Glyphosate disrupts redox status and up-regulates metallothionein I and II genes expression in the liver of adult rats. Alleviation by quercetin.

The present work evaluated the possible protective effects of quercetin against glyphosate-induced hepatotoxicity in adult rats. Rats were randomly divided into three groups: a control group (C), a glyphosate-treated group (Gly) and a group treated with both glyphosate and quercetin (Gly+QE). During the experimental period (15 days), glyphosate (50 mg/kg b.w.) was administered every two days by intraperitoneal way while quercetin (20 mg/kg b.w./day) was administered daily by gavage. Glyphosate-induced hepatic oxidative stress was evidenced by the increased levels of malondialdehyde, hydrogen peroxide, advanced oxidation protein products and protein carbonyls with a significant decrease in enzymatic (superoxide dismutase, catalase, glutathione peroxidase) and non-enzymatic (non-protein thiols, glutathione, vitamin C) antioxidants. Plasma biomarkers of hepatotoxicity (AST, ALT, ALP, γ-GT, albumin) were also altered. Moreover, glyphosate induced DNA damage, up-regulated metallothionein (MT I and MT II) genes expression and provoked histopathological changes in rats' liver. Quercetin supplementation to glyphosate-treated rats markedly ameliorated all the parameters indicated above as well as the liver histoarchitecture. Therefore, quercetin might have beneficial effects against glyphosate-induced hepatotoxicity in rats.

Nitric-oxide-mediated zinc release contributes to hypoxic regulation of pulmonary vascular tone.

The metal binding protein metallothionein (MT) is a target for nitric oxide (NO), causing release of bound zinc that affects myogenic reflex in systemic resistance vessels. Here, we investigate a role for NO-induced zinc release in pulmonary vasoregulation. We show that acute hypoxia causes reversible constriction of intraacinar arteries (<50 microm/L) in isolated perfused mouse lung (IPL). We further demonstrate that isolated pulmonary (but not aortic) endothelial cells constrict in hypoxia. Hypoxia also causes NO-dependent increases in labile zinc in mouse lung endothelial cells and endothelium of IPL. The latter observation is dependent on MT because it is not apparent in IPL of MT(-/-) mice. Data from NO-sensitive fluorescence resonance energy transfer-based reporters support hypoxia-induced NO production in pulmonary endothelium. Furthermore, hypoxic constriction is blunted in IPL of MT(-/-) mice and in wild-type mice, or rats, treated with the zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN), suggesting a role for chelatable zinc in modulating HPV. Finally, the NO donor DETAnonoate causes further vasoconstriction in hypoxic IPL in which NO vasodilatory pathways are inhibited. Collectively, these data suggest that zinc thiolate signaling is a component of the effects of acute hypoxia-mediated NO biosynthesis and that this pathway may contribute to constriction in the pulmonary vasculature.

Comparative toxicity of cadmium in the commercial fish species Sparus aurata and Solea senegalensis.

The induction of metallothionein-like proteins (MTLPs) as well as cadmium levels (Cd) was studied in tissues of gilthead sea bream Sparus aurata and flatfish Solea senegalensis after an intraperitoneal (i.p.) injection of cadmium (2.5 mg kg(-1) body weight). The liver, gills, intestine and blood of S. aurata as well as the liver and intestine of S. senegalensis were collected for analysis at 0, 3 and 6 days after the injection. Cd levels significantly increased in all tissues of the treated animals, and the highest accumulation was found in the liver in both species (p<0.05). Cadmium administration provoked significant inductions of MTLP synthesis in the liver and intestine of S. aurata (p<0.05), while no increase in these protein levels was recorded in tissues of S. senegalensis. Our findings imply that MTLP synthesis in S. aurata probably led to an enhanced ability to cope with the toxicant, whereas the MTLP induction in S. senegalensis was inhibited by the accumulated cadmium, as reflected by the mortality rate of this species. Under the experimental conditions, hepatic MTLPs appear to be a good indicator of the Cd level in S. aurata; however, no link was found between the cadmium concentration and MTLP induction in S. senegalensis, which suggests that MTLPs did not play a main role in detoxification by metal sequestration.

Metal and metallothionein content in bullfrogs: study of a whole watershed impacted by agricultural activities.

Among the various anthropogenic activities potentially affecting amphibian populations, agriculture has often been evoked. Fertilizers used in agriculture are documented sources of metals that may contaminate nearby rivers. Bullfrogs (Rana catesbeiana) were collected within the Yamaska River basin (Quebec, Canada) along a gradient of agricultural land use intensity. Hepatic, renal and intestinal metal content, along with hepatic metallothionein content, were determined. In general, dissolved metal concentrations and accumulated metal concentrations in bullfrogs varied little among sites. Hepatic Cu and Zn concentrations were significantly different among subwatersheds and were in accordance with the gradient in agricultural activities in at least one of the three years studied. Liver metallothionein content did not vary significantly among the subwatersheds, but they were correlated with Cu content within two sites and Cd content at one site. We conclude that R. catesbeiana could be used as a biomonitor for Cu contamination and potentially for other metals.

Evaluation of the potential of the common cockle (Cerastoderma edule 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.

Cadmium accumulation and biochemical responses in Sparus aurata following sub-lethal Cd exposure.

Cadmium (Cd), a heavy metal with limited biological function, is widely distributed in the aquatic environment as a result of natural and anthropogenic activities. The effect of 4 and 11 days exposure of gilthead sea bream Sparus aurata to sub-lethal concentrations of Cd was evaluated as levels of Cd content and Cd-metallothionein (MT) presence in different organs. The possible genotoxic effect was also evaluated in erythrocytes by using the "comet assay", a promising tool for estimating DNA damage at the single-cell level. The results obtained show that in the controls, Cd content was significantly higher in gills compared to in liver, but the treatment of fish with 0.1mg/l Cd induced a stronger accumulation of metal in liver depending on the length of the exposure period. Cd traces were found in plasma, muscle and kidney. Cd forms complexes in the cytosol with MT only in the liver but Cd-MT content significantly increased after 11 days of exposure to the metal, while after 4 days of treatment the protein level was similar to the control. The "comet assay" performed on S. aurata eryhtrocytes isolated from fish treated for 4 and 11 days with 0.1mg/l Cd, showed that there was no DNA damage at both exposure periods.

Biomarkers of exposure to metal contamination and lipid peroxidation in the benthic fish Cathorops spixii from two estuaries in South America, Brazil.

Biomarkers as lipid peroxidation, metallothionein and delta-aminolevulinic acid dehydratase were determined in Cathorops spixii to compare the biological responses of this fish from estuaries with distinct anthropogenic influence. Three areas were selected in two estuaries in accordance with the levels of contamination for the polluted (Santos/São Vicente) and with the hydrodynamic characteristics for the non-polluted (Cananéia) estuary. Water characteristics and mercury levels in C. spixii confirmed a high human influence in the polluted system. In general, the biomarkers showed differences between the estuaries, suggesting disturbances in the specific cell mechanisms due to the presence of multiple xenobiotics in the contaminated system. Therefore, these biomarkers are recommended to promote more accurate information about the exposure to pollutants. Additionally, the study of the effect of the multiple xenobiotics on resident species such as the benthic fish C. spixii can favor a better assessment of the environmental quality of these systems.

Biochemical endpoints on juvenile Solea senegalensis exposed to estuarine sediments: the effect of contaminant mixtures on metallothionein and CYP1A induction.

Juvenile Solea senegalensis were exposed to fresh sediments from three stations of the Sado estuary (Portugal) in 28-day laboratory assays. Sediments revealed distinct levels of total organic matter, fine fraction, redox potential, trace elements (arsenic, cadmium, chromium, copper, nickel, lead and zinc) and organic contaminants (polycyclic aromatic hydrocarbons, polychlorinated biphenyls and a pesticide: dichloro diphenyl trichloroethane). Organisms were surveyed for contaminant bioaccumulation and induction of two hepatic biochemical biomarkers: metallothionein (MT) and cytochrome P450 (CYP1A), as potential indicators of exposure to metallic and organic contaminants, respectively. Using an integrative approach it was established that, although bioaccumulation is in general accordance with sediment contamination, lethality and biomarker responses are not linearly dependent of the cumulative concentrations of sediment contaminants but rather of their bioavailability and synergistic effects in organisms. It is concluded that metals and organic contaminants modulate both MT and CYP1A induction and it is suggested that reactive oxygen species may be the link between responses and effects of toxicity.

S-glutathionylation of metallothioneins by nitrosative/oxidative stress.

Cystein residues within metallothionein (MT) structure have been shown to be particularly prone to S-nitrosylation. The objective of this study was to examine the possibility that MTs undergo S-glutathionylation under nitrosative/oxidative stress. MT from rabbit liver was treated with different concentrations of GSNO, diamide plus GSH or H(2)O(2) plus GSH. Parallel sets of samples were treated with 10mM DTT for 30min at 37 degrees C to reduce mixed disulphides. Incubations were then processed for Western blot or dot-immunobinding assay. Western blot with anti-MT or anti-GSH were also performed on peripheral blood mononuclear cell extracts. Structural aspects of S-glutathionylation of MTs were also examined. Treatment with GSNO, diamide/GSH or H(2)O(2)/GSH induced a dose-dependent increase in the levels of MT S-glutathionylation. This effect was completely reversed by treatment with the reducing agent DTT, indicating that S-glutathionylation of MT protein was related to formation of protein-mixed disulphides. Structural analysis of rat MT indicated that Cys residues located in the N-terminal domain of the protein are the likely targets for S-glutathionylation, both for their solvent accessibility and electrostatics induced reactivity. S-Glutathionylation of MT, given its reversibility, would provide protection from irreversible oxidation of Cys residues, thus representing a mechanism of high potential biological relevance.

Effect of zinc supplementation on plasma IL-6 and MCP-1 production and NK cell function in healthy elderly: interactive influence of +647 MT1a and -174 IL-6 polymorphic alleles.

Pro-inflammatory cytokine response and NK activity are controlled by the availability of zinc ion, whose intra-cellular transport is regulated by metallothioneins. In order to closely examine the importance of circulating zinc in the modulation of immune response during ageing, in the balance of Th2/Th1 equilibrium and finally in the reversibility of systemic low grade inflammation, we evaluated the changes occurring in plasma IL-6 and MCP-1 concentrations and NK lytic activity in a healthy low grade inflamed elderly population, following zinc-aspartate supplementation. In addition, we aimed to highlight the potential interaction among circulating zinc increments, changes in immunological parameters and +647 MT1a and -174 IL-6 polymorphic alleles. Thirty-nine healthy individuals (60-83 years) from the ZINCAGE cohort (previously typed for +647 MT1a and -174 IL-6 polymorphisms) were supplied with zinc-aspartate. Blood samples collected before and after supplementation underwent basal laboratory determinations (circulating zinc, albumin and C-reactive protein) and immunological studies (plasma IL-6 and MCP-1 and NK lytic activity). Zinc supplementation in subjects with low or borderline-normal circulating zinc increased the concentration of this ion and modulated plasmatic IL-6 and MCP-1 as well as NK lytic activity. An interactive effect of polymorphic alleles of MT1a and IL-6 genes on zinc, IL-6, MCP-1 and NK activity was evidenced following supplementation, indicating the genetic background as one of the determinants for identifying groups of subjects that can take advantage of therapeutic intervention.

Covalent arylation of metallothionein by oxidized dopamine products: a possible mechanism for zinc-mediated enhancement of dopaminergic neuron survival.

Metallothioneins are a group of low molecular weight proteins which can be induced upon exposure to metal ions, including Zn(II). These cysteine-rich proteins are believed to have antioxidant-like properties due to their ability to scavenge free radicals with their multiple sulfhydryl groups. Dopamine is a neurotransmitter that can form toxic quinone and semi-quinone products in an oxidative environment. While Zn(II) is known to be toxic to some neuron subtypes, here we report a beneficial effect of Zn(II) on dopaminergic neurons and identify a mechanism through which metallothionein may scavenge toxic dopamine oxidation products. Cultured embryonic neurons were treated with Zn(II), and the number of dopaminergic neurons surviving after two or three weeks in culture was determined. We demonstrate that under these conditions metallothionein is upregulated and is able to form covalent arylation products with dopamine and 6-hydroxydopamine both in vitro and in culture. These experiments suggest that Zn(II) enhances the survival of dopaminergic neurons, and we propose that as a mechanism, upregulated metallothioneins form covalent adducts with both dopamine and 6-hydroxydopamine, resulting in the observed neuroprotective effect of Zn(II) on these cells. As Zn(II) homeostasis and modulation of metallothionein expression are hallmarks of neurodegeneration, these studies may have significant implications for understanding the underlying basis of degenerative diseases involving dopaminergic neurons, including Parkinson's disease.

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 (0.0005-0.1mg/L) to determine if a concentration-response occurred. Total metal levels ranged from 527 to 33.5mg/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.

The influence of metallothionein on exposure to metals: an in vitro study on cellular models.

In the present study, the interactions between zinc (Zn) and copper (Cu) or iron (Fe) have been examined. Rat hepatoma cell line H4-II-E-C3, fibroblast cell line mutant MT-/-, and wild-type MT+/+ cells treated with ZnSO4 or CuSO4 or FeSO4 or CuSO4+ZnSO4 or ZnSO4+FeSO4 for different times have been employed to study the effect of metallothionein (MT), glutathione (GSH) and metal (Cu, Fe and Zn) accumulation during cellular adaptation to supraphysiological metal concentrations. To investigate the different biological functions in the processes of metal homeostasis and detoxification, the levels of both MT-1 and MT-2 mRNAs have been evaluated. The three cell lines responded differently to metal treatments suggesting that the uptake and storage of these metals are affected by the specific cellular model and MT presence. In particular, Zn treatment significantly decreased Fe accumulation (p<0.05), whereas MT induced by Zn increased intracellular Cu content (p<0.05). Moreover, in H4-II-E-C3 cells administration of metals resulted in a rapid and transient induction of MT (p<0.05) and in GSH accumulation (p<0.05) suggesting synergistic interactions in which both appear essential for a protective regulatory function against the redox activity of metals. Taken together these results demonstrate that Zn affects the cellular levels of Cu and Fe by competition with the same ligand sites and/or by coordinate regulation of MT and GSH content.

Changes of heavy metal, metallothionein and heat shock proteins in Sertoli cells induced by cadmium exposure.

In this study, we examined the levels of Cadmium (Cd), iron (Fe) and zinc (Zn), which were considered to be involved in Sertoli cell damage caused by Cd exposure. We also examined metallothionein (MT), heat shock protein 70 (Hsp70) and heme oxygenase-1 (HO-1) expressions in Sertoli cells induced by Cd exposure. Evaluation by the in-air micro-particle induced X-ray emission (PIXE) method revealed that Cd and Fe distribution was increased in the cytoplasm of Sertoli cells after Cd exposure. By contrast, Zn was decreased in the cytoplasm of Sertoli cells after Cd exposure. It was suggested that the target of Cd toxicity was the cytoplasm of Sertoli cells, Fe was considered to enhance damage to Sertoli cells caused by Cd exposure. The DNA fragmentation rate was determined by ELISA after Cd exposure to Sertoli cells. It remained essentially unchanged with 2.5 microM Cd exposure of Sertoli cells; however, MT, Hsp70 and HO-1 were significantly increased by Cd exposure. As a result, Cd-induced MT was protected Sertoli cells against apoptosis, and Cd-induced HO-1 was involved in protection against oxidative stress. Incidentally, MT, Hsp70 and HO-1 showed similar responses to Cd exposure.

Effects of physiological concentrations of heavy metals both individually and in mixtures on the viability and function of peripheral blood human leukocytes in vitro.

Among environmental contaminants recognized for their toxicity and global distribution, heavy metals are elements known to exert serious ecological consequences. Published experiments on the immunotoxic effects of metals such as methylmercury (MeHg), cadmium (Cd), and lead (Pb) were often conducted at concentrations higher than those present in the environment or those in human blood. In the present study the in vitro effects on human blood of environmentally relevant concentrations of MeHg (33-200 mug/L), Cd (3.1-16 mug/L), and Pb (75-207 mug/L) were assessed individually and in mixtures on the viability and immune competence of peripheral blood leukocytes (PBLs). At MeHg concentrations of 120 and 200 mug/L both lymphocyte proliferation, as measured by [(3)H]thymidine incorporation, and natural killer (NK) cytotoxity activity, as determined by dioctadecyloacarbocyanine, were suppressed. Our results showed an increase of intracellular thiols in lymphocytes and in monocytes at all the concentrations of metals tested. A decrease in the level of metallothionein (MT) was seen in monocytes in presence of Hg at concentration of 120 mug/L and higher. For lymphocytes, a significant increase of MT in groups containing the lower concentrations of Cd, and Hg was noted. In summary, it appears that Hg represents the most toxic metal at environmentally relevant concentrations on human peripheral mononuclear cells. The effects of Hg exposure were greater on lymphocytes and NK cells than on monocytes.

Dexamethasone Inhibits Copper-Induced Alpha-Synuclein Aggregation by a Metallothionein-Dependent Mechanism.

Intracellular aggregates of α-synuclein are the pathological hallmark of Parkinson's disease (PD) and dementia with Lewy bodies (DLB), being linked to neurotoxicity. Multiple triggers of α-synuclein aggregation have been implicated, including raised copper. The potential protective role of the endogenous copper-/zinc-binding proteins, metallothioneins (MT), has been explored in relation to copper-induced α-synuclein aggregation. Up-regulated endogenous expression of MT was induced in SHSY-5Y cells by the synthetic glucocorticoid analogue, dexamethasone. After treatment to induce endogenous MT expression, immunofluorescence confocal microscopy was used to quantify protein aggregates in cells with/without copper treatment. MT induction resulted in significant (p < 0.01), dose-dependent up-regulation of MT expression and significant reduction in Cu-dependent α-synuclein intracellular aggregates (p < 0.01) that could be suppressed by MT-specific siRNA. Ubiquitous (MT-2) and brain-specific (MT-3) isoforms were investigated by transient transfection of the GFP-fusion proteins, observing equivalent α-synuclein aggregate suppression by each. These studies indicate MT induction could have potential in PD/DLB neuroprotective therapy by suppressing α-synuclein aggregation.

Response of phase I and II detoxification enzymes, glutathione, metallothionein and acetylcholine esterase to mercury and dimethoate in signal crayfish (Pacifastacus leniusculus).

Metals and pesticides are common pollutants and the modulation of biomarkers can indicate sub-lethal influences on the physiology of organisms inhabiting impacted aquatic systems. We examined the effects of mercury and the organophosphate pesticide dimethoate on EROD, MROD, glutathione S-transferase (GST), acetylcholine esterase (AChE), metallothionein (MT) and glutathione (GSH) in the signal crayfish (Pacifastacus leniusculus). Crayfish were injected with mercury chloride or dimethoate (0.3, 0.6, 0.9 µg kg-1) and dissected after 72 h. EROD activity in the hepatopancreas did not change in response to mercury chloride treatment but exhibited a dose dependent decrease at all concentrations of dimethoate tested. MROD (hepatopancreas) exhibited a significant decrease at the 0.9 µg kg-1 treatment for both chemicals. GST (hepatopancreas) demonstrated a significant dose dependent decrease at all concentrations of both mercury chloride and dimethoate. AChE (tail muscle) decreased at the 0.6 and 0.9 µg kg-1 concentrations of dimethoate and 0.9 µg kg-1 mercury chloride. In gill tissue, MT increased in response to 0.3 and 0.6 µg kg-1 of mercury chloride but no effect was observed at the 0.9 µg kg-1 concentration of mercury chloride or any concentrations of dimethoate tested. MT did not change in response to mercury or dimethoate in tail tissue. Furthermore, neither chemical modulated GSH concentrations. Our results indicate that, apart from GSH, these markers are sensitive to the pollutants tested and that animals exposed in the wild are potentially compromised in their ability to detoxify environmental contaminants and carry out normal cellular processes.

Coordinated responses of phytochelatin synthase and metallothionein genes in black mangrove, Avicennia germinans, exposed to cadmium and copper.

To evaluate the role of phytochelatins and metallothioneins in heavy metal tolerance of black mangrove Avicennia germinans, 3-month-old seedlings were exposed to cadmium or copper for 30 h, under hydroponic conditions. Degenerate Mt2 and PCS primers were synthesized based on amino acid and nucleotide alignment sequences reported for Mt2 and PCS in other plant species found in GenBank. Total RNA was isolated from A. germinans leaves and two partial fragments of metallothionein and phytochelatin synthase genes were isolated. Gene expression was evaluated with reverse transcripatase-polymerase chain reaction (RT-PCR) amplification technique. Temporal analysis showed that low Cd2+ and Cu2+ concentrations caused a slight (but not significant) increase in AvMt2 expression after a 16 h exposure time, while AvPCS expression showed a significant increase under the same conditions but only after 4h. Results strongly suggest that the rapid increase in AvPCS expression may contribute to Cd2+ and Cu2+ detoxification. Moreover, we found that A. germinans has the capacity to over-express both genes (AvMt2 and AvPCS), which may constitute a coordinated detoxification response mechanism targeting non-essential metals. Nonetheless, our results confirm that AvPCS was the most active gene involved in the regulation of essential metals (e.g., Cu2+) in A. germinans leaves.

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.

Tilapia metallothionein genes: PCR-cloning and gene expression studies.

Genomic PCR reactions were performed to isolate gene sequences of tilapia metallothionein (tiMT) from Oreochromis mossambicus and Oreochromis aureus. Two AP1 binding sites, four metal responsive elements, and a TATA box are the major cis-acting elements identified in the 800-bp 5' flanking region of the tiMTs obtained in this study. The tiMT gene promoter cloned from O. aureus was characterized in vitro using PLHC-1 cell-line, a hepatocellular carcinoma of a desert topminnow (Poecciliopsis lucida), following the administrations of Cd2+, Co2+, Cu2+, Ni2+, Pb2+ and Zn2+. Only Cd2+, Pb2+ and Zn2+ were able to induce the transcription of tiMT gene promoter in PLHC-1 cells in a dose-dependent manner. Zn2+ had the highest fold induction of tiMT gene promoter activity. Deletion mutants were tested for their abilities to drive the transcription of reporter gene following Cd2+ and Zn2+ administrations. However, Cu2+ and Ni2+ also induced the production of hepatic MT mRNA in vivo. Northern blot analysis showed that liver gave the highest fold induction of MT gene expression following the administration of heavy metal ions. These data indicated that hepatic MT mRNA level in tilapia is a potential sensitive biomarker of exposure to various metal ions including Cu2+, Cd2+, Ni2+, Pb2+, Hg2+ and Zn2+ ions.