Waterborne ethynylestradiol induces vitellogenin and alters metallothionein expression in lake trout (Salvelinus namaycush)

Estrogenic contaminants isolated from waters receiving sewage treatment plant effluents are known to induce the egg yolk precursor vitellogenin (VTG) in male fish. Levels of the metal binding protein metallothionein (MT) have also been shown to be affected by estrogens in fish. It has been postulated that MT declines in estrogen exposed fish to facilitate transfer of the essential metal Zn to cellular components required for VTG synthesis. To examine the changes in MT and VTG concentrations in fish exposed to an estrogen contaminant, lake trout (Salvelinus namaycush) were exposed to waterborne ethynylestradiol at 0, 4, 40 or 400 ng/l(-1) for 21 days. Blood and tissues were collected after 21 days of exposure to measure circulating levels of VTG as well as MT concentrations in liver and kidney. VTG increased in male and female fish from all three exposure groups compared to control fish. MT in liver significantly decreased in males and females compared to the controls, in the two highest exposures. MT in kidney was significantly higher in both sexes of fish exposed to the two highest concentrations of ethynylestradiol. These data are supportive of a relationship between estrogen exposure and the regulation of MT. Further studies to examine the specific links between estrogen exposure, VTG induction and regulation of essential metals like Zn are required.     

Induction of metallothionein synthesis in cultured human trophoblasts by cadmium and zinc

A system of primary cultures of human chorionic trophoblasts has been used for studying the effects of heavy metals on human reproductive tissue. Using this system, changes in cellular concentration of metallothionein (MT) in response to exposure to Cd or Zn were determined. Trophoblasts were isolated from term chorion leave, grown in RPMI-1640 medium, and exposed to Cd or Zn. Cellular content of MT was measured using the Cd/heme radioassay. MT increased in a concentration- and time-dependent manner after exposure to either metal. Cd increased the content of MT in trophoblasts at concentrations as low as 0.5 microM during a 24-h exposure. Moreover, extending exposure to Cd (2 microM) to 72 h resulted in a 3-4-fold increase in the concentration of MT. On a molar basis, Zn was not as potent a stimulus for MT synthesis as Cd, and required a concentration of 2.5 microM to increase the concentration of MT over a 24-h period. However, a 48- or 72-h exposure to Zn (10 microM) increased concentrations of MT nearly 8-fold over control values. Simultaneous exposure to Cd (2 microM) and inhibitors of protein synthesis, cycloheximide and actinomycin D, prevented the typical increase in MT concentration, suggesting that the metals act to increase the synthesis of MT. In another series of experiments, trophoblasts were exposed to Cd (2 microM) for 24 h, after which the cells were challenged with cytotoxic concentrations of Cd. Cells pretreated with Cd and then challenged with toxic concentrations of Cd had higher levels of MT and showed less toxicity, as indicated by leakage of lactic dehydrogenase. These results suggest that MT serves to sequester the metals in trophoblasts and reduce the toxicity of heavy metals. Thus, this system should be useful for studying the effects of heavy metals and characterizing the induction of MT in human reproductive tissue in vitro.     

Cellular biomarkers of exposure and biological effect in hepatocytes of turbot (Scophthalmus maximus) exposed to Cd, Cu and Zn and after depuration

Cellular biomarkers of exposure and biological effects were measured in hepatocytes of turbot exposed to either Cd, Cu or Zn at concentrations of 1 and 10 mg/l seawater for 7 days and after depuration for 14 days. Metal content in hepatocyte lysosomes was determined by image analysis after autometallography (AMG) as volume density of autometallographed black silver deposits (Vv(BSD)). Metallothionein (MT) levels were quantified on liver sections by microdensitometry after immunohistochemical staining with a polyclonal anti cod-MT antibody (MT-OD), and in the cytosolic fraction of hepatocytes by difference pulse polarography (MT-DPP). Lysosomal structural changes (lysosomal volume, surface and numerical densities--Vv(LYS), Sv(LYS) and Nv(LYS-), and surface-to-volume ratio S/V(Lys)) were quantified by image analysis after demonstration of beta-glucuronidase activity on liver cryotome sections. Vacuolisation produced by metal-exposure in hepatocytes was quantified by stereology as volume density of vacuoles (Vv(VAC)). Exposure time and metal concentrations significantly affected Vv(BSD) in lysosomes, MT levels and the degree of vacuolisation after 1 h and 1 day exposure to the three metals. The highest Vv(BSD), MT and Vv(VAC) values were recorded after 7 days exposure in all cases. MT-OD and MT-DPP were significantly correlated with Vv(BSD). Vv(LYS) in hepatocytes increased significantly after exposure to the metals. Exposure biomarkers returned to control values after depuration with the exception of those turbots that had been exposed to 10 mg Cd/l. Alike, Vv(LYS) and Sv(lys) (Cu exposure) and Nv(LYS) (Cd and Zn exposures) returned to control values after depuration. It has been therefore demonstrated that the biomarkers used are reversible and return towards control levels once metal exposure ceases. Overall, it is concluded that Vv(BSD), MT-levels and lysosomal responses are valuable biomarkers to assess metal exposure and its effects in turbot, although in quantitative terms the biomarker response varied between metals.     

A bioassay for metals utilizing a human cell line

The purpose of this study was to assess the ability of the HepG2 cell line to function as a bioassay for metal contamination in sediments, using metallothionein (MT) as a biomarker of exposure. Sediments were collected from the eastern and western ends of Lake Erie, extracted using EPA method 200.7, and analyzed for cadmium (Cd), mercury (Hg) and lead (Pb) levels using ICP-AES. Sediment extracts were neutralized then used at a 2.5% final concentration in the exposure medium. MT levels were measured using the cadmium–hemoglobin affinity assay after a 48 h exposure. Fortified blanks from the ICP protocol served as positive controls. Also, HepG2 cells were exposed to Cd, Pb or combinations of Cd and Pb to determine whether or not induction of MT observed in cells exposed to sediment extracts was due to a single metal, combinations of metals, pH, or some other factor. Additionally, cells were exposed to a range of Cd concentrations approximating the levels found in the extracts (0.0005–0.1 mg/L) to determine if a concentration-response occurred. Total metal levels ranged from 527 to 33.5 mg/kg with lead the predominant metal, accounting for 100–88.9% of the total quantifiable metals in the sediments. The biomarker response (MT induction) was strongly correlated (r² = 0.9919, r² = 0.990) with total metal and lead levels in the sediments, respectively, which supports recent field studies indicating the biomarker can discern differences in the strength of the inducing agent. Statistically significant MT induction was associated with sediments which contained measurable Cd concentrations and no significant differences were observed when comparing Cd only and Cd + Pb exposed cells indicating no interactions between Cd and Pb were occurring and supporting our finding that Cd was the main inducing agent in sediment extracts. MT levels also increased significantly in a concentration-dependent manner when cells were exposed only to Cd. Results suggest this human bioassay and the MT biomarker of exposure may be useful for monitoring complex metal mixtures in aquatic sediments.     

Inducibility of metallothionein biosynthesis in the whole soft tissue of zebra mussels Dreissena polymorpha exposed to cadmium,copper, and pentachlorophenol

Freshwater mussels Dreissena polymorpha (Pallas, 1771) were exposed to the elevated concentrations of Cd (10, 50, 100, and 500 μg/L), Cu (10, 30, 50, and 80 μg/L), and an organochlorinated pesticide, pentachlorophenol (PCP) (1, 10, and 100 μg/L). Induced synthesis of biomarker metallothionein (MT) and changes in concentrations of cytosolic Cd, Cu, and Zn in the whole soft tissue of mussels were monitored after a 7‐day laboratory exposure to the contaminants. A clear dose‐dependent elevation in the MT concentration was observed after exposure to Cd at doses of 10–100 μg/L, and this increase of MT content was accompanied with a linear increase of cytosolic Cd. Cd concentration of 500 μg/L caused no additional increase of MT and Cd in mussel cytosol, suggesting possible toxic effects due to exceeding cellular inducible/defense capacity. Cu exposure resulted with variable changes in MT concentrations, with no clear linear relationship between MT and Cu concentrations in water, although a progressive dose‐dependent accumulation of Cu in the soluble fraction of mussel tissues was recorded. A decrease of cytosolic Zn was evident at higher exposure concentrations of both metals used. PCP in concentrations applied was unable to induce MT synthesis, but the higher concentrations of PCP influenced the cytosolic metal concentrations. In conclusion, the results obtained confirm the specificity of MT induction in D. polymorpha as an biological response on metal stimulation, especially by cadmium, being more closely correlated to MT than copper within the ecologically relevant concentration range. The strong induction potential of cadmium as well as an absence of MT induction following exposure to PCP as an organic chemical contaminant are supporting evidences for usage of zebra mussel MT as a specific biomarker of Cd exposure in biomonitoring programs. © 2009 Wiley Periodicals, Inc. Environ Toxicol, 2010.     

Responses of metallothionein and reduced glutathione in a freshwater fish Oreochromis niloticus following metal exposures

In this study, levels of reduced glutathione (GSH) and metallothionein (MT) which are known to be biomarker of metal exposures were measured in a freshwater fish Oreochromis niloticus following exposure to 0, 5, 10 and 20μM concentrations of Cu, Zn, Cd and Pb for 14 days. Metals and GSH were measured in the liver, gill, intestine, muscle and blood, and MT in the liver. Copper accumulation occurred only in the gill, while Zn accumulation occurred only in the muscle. Lead accumulated in the liver and gill, whereas Cd accumulated in all the tissues. Metal exposures did not alter GSH levels in the blood, muscle and gill, but its levels increased in the liver following Cd, Zn and Cu exposures. MT levels in the liver increased only in Cd-exposed fish. The results showed that there was no significant change in tissue GSH levels following metal exposures, except in the liver. The levels of liver GSH increased significantly by all the metals, except lead. Data indicated that only the liver may be suitable indicator tissue to determine the response of GSH and MTs to metal exposure in environmental monitoring studies.     

Induction and binding of Cd, Cu, and Zn to metallothionein in carp (Cyprinus carpio) using HPLC-ICP-TOFMS.

The binding of Cd, Cu, and Zn to metallothionein in carp was studied under control and acute Cd exposure scenarios. Carp were exposed to different Cd concentrations for 96 h. Total (Cu, Cd, Zn)-MT levels were determined by the cadmium thiomolybdate saturation assay. Total tissue and cytosolic Cd, Cu, and Zn concentrations were determined by ICP-MS. The cytosolic metal speciation was determined by high pressure liquid chromatography (size-exclusion [SE] in combination with anion exchange [AE]) directly coupled to an inductively coupled plasma time of flight mass spectrometer (ICP-TOFMS). This coupled technique allows the chromatographic separation and online determination of the metals associated to the protein fractions separated. Very strong differences in the tissue compartmentalization and cytosolic speciation of the metals were observed. For example, over 30% of cytosolic zinc was bound to MT in liver while this was only 2% in the kidneys although total cytosolic levels were considerably higher. Induction of metallothionein during cadmium exposure was also tissue specific, displaying different response patterns in gills, liver, and kidney. Cadmium accumulated much stronger in liver and kidney compared to the gills and the latter also showed much lower MT levels. The renal MT-induction was more sensitive to Cd exposure than the hepatic MT induction since a significant increase of Cd-MT and total MT levels occurred at lower tissue Cd concentrations in the kidney in comparison to the liver, except for the highest Cd exposure level where a drastic 10-fold increase in hepatic Cd-MT was observed. At this Cd exposure level also an apparent spill over of zinc to the high molecular weight fraction was observed in the kidneys.     

Metallothionein gene and protein expression as a biomarker for metal pollution in natural gudgeon populations

Gudgeons (Gobio gobio) from historically Cd and Zn contaminated sites in Flanders (Belgium) were found to be resistant to elevated Cd levels. In previous work, this increased resistance was largely explained by increased metallothionein (MT) expression. Recently, environmental cleanup efforts resulted in a significant decrease in Cd concentrations in the surface water. In this study, we evaluated the use of hepatic metal and metallothionein (MT) concentrations as biomarkers of metal exposure before and after the cleanup. Hepatic MT mRNA levels were determined after the environmental metal levels decreased in order to assess the applicability of MT gene expression as an environmental biomarker in natural fish populations. Our data show that both metallothionein protein and gene expression have the potential to be sensitive biomarkers for metal exposure. Significant correlations were found (a) among accumulated metal concentrations and both MT protein and mRNA levels, and (b) between MT protein and mRNA levels. However, our data illustrated that while MT protein and gene expression give a quantitative picture of metal load at a single time point, quantitative information in natural populations cannot always be obtained when different time points (including different years) are compared, since MT gene and protein expression are affected by many other factors in addition to the metal load. Furthermore, the result of the environmental cleanup was reflected in a decrease of hepatic Cd concentrations. Zn remained the most important factor determining MT concentrations. Finally, two differently sized MT mRNAs were amplified to test the hypothesis that 3'-UTR length can offer a protective advantage in conditions of environmental stress. Our data provided no evidence to support this hypothesis. In contrast, the ratio of the long mRNA variant relative to total MT mRNA was surprisingly constant, and independent of exposure history.     

Zinc as a countermeasure for cadmium toxicity

Cadmium(Cd)is an important environmental pollutant and long-term Cd exposure is closely related to autoimmune diseases,cancer,cardiovascular diseases(CVD),and hepatic dysfunction.Zinc(Zn)is an essential metal that plays key roles in protein structure,catalysis,and regulation of their function.Numerous studies have shown that Zn can reduce Cd toxicity;however,the underlying mechanisms have not been extensively explored.Preclinical studies have revealed direct competition for sarcolemmal uptake between these two metals.Multiple sarcolemmal transporters participate in Cd uptake,including Zn transporters,calcium channels,and DMT1(divalent metal transporter 1).Zn also induces several protective mechanisms,including MT(metallothionein)induction and favorable redox homeostasis.This review summarizes current knowledge related to the role of Zn and metal transporters in reducing Cd toxicity and discusses potential future directions of related research.     

Tissue‐specific copper accumulation,zinc levels,induction, and purification of metallothionein in freshwater crab Sinopotamon henanense exposed to subacute waterborne copper

Copper (Cu) is one of the most important essential metals for crustaceans, buttoxic in excess. Metallothioneins (MT) are a family of low molecular weight, cysteine‐rich, metal‐binding proteins, which play important roles in metal homeostasis, detoxification, and cytoprotection. In the present study, Sinopotamon henanense were exposed to 0 (controls), 2.86, and 14.3 mg L^?1 waterborne Cu, Cu accumulation, zinc (Zn) levels and MT induction in gills and hepatopancreas were determined with Cd/Hemoglobin saturation assay and atomic absorption spectrophotometry method. Results showed that Cu accumulation and MT levels were both tissue‐specific and revealed some time‐dependent and dose‐dependent, respectively. The highest Cu accumulations of 82.10 ± 16.38 μg g^?1 w wt were observed in the gill after 15 days of 14.3 mg L^?1 Cu exposure, the peak MT induction of 136.16 ± 19.39 μg g^?1 w wt were observed in the hepatopancreas after 3 day of 14.3 mg L^?1 Cu exposure.In addition, the essential metal homeostasis of Zn was disturbed in some ways by subacute Cu exposure. The calculated ratios of actual Cu to theoretical maximum metal bound by MT indicating that the hepatopancreas had much greater Cu‐binding potentials than the gills. Positive correlation were shown between MT induction and Cu accumulation both in hepatopancreas and gills, indicating that MT induction in S. henanense can be considered as a biomarker for subacute waterborne Cu pollution. Furthermore, the Cu induced MT (CuMT) from S. henanense was purified using acetone precipitation (50–80%), followed by gel filtration chromatography and anion exchange chromatography. SDS‐PAGE and time‐of‐flight mass spectrometry analysis showed that S. henanense CuMT possess two isoforms and both mainly existed as monomer and dimmer forms. These present studies will be helpful to increase the database information of heavy metal‐induced MT in terms of crustaceans. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 407–417, 2014.     

Evaluation of the potential of the common cockle (<Emphasis Type="Italic">Cerastoderma edule</Emphasis> L.) for the ecological risk assessment of estuarine sediments: bioaccumulation and biomarkers

Common cockles (Cerastoderma edule, L. 1758, Bivalvia: Cardiidae) were subjected to a laboratory assay with sediments collected from distinct sites of the Sado Estuary (Portugal). Cockles were obtained from a mariculture site of the Sado Estuary and exposed through 28-day, semi-static, assays to sediments collected from three sites of the estuary. Sediments from these sites revealed different physico-chemical properties and levels of metals and organic contaminants, ranging from unimpacted (the reference site) to moderately impacted, when compared to available sediment quality guidelines. Cockles were surveyed for bioaccumulation of trace elements (Ni, Cu, Zn, As, Cd and Pb) and organic contaminants (PAHs, PCBs and DDTs). Two sets of potential biomarkers were employed to assess toxicity: whole-body metallothionein (MT) induction and digestive gland histopathology. The bioaccumulation factor and the biota-to-soil accumulation factor were estimated as ecological indices of exposure to metals and organic compounds. From the results it is inferred that C. edule responds to sediment-bound contamination and might, therefore, be suitable for biomonitoring. The species was found capable to regulate and eliminate both types of contaminants. Still, the sediment contamination levels do not account for all the variation in bioaccumulation and MT levels, which may result from the moderate metal concentrations found in sediments, the species’ intrinsic resistance to pollution and from yet unexplained xenobiotic interaction effects.     

Metallothionein induction in aquatic oligochaete tubifex tubifex exposed to herbicide isoproturon

Metallothioneins (MTs) are low-molecular-weight proteins mainly involved in metal ion detoxification. Recently it has been demonstrated that MTs participate in several cellular functions such as regulation of growth and antioxidative defenses. Moreover, pesticides can induce their synthesis. The aim of the current work was to determine the effects of isoproturon, either pure or formulated as Matin (suspension containing an isoproturon concentration of 500 g. L(-1)), on the metallothionein and total protein contents of the aquatic worm Tubifex tubifex. MT levels in exposed worms increased significantly after 7 and 15 days of exposure to a concentration of the herbicide of 50 mg. L(-1). Isoproturon reduced the metal (Cu, Zn, and Cd) content of metallothioneins, and it also increased the total protein content of the worms. These results suggest that MT induction may not be considered a specific biomarker of metal exposure but that it can be used as a nonspecific biomarker of the effect of isoproturon effect in aquatic worms.     

Protective effects of zinc on cultured rat primary hepatocytes to metals with low affinity for metallothionein.

The purpose of this study was to determine if Zn pretreatment could protect rat primary hepatocyte cultures from the cytotoxicity of five metals that have little or no affinity for metallothionein (MT). Hepatocytes were grown in monolayer cultures for 22 h and subsequently treated with ZnCl2 (100 microM) for 24 h; which increased the MT concentration 15-fold. Following Zn pretreatment, hepatocytes were exposed to various concentrations of Mn, V, Cr, Se, or Fe for an additional 24 h. Cytotoxicity was assessed by enzyme leakage and loss of intracellular K+. The toxicity of all five metals was significantly reduced in the Zn-pretreated cells. Zn pretreatment had no appreciable effect on the hepatocellular uptake (1-24 h) of Mn or Se. Zn pretreatment also did not increase the distribution of Mn or Se to the cytosol and neither metal was bound to MT, suggesting the protection was not due to their binding to MT. However, Zn pretreatment significantly decreased Mn-, Cr-, and V-induced cellular glutathione depletion. In summary, Zn pretreatment of rat primary hepatocyte cultures protects against Cr-, Mn-, Fe-, Se-, or V-induced hepatotoxicity. This protection does not appear to be related to MT induction but may be due to Zn-induced thiol or membrane stabilization and/or other biological changes produced by Zn.     

Effect of heavy metal exposure on blood haemoglobin concentration and methemoglobin percentage in Lumbricus terrestris

The earthworm haemoglobin (Hb) is a large extracellular hemoprotein flowing in a closed circulatory system. In spite of the fundamental role of this respiratory pigment in earthworm physiology, little is known about its sensitivity to environmental pollutants. The aim of the present work was to investigate the possible effect of heavy metal (cadmium, copper, mercury) exposure on Hb concentration and oxidation state (methemoglobin formation) in the earthworm Lumbricus terrestris. In addition, the tissue concentration of metallothioneins, a well-known biomarker of heavy metal exposure, was determined as an indicator of metal uptake. The animals were exposed to increasing concentrations of Cd, Cu and Hg utilizing the standard acute toxicity test, “Filter paper test” for 48 h. Exposure to heavy metals (10⁻⁵–10⁻³ M for Cd, 10⁻⁴–10⁻³ M for Hg, and 10⁻⁴–10⁻² M for Cu) was found to increase haemoglobin concentration in L. terrestris, although the magnitude of such an increase was dependent on the metal. In addition, metal exposure led to the formation of methemoglobin. Compared to other known biological responses to heavy metals, such as metallothionein induction, methemoglobin increase showed a higher sensitivity and a higher percentage variation in exposed organisms, showing to be a possible suitable biomarker of exposure/effect to be included in a multi biomarker strategy in earthworm in soil monitoring assessment.     

Biomarker study of a municipal effluent dispersion plume in two species of freshwater mussels

The toxicological effects of a primary-treated municipal effluent plume were investigated in two species of freshwater mussels, Elliptio complanata and Dreissena polymorpha, exposed for 62 days at sites upstream and downstream of an effluent outfall in the St. Lawrence River (Quebec, Canada). Levels of metallothioneins (MT), cytochrome P4501A1 activity, DNA damage, total lipids, relative levels of vitellins, and phagocytic activity (in E. complanata hemocytes) were determined after the exposure period. A parallel analysis measured heavy metals and coprostanol in mussel tissues. The results show that significant levels of coprostanol and some metals (specifically, Cu, Hg, Sb, Se, and Zn) had accumulated in mussels caged 5 km downstream of the effluent plume. Mixed-function oxidase activity, MT in gills, total lipids, DNA damage (in D. polymorpha only), and total hemolymph bacteria (in E. complanata only) had increased in these mussels, while levels of total cadmium (Cd), MT in digestive glands or whole soft tissues, phagocytic activity, and DNA damage in the digestive gland (in E. complanata only) were diminished. The exposure of mussels to surface waters contaminated by a municipal effluent led to many stress responses, depending on both the tissues and the species being examined.     

Involvement of cardiac metallothionein in prevention of adriamycin induced lipid peroxidation in the heart

The effect of pretreatment of mice with heavy metals: Zn, Cu, Bi, Co, Cd or Hg, against lethal and cardiac toxicity of adriamycin (ADR) was investigated. The lethal toxicity of ADR was significantly reduced by pre-administration of these metals except for Cu. The levels of both malondialdehyde and conjugated dienes, determined as indicators of lipid peroxidation in the heart, markedly increased with ADR administration, but the increases were significantly prevented by the pre-administration of Zn, Bi or Cd. The survival rate of mice after ADR injection was significantly correlated with metallothionein (MT) levels in the hearts which was increased by the injection of each metal, but was not correlated with those in the liver and kidneys. A significant negative correlation between the cardiac MT concentrations and the levels of lipid peroxidation in the hearts was also observed. These results suggest that increased MT synthesis in the heart may be involved in the protective effect of the heavy metals tested against the lethal and cardiac toxicities of ADR.     

Metal accumulation and metallothionein in two populations of brown trout, Salmo trutta, exposed to different natural water environments during a run-off episode

Cd, Cu and Zn concentrations were measured in ambient water as well as in gills, liver and kidney tissues of two natural populations of brown trout (Salmo trutta) during a run-off episode in two rivers with different metal compositions due to mining pollution. Metallothionein (MT) was also measured in these tissues. The two rivers, Rugla (Cu contaminated) and Naustebekken (Cd and Zn contaminated), are located in two neighboring drainage basins separated by the topographic divide near the city of Røros in the County of Sør-Trøndelag, Norway. In Rugla, the Cu concentration increased from 15 μg/l at the low water level to 41 μg/l during the run-off episode. In Naustebekken, corresponding values for Cd were 90–170 ng/l and those for Zn were 49–91 μg/l. Gill concentrations of Cu and Cd/Zn MT in both populations of native trout clearly reflect the presence of these metals in the rivers during the run-off, in accordance with the hypothesis of protection caused by MT induction. When Rugla trout were transferred to Naustebekken and vice versa, both the amounts of MT itself and the Cu contents reflected the concentration of this metal in the new environment, indicating that MT induction also protects against acutely increased metal levels. The measured levels of MT in both native and transferred trout can account for all the Cd present in the tissues, but not for all of the Cu and Zn. The capacity of MT to regulate Cd and Cu in the trout populations in their natural habitat therefore seems clearly present. Our data also indicate that the MT I and II isoforms may bind metals selectively.     

Metallothionein induction attenuates the effects of glutathione depletors in rat hepatocytes.

Metallothionein (MT) is a protein involved in heavy metal homeostasis and detoxification. According to several studies, MT could be involved in the antioxidant defense system, in which glutathione (GSH) is an essential component. The aim of this study was to verify the implication of MT in the antioxidant defense system in isolated rat hepatocytes. For this purpose, hepatocyte cultures were exposed to treatments known to modify MT or GSH levels. Zinc (Zn) was used as an inducer of MT while diethyl maleate (DEM) and buthionine sulfoximine (BSO) were used as GSH depletors. GSH, MT, and antioxidant enzyme activities were measured under conditions of MT induction and GSH depletion. Induction of MT synthesis through an 18-hour exposure to Zn (20 microM), did not result in any significant change in GSH levels or in activities of the antioxidant enzymes, glutathione-peroxidase (GSH-Px), catalase, and superoxide dismutase (SOD). DEM caused GSH depletion in cells, whether they were exposed to Zn or not, that lasted one h; after that time, GSH rose back to basal levels. BSO also caused GSH-depletion in cells exposed or unexposed to Zn, and no recovery in GSH levels was detectable during the entire period of exposure (12 h). However, GSH depletion induced by both DEM or BSO was attenuated in Zn-treated hepatocytes. Moreover, DEM and BSO exposures led to a depletion of MT levels in Zn-treated hepatocytes, indicating a link between GSH and MT metabolism. In cells unexposed to either Zn, DEM or BSO, there was an increase in GSH-Px and SOD activities after 6 and 12 h of incubation, respectively. Under the same conditions, catalase activity was inhibited after 6 h of incubation and returned to the activity found at t = 0 after 12 h of incubation. DEM and BSO treatments had no significant effect on GSH-Px or SOD activities although they led to inhibition of catalase activity. Taken together, our data indicate that MT induction, which creates a new pool of thiol groups in the cell cytosol, can attenuate GSH depletion induced by DEM or BSO. It appears that catalase is most sensitive to oxidative stress and that MT induction can antagonize the deleterious effects of such stress on the enzyme. This study supports the view that MT is part of the hepatocyte antioxidant-defense-system.     

Induction of metallothionein in rat primary hepatocyte cultures: evidence for direct and indirect induction

The ability of a number of metals and organic chemicals to induce metallothionein (MT) synthesis in primary cultures of rat hepatocytes was tested to determine whether MT induction in vivo results from a direct effect of the agent on the liver or as a result of an indirect, physiologic response to the agent. Hepatocytes were exposed to metals [zinc (Zn), cadmium (Cd), mercury (Hg), manganese (Mn), lead (Pb), cobalt (Co), nickel (Ni), and vanadium (V)] or organic compounds [ethanol, urethane, L-2-oxothiozolidine 4-carboxylate (L-OTCA), or dexamethasone] and were assayed for metallothionein by the Cd/hemoglobin radioassay. Cell viability was monitored by protein synthesis activity and cellular K+ concentration. Increases in MT concentrations were noted for Zn (22-fold), Hg (6.4-fold), Cd (4.8-fold), Co (2.4-fold), Ni (2.2-fold), and dexamethasone (4.5-fold). However, even at maximum tolerated concentrations, Mn, Pb, V, ethanol, urethane, and L-OTCA did not increase MT. The results indicate that Zn, Cd, Hg, Co, Ni and dexamethasone induce MT in vitro and thus are direct inducers of MT synthesis in hepatic tissue. In contrast, Mn, Pb, ethanol, urethane and L-OTCA, which did not increase the MT content of hepatocytes, apparently do so in vivo by an indirect mechanism.