 mg MeHg kg−1 after 2 months of exposure. At the end of the experiment, blood haematology was significantly affected in fish fed 5 mg MeHg kg−1 and these fish exceeded the current food safety limit for mercury. Tissue MT induction and intestinal cell proliferation appeared to be useful and quantifiable early indicators of toxic mercury exposures. Based on the absence of induction of these early biological markers such as MT and cell proliferation, nonobserved effect levels (NOELs) could be set to 0.5 mg Hg kg−1 for dietary methylmercury and 1 mg Hg kg−1 for inorganic mercury. Lowest observed effect levels (LOELs) levels could be set to 5 mg kg−1 for methylmercury and 10 mg Hg kg−1 for inorganic mercury.

Quantification of Metallothionein-like Proteins in the Mussel Mytilus galloprovincialis Using RP-HPLC Fluorescence Detection

A HPLC-fluorescence method, using the fluorophore SBD-F (ammonium-7-fluorobenz-2-oxa-1,3-diazole-4-sulfonate), was adapted for the quantification of metallothioneins and their isoforms from the Moroccan mussel Mytilus galloprovincialis. The method was first optimized using a rabbit liver metallothionein. The effects of EDTA, tris(2-carboxyethyl)phosphine, and SBD-F on the labeling efficiency were studied. The optimized method was then applied to evaluate the amount of metallothionein in the mussels either exposed to cadmium in the laboratory or collected from the Casablanca coast, Morocco. The concentrations of metallothioneins measured in the field samples describe the degree of contamination of the sites and are reflected by distinct isoform patterns.

Effects of zinc and cadmium on HgCl 2-δ-ALA-D inhibition and Hg levels in tissues of suckling rats

The effects of CdCl 2 and ZnCl 2 pretreatments on the inhibition of δ-ALA-D (δ-aminolevulinic acid dehydratase) activity and Hg contents in liver and kidneys of suckling rats intoxicated with HgCl 2 were investigated. Zn-pretreatment prevented the effects of mercury at a higher magnitude than CdCl 2. Hepatic and renal δ-ALA-D activities were significantly inhibited by HgCl 2 and prior exposure to CdCl 2 partially prevented the renal effect of mercury but not altered the mercury levels in both tissues. Pretreatment with ZnCl 2 abolished mercury-induced δ-ALA-D-inhibition in kidneys and liver and induced an increase in renal (about three times) and a decrease in hepatic (to one-third) Hg contents when compared to the group injected only with mercury. In face of zinc effects to prevent Hg-δ-ALA-D inhibition and to alter Hg-deposition levels in kidney and liver, these results suggest that these effects may be partially due to the synthesis of metallothioneins (MT). In fact, liver MT content presented by animals pretreated with zinc was significantly greater than control and Hg-treated groups, but the increase showed by renal tissue (about 60%) was not significant. Although the MT is rich in cysteine (-SH) and consequently can form a great number of MT–Hg complex, other mechanisms should be also involved in zinc protection on mercury toxicity.

Helicobacter-induced gastritis in mice not expressing metallothionein-I and II.

Helicobacter pylori a primary cause of gastritis and peptic ulcer disease, is associated with increased production of reactive oxygen species within the gastric mucosa. Metallothionein (MT), a low-molecular-weight, cysteine-rich, metal-binding ligand, has been shown to sequester reactive oxygen species and reduce tissue damage. This study investigates the role of MT in H. pylori-induced gastritis in mice. Control (MT+/+) and MT-null (MT-/-) mice were inoculated with either 1 x 108H. pylori or H. felis, and were infected for 4, 8 and 16 weeks or 8 weeks, respectively. H. pylori load was determined by culture. Myloperoxidase activity and MT levels were also determined. The stomachs of H. felis-infected mice were more severely inflamed than those of H. pylori-infected mice. H. felis-induced gastritis was more severe (p =.003) in MT-/- than in MT+/+ mice. MT-/- mice also had higher (60%; p <.05) H. pylori loads than MT+/+ mice 4 weeks after infection but not 8 or 16 weeks after infection. Myloperoxidase activity with H. pylori was similar between MT+/+ and MT-/- mice. Thirty-three per cent greater (p <.05) myloperoxidase activity was observed in MT-/- than in MT+/+ mice infected with H. felis. In MT+/+ mice infected with H. pylori, liver MT was increased by 33 and 39% (p <.05) at 8 and 16 weeks, respectively, whereas gastric MT increased by 46% (p <.05) at 4 weeks and declined to baseline levels at 8 and 16 weeks. Mice lacking MT are more susceptible to H. pylori colonization and gastric inflammation, indicating that MT may be protective against H. pylori-induced gastritis.

A microscaled mercury saturation assay for metallothionein in fish.

A mercury (Hg) saturation assay for measuring metallothionein (MT) in fish liver was modified by optimizing binding conditions to minimize the mercury and tissue consumed. The revised method uses stable Hg at low concentrations instead of 203Hg. At the reduced Hg concentrations used, MT concentrations in livers homogenized in saline appeared to increase systematically with dilution in both bluegill sunfish (Lepomis macrochirus) and largemouth bass (Micropterus salmoides). This error suggested a binding limitation due to sulfhydryl oxidation or competition for and removal of mercury by non-MT proteins. Homogenizing tissues in trichloroacetic acid (TCA) eliminated the interference. To further evaluate the method, the protocol was tested in the laboratory and field. Metallothionein in bluegill injected with 0.6 mg/kg zinc chloride increased at a rate of 0.03 nmole MT/g liver/h (r2 = 0.53, p = 0.001). Linearity improved when data were corrected for protein content (r2 = 0.74, p < 0.0001). Metallothionein levels in bluegill from a coal ash-contaminated environment were significantly increased over that of hatchery-reared sunfish (F = 20.17, p = 0.0003). The microscaled procedure minimizes concerns related to radioisotope use and waste generation while retaining the high sensitivity of the 203Hg assay.

Properties of the reaction of chromate with metallothionein.

The reactivity of chromate or Cr(VI) with rabbit liver metallothionein (MT) was explored in this study. Zn(7)-MT reacts very slowly with Cr(VI) in a process characterized by a second-order rate constant of 3.9 x 10(-)(4) M(-)(1) s(-)(1). During the reaction, Zn(2+) was released from the protein. In contrast, apo-MT reduces chromate quicker and in this reaction is much more effective as a reducing agent, when compared to Cys or GSH. The kinetics are consistent with a reaction pathway involving an initial binding step followed by the reduction of Cr(VI). In the process, MT sulfhydryl groups were oxidized at the same rate that Cr(VI) disappeared. A Cr(V) intermediate was detected by EPR spectroscopy immediately upon mixing apo-MT with Cr(VI). The Cr(V) signal decayed during the reaction but was quite stable and could be observed for hours once the supply of thiols was depleted. The g values for the Cr(V) species were 2.014 and 1.987. The kinetics of the reaction of Cr(VI) and the concentration of the intermediate Cr(V) signal were independent of the oxygen concentration and were unaffected by the presence of superoxide dismutase, catalase, or DMSO. In the presence of oxygen, oxy radicals were generated according to ESR spin-trapping experiments with 5,5'-dimethyl-1-pyrroline-N-oxide. Superoxide dismutase decreased and catalase or DMSO largely inhibited the formation of the spin-trapped adduct. Cr(III), the presumed final species of the Cr(VI) reduction, formed a stable complex with apo-MT in the absence of oxygen with an average stoichiometry of two Cr ions bound per protein molecule. Upon addition of O(2), the complex slowly dissociated.

Zinc treatment prevents lipopolysaccharide‐induced teratogenicity in mice

During pregnancy, exposure to lipopolysaccharide (LPS) can lead to abortion, preterm delivery, and teratogenicity. The mechanisms underlying these effects are unclear. Both LPS and ethanol are potent inducers of liver metallothionein (MT), a key Zn binding protein. The teratogenic effects of ethanol have been linked to MT-induced changes in maternal-fetal Zn homeostasis, leading tofetal deficiency. This study was designed to assess whether the teratogenic effects of LPS are also related to MT induction and changes in Zn homeostasis. Non-pregnant normal (MT +/+) and MT-null (MT -/-) mice were injected subcutaneously with 0.5 microg/gm LPS and killed over 48 hr. In MT +/+ mice, liver MT concentrations were elevated from 6 hr, and were maximal at 24 hr (30-fold basal), whereas liver Zn levels were also increased from 6 hr. Plasma Zn concentrations decreased by 80% at 6 hr, and were below normal between 6 and 24 hr. In MT -/- mice, plasma Zn levels were increased from basal between 6 and 16 hr. Dams were injected with LPS, saline, or LPS and ZnSO4 (2 microg/gm, MT +/+ only) on Day 8 of gestation (GDS), killed on GD18, and the fetuses examined for malformations. External abnormalities were most prevalent in fetuses from MT +/+ dams exposed to LPS, where 34% of fetuses in each litterwere affected. MT +/+ dams treated with LPS and ZnSO4, and MT -/- dams treated with LPS had litters in which 5.4 and 4.8% of fetuses were abnormal respectively. The findings of this study strongly support the hypothesis that LPS teratogenicity is mediated at least in part by MT-induced changes in maternal Zn homeostasis,which compromises fetal Zn supply.

Sample stacking capillary electrophoresis with ICP-(Q)MS detection for Cd, Cu and Zn speciation in fish liver metallothioneins

Speciation of different metal isoforms present in metallothioneins (MTs) of living organisms requires both a high efficiency separation technique, e.g., CE, and a multielement, specific and highly sensitive detector (e.g. ICP-MS). To cope with naturally occurring low metal-MTs levels in biological systems and compensate for CE lack of sensitivity a large volume sample stacking (LVSS) methodology for CE-ICP-MS measurements is presented here. Two different CE-ICP-MS interfaces, including the use of a Babington and a HEN nebulizer, are investigated in detail and compared in terms of repeatability (%RSD) and limit of detection (LOD). By using the LVSS technique developed here the LODs obtained were improved 9–13 times. Finally, the proposed methodology was successfully applied to metal speciation studies of MT isoforms present in eel liver, both in control and Cd-induced groups, revealing a clear increase in Cd isoform signals by this metal exposure. Clear differences in profiles and signal intensities of the isoforms detected in both groups were also observed when Cu and Zn are monitored.

Zinc Supplementation at the Time of Ethanol Exposure Ameliorates Teratogenicity in Mice

Background: We have previously demonstrated that ethanol teratogenicity in mice is related to the maternal expression of metallothionein (MT), a zinc (Zn)-binding protein. Ethanol induces maternal liver MT, which causes plasma Zn concentrations to decrease as Zn moves into the liver. During pregnancy it is suggested that this change decreases fetal Zn supply and contributes to abnormal development. Here we investigated whether maternal Zn supplementation at the time of ethanol exposure reduces teratogenicity. Methods: Mice were injected with 25% ethanol (0.015 ml/g intraperitoneally at 0 and 4 hr) and ZnSO4 (2.5 μgZn/g subcutaneously at 0 hr) and were killed over 16 hr to ascertain changes in plasma Zn. Plasma Zn concentrations peaked at 2 hr, where levels were 5-fold normal and then returned toward normal over 14 hr. Pregnant mice were treated in a similar manner on gestation day 8 with saline, saline + Zn, ethanol + Zn, or ethanol alone, and fetal abnormalities were assessed on gestation day 18. Results: External abnormalities were most prevalent in offspring from dams treated with ethanol. Zn treatment at the time of ethanol exposure reduced the incidence of fetal abnormalities to basal levels. Litters from dams treated with ethanol + Zn contained more fetuses and fewer fetal resorption sites compared with those from ethanol-treated dams. Conclusions: These findings demonstrate that Zn supplementation at the time of ethanol exposure significantly negates the deleterious effects of ethanol on the fetus.

Metallothionein induction related to hepatic structural perturbations and antioxidative defences in roach (Rutilus rutilus) exposed to the fungicide procymidone

A variety of stresses, hormones, glucocorticoids and cytokines are known to induce metallothioneins (MTs) in animals. The aim of this study was to investigate the effects of chemical stress induced by the dicarboximide fungicide procymidone on hepatic structure, MT content and antioxidative defences (catalase and glutathione reductase activities and glutathione content) in the common fish Rutilus rutilus. Catalase and glutathione reductase activities remained stable throughout the experiment. Four days of exposure to 0.2 or 0.4 mg l−1 of procymidone induced an obvious increase in liver MT content, perturbation of metal MT contents, and an increase in hepatic glutathione content. After 14 days' exposure, obvious and large structural alterations of the hepatic parenchyma occurred simultaneously with a decrease in MT and glutathione content. These events were interpreted as degeneration of the liver. Fish exposed for 14 days to procymidone and then placed for 14 days in clean water showed nearly complete decontamination of the liver, but MT concentrations remained high. The toxicological significance of these events is discussed.

Zinc Supplementation at the Time of Ethanol Exposure Ameliorates Teratogenicity in Mice

Background: We have previously demonstrated that ethanol teratogenicity in mice is related to the maternal expression of metallothionein (MT), a zinc (Zn)‐binding protein. Ethanol induces maternal liver MT, which causes plasma Zn concentrations to decrease as Zn moves into the liver. During pregnancy it is suggested that this change decreases fetal Zn supply and contributes to abnormal development. Here we investigated whether maternal Zn supplementation at the time of ethanol exposure reduces teratogenicity.Methods: Mice were injected with 25% ethanol (0.015 ml/g intraperitoneally at 0 and 4 hr) and ZnSO4 (2.5 μgZn/g subcutaneously at 0 hr) and were killed over 16 hr to ascertain changes in plasma Zn. Plasma Zn concentrations peaked at 2 hr, where levels were 5‐fold normal and then returned toward normal over 14 hr. Pregnant mice were treated in a similar manner on gestation day 8 with saline, saline + Zn, ethanol + Zn, or ethanol alone, and fetal abnormalities were assessed on gestation day 18.Results: External abnormalities were most prevalent in offspring from dams treated with ethanol. Zn treatment at the time of ethanol exposure reduced the incidence of fetal abnormalities to basal levels. Litters from dams treated with ethanol + Zn contained more fetuses and fewer fetal resorption sites compared with those from ethanol‐treated dams.Conclusions: These findings demonstrate that Zn supplementation at the time of ethanol exposure significantly negates the deleterious effects of ethanol on the fetus.