Metallothionein cDNA Research Articles

A novel biomarker for marine environmental pollution: A metallothionein from Mytilus coruscus

As one of the key enzymes that maintain the dynamic balance of metal content in animals and take part in the process of heavy metals detoxification, metallothionein (MT) is also associated with the process of immune response. In this study, the full-length metallothionein cDNA sequence of 324 bp from Mytilus coruscus was obtained (named as McMT) with a 222bp ORF encoding 73 amino acid residues in length and its theoretical pI/MW was 7.75/7.21 kDa. Blastp showed that the deduced amino acid sequence of McMT had significant homology with metallothionein from many animals, plants and bacteria, especially with other known MT of mollusca, for example Mytilus edulis and Mytilus galloprovincialis and they fell into the same evolutionary branch. Real-time PCR was used to examine the transcript pattern of McMT in hemocyte, gill, hepatopancreas, gonad, adductor and mantle. The highest transcript level appeared in hepatopancreas, which showed its multiple physiological roles with relation to the tissue type and the maximum expression in hepatopancreas could contribute to detoxification. Under the stress of heavy metal exposure for 15 days, the time-dependent expression pattern of McMT was observed in each treated group, but the highest expression appeared at different times for various concentrations of Cu2+ and Pb2+. The temporal expression of McMT after bacteria (Vibrio parahemolyticus and Aeromonas hydrophila) challenge firstly rose and then went down, and the expression peak appeared at 4h after V.parahemolyticus infection with 40-fold and 100-fold under A.hydrophila stress at 24h. The results suggested that McMT played a significant role in antioxidation and the expression of McMT can be used as a biomarker for monitoring the situation of marine pollution.

Metallothionein cDNA Cloning and Metallothionein Expression in Sea Cucumber (Apostichopus japonicus, Slenka) from the WeiHai Coast of China

The aims of this work were (1) the identification of the metallothionein (MT) gene cDNA sequence in Apostichopus japonicus and (2) the quantification of MT mRNA expression in the intestine, body wall and respiratory tree of A. japonicas after exposure to cadmium or zinc. Using RT-PCR and RACE-PCR, we cloned the MT complete cDNA sequence of A. japonicus using the RNA extracted from Cadmium-exposed intestines. The 1351 bp MT cDNA consisted of a 126 bp 5’ untranslated region, a 1069 bp 3’ untranslated region, and a 156 bp open reading frame coding for 51 amino acids, including 16 cysteines. The MT sequence of A. japonicus was 47% identical to the sequence of MT from Strongylocentrotus purpuratus. Through real-time quantitative PCR analysis, we found that the MT expression levels were significantly higher when the specimens were exposed to heavy metals, and the response to combined metal ions exposure occurred earlier than that for single metal ion exposure. Additionally, the induction of MT transcription was more responsive to zinc and can be envisaged as a potential biomarker for zinc contamination.

Molecular cloning, characterization and expression analysis of metallothionein in the liver of the teleost Acrossocheilus fasciatus exposed to cadmium chloride

Metallothionein (MT) has a characteristic molecular structure with a cysteine-rich content. This unique structure provides metal-binding and redox capabilities and promoting metal homeostasis and detoxification in living animals. In order to evaluate the effects of cadmium (Cd) on hepatic MT expression in the liver of Acrossocheilus fasciatus, we obtained the complete cDNA of the A. fasciatus liver MT for the first time. The MT cDNA contains a 605-bp sequence, which codes for 60 amino acids. Protein alignment showed that the similarity between MT protein sequences of A. fasciatus and those of other vertebrates (especially teleosts) was very high and a cysteine residue structure was also conserved. MT was detected in the liver, kidney, gill, testis, muscle, spleen, heart and brain tissues of A. fasciatus by tissue-specific expression analysis. After Cd exposure, Cd/hemoglobin saturation assay, immunohistochemistry and reverse-transcription quantitative PCR (RT-qPCR) were used to describe MT expression in liver tissue. These techniques indicate a sensitive response by liver MT to Cd exposure. The results suggest that A. fasciatus MT may play an important role in the detoxification processes in the liver, and also would be a useful biomarker for monitoring metal pollution in aquatic environments. In addition, A. fasciatus could be regarded as one candidate for a model species for bony fishes in ecotoxicology.

A copper-induced metallothionein gene from Exopalaemon carinicauda and its response to heavy metal ions

A full-length copper-induced metallothionein (EcMT-Cu) cDNA was obtained from Exopalaemon carinicauda (Holthuis) and it contained a 198bp open reading frame that encoded a peptide with 65 amino acid residues. Twenty-one cysteines were found in deduced amino acid sequence and the cysteine (Cys)-rich characteristic was also reported in different types of metallothioneins from other species. EcMT-Cu mRNA expression profile showed that it is the hepatopancreas specific gene. The expression of EcMT-Cu was extremely different when shrimp were exposed to seawater containing 50μM CuSO4 or 2.5μM CdCl2. The expression of EcMT-Cu in shrimp was significantly up-regulated at 12 and 24h after exposure to CuSO4, however, its expression was not induced compared to that of pretreatment (p>0.05) when shrimp were exposed to CdCl2. The transcript of EcMT-Cu was found to be extremely low at gastrula and nauplius stage and expression of EcMT-Cu could be detected from egg protozoa stage.

A cadmium metallothionein gene of ridgetail white prawn Exopalaemon carinicauda (Holthuis, 1950) and its expression

Metallothioneins (MTs) are a group of low molecular weight cysteine-rich proteins capable of binding heavy metal ions. A cadmium metallothionein (EcMT-Cd) cDNA with a 189 bp open reading frame (ORF) that encoded a 62 amino acid protein was obtained from Exopalaemon carinicauda. Seventeen cysteines were in the deduced amino acid sequence, and the cysteine (Cys)-rich characteristic was revealed in different metallothioneins in other species. In addition, the deduced amino acid sequence did not contain any aromatic amino acid residues, such as tyrosine (Tyr), tryptophan (Trp), and phenylalanine (Phe). EcMT-Cd mRNA was expressed in all tested tissues (the ovary, muscle, stomach, and hepatopancreas), and its expression profiles in the hepatopancreas were very different when shrimps were exposed to seawater containing either 50 mu mol/L CuSO4 or 2.5 mu mol/L CdCl2. The expression of EcMT-Cd was significantly up-regulated in shrimp exposed to CuSO4 for 12 h and down-regulated in shrimps exposed to CdCl2 for 12 h. After 24 h exposure to both metals, its expression was down-regulated. By contrast, at 48 h the EcMT-Cd was up-regulated in test shrimps exposed to CdCl2. The transcript of EcMT-Cd was very low or even absent before the zoea stage, and the expression of EcMT-Cd was detected from mysis larvae-I, then its expression began to rise. In conclusion, a cadmium MT exists in E. carinicauda that is expressed in different tissues and during different developmental stages, and responds to the challenge with heavy metal ions, which provides a clue to understanding the function of cadmium MT.

Effects of cadmium exposure on sea urchin development assessed by SSH and RT-qPCR: metallothionein genes and their differential induction

In order to study the defense strategies activated by Paracentrotus lividus embryos in response to sub-lethal doses of CdCl2, we compared the induced transcripts to that of control embryos by suppression subtractive hybridization technique. We isolated five metallothionein (MT) cDNAs and other genes related to detoxification, to signaling pathway components, to oxidative, reductive and conjugative biotransformation, to RNA maturation and protein synthesis. RT-qPCR analysis revealed that two of the five P. lividus MT (PlMT7 and PlMT8) genes appeared to be constitutively expressed and upregulated following cadmium treatment, whereas the other three genes (PlMT4, PlMT5, PlMT6) are specifically switched-on in response to cadmium treatment. Moreover, we found that this transcriptional induction is concentration dependent and that the cadmium concentration threshold for the gene activation is distinct for every gene. RT-qPCR experiments showed in fact that, among induced genes, PlMT5 gene is activated at a very low cadmium concentration (0.1 μM) whereas PlMT4 and PlMT6 are activated at intermediate doses (1-10 μM). Differently, PlMT7 and PlMT8 genes increase significantly their expression only in embryos treated with the highest dose (100 μM CdCl2). We found also that, in response to a lethal dose of cadmium (1 μM), only PlMT5 and PlMT6 mRNA levels increased further. These data suggest a hierarchical and orchestrated response of the P. lividus embryo to overcome differential environmental stressors that could interfere with a normal development.

Assessment of potential biomarkers, metallothionein and vitellogenin mRNA expressions in various chemically exposed benthic Chironomus riparius larvae

The objective of this study was conducted to identify the possibility of using Chironomus metallothionein (MT) and vitellogenin (VTG) as biomarkers of stress caused by endocrinedisrupting chemicals (EDCs), heavy metals, herbicides and veterinary antibiotics. We characterized the MT and VTG cDNA in Chironomus riparius and evaluated their mRNA expression profiles following exposure to different environmental pollutants. The gene expression analysis showed that the MT mRNA levels increased significantly after long-term exposure to cadmium (Cd), copper (Cu), Lead (Pb), di(2-ethylhexyl) phthalate (DEHP), and 2,4-dichlorophenoxyacetic acid (2,4-D). Moreover, the VTG mRNA expression increased significantly in C. riparius larvae exposed to BPA, NP, DEHP, Cd, 2,4-D and fenbendazole. Evaluation of the long-term effects of environmental pollutants revealed up regulation of Chironomus MT mRNA in response to DEHP exposure among EDCs, and the level of the VTG mRNA was increased significantly following treatment with Cd and herbicide 2,4-D at all concentrations in a dose-dependent manner. These results indicate that VTG could be used as a potential biomarker of herbicide and Cd as well as EDCs, while MT was a potential biomarker of heavy metals such as Cd, Cu, and Pb in aquatic environments.

Sequencing and Expression of Metallothionein mRNA in Freshwater Murrel after Cadmium Exposure

Metallothionein (MT) is a cysteine-rich, low molecular weight metal sequestering protein that has been shown to be involved in essential metal homeostasis and in detoxification of heavy metals. In the study sequences of MT genomic DNA and cDNA of freshwater murrel, Channa punctata (Bloch) were reported. cDNA was verified as the C. punctata MT gene by the characteristics of its predicted translation product, namely a high cysteine content, conserved Cys-Cys, Cys-X-Y-Cys, and Cys-X-Cys motifs, and a molecular weight of around 6 kDa. The quantitative real-time reverse-transcriptase polymerase chain reaction (q-RT-PCR) study was carried out in liver, kidney and gill tissues after exposure of the organism to waterborne cadmium for 8 h. At all the time points (viz. 1, 2, 4 & 8 h), a significant up regulation of MT transcript level was observed only in the liver. The results of time kinetics study indicated that expression pattern of C. punctata MT mRNA was found to be in tissue specific manner and the transcript level in the liver could be used as a biomarker of heavy metal contamination in water bodies.

Expression and function analysis of metallothionein in the testis of stone crab Charybdis japonica exposed to cadmium

Metallothionein (MT) participates in metallic homeostasis and detoxification in living animals. Previous studies have focused mainly on the functions of MT in vertebrates, but the functions of MT during spermiogenesis in invertebrates remain unclear. In order to investigate the functions of MT during spermiogenesis in the Japanese stone crab (Charybdis japonica), we identified the C. japonica MT complete cDNA sequence from the total RNA of the testis using RT-PCR and RACE. The 587bp MT cDNA contains: an 80bp 5′ untranslated region, a 333bp 3′ untranslated region, and a 174bp open reading frame. MT has 57 amino acids including 19 cysteines. The protein alignment between MT sequences of C. japonica and other crabs shows a high similarity and a strong identity in cysteine residues vital for the metal-binding affinity of MT. After the cadmium (Cd) exposure, the testis displays both abnormal morphology and MT mRNA expression both of which indicate a sensitive response of testis MT to Cd. Therefore, we suggest that MT is an excellent biomarker candidate for evaluating Cd pollution.

The yellow catfish, Pelteobagrus fulvidraco (Siluriformes) metallothionein cDNA: molecular cloning and transcript expression level in response to exposure to the heavy metals Cd, Cu, and Zn

Metallothionein (MT) has been used extensively as a potential molecular biomarker to detect heavy metal pollution in aquatic organisms. In order to investigate the modulation effect of heavy metals and to establish suitable biomarkers for the monitoring of heavy metal pollution, Pelteobagrus fulvidraco metallothionein gene was characterized as the first report in the family Bagridae. Pf-MT transcript was detected at high levels in liver, gonad, kidney, and brain compared to other tissues. A time-course study in response to waterborne Cd (5ppm) revealed that a significant increase in the Pf-MT transcript abundance was observed at 6h in gill, kidney, and liver. These elevated levels were kept for 96h, implying that Cd distributed fast into different organs and was involved in the tissue-specific induction pattern. We observed a significant Pf-MT transcript increase in liver tissues at 48h, followed by gill at 12h and intestine at 48h after Cd exposure. This indicates hepatic MT expression as a potential biomarker of acute Cd exposure in this species. Cd-binding ability of recombinant Pf-MT protein provided evidence for sensitivity to Cd and other heavy metal exposure. In the case of Zn exposure (1ppm), a significant increase in Pf-MT transcript abundance was observed at 12h, and a peak induction level reaching sixfold at 24h was kept until 48h, showing similar transcript induction patterns with Cd. A high level of Pf-MT mRNA after exposure to Cu (1ppm) was observed at 12h that gradually increased until 96h with a 12-fold induction, revealing a long-lasting induction and somewhat dissimilar pattern compared to other metals in liver. Our results demonstrate that Pf-MT can be induced by heavy metals in a tissue-specific and metal-specific manner and plays probably a conserved role in metal detoxification. This study provides new information on P. fulvidraco metallothionein gene for the use of biomarkers indicating metal pollution in fish.

Metallothionein cDNA cloning, metallothionein expression and heavy metals in Scapharca inaequivalvis along the Northern Adriatic coast of Italy

The aims of this work were: (1) identification of the metallothionein (MT) gene coding sequence in order to prepare an MT probe in Scapharca inaequivalvis and (2) quantification of Cd, Zn, Cu, MT and MTmRNA expression in tissues of molluscs from three areas along the Northern Adriatic coast of Italy. By RT-PCR we cloned the MTcDNA of S. inaequivalvis using the RNA extracted from hepatopancreas of specimens exposed to Cd. The 61 amino acids sequence of MT was deduced and was 70% identical to S. brughtonii MT. Cd concentration in molluscs from the wild was significantly higher in gills from specimens sampled near Ravenna. Zn concentration in the same tissue was significantly higher in Ravenna with respect to Porto Garibaldi while no difference with respect to Cesenatico was detected. Cu levels showed significant differences among sites in gills and mantle whereas values in the hepatopancreas were similar in all sites. The low MT levels were indicative of a low metal exposure; few differences were found in MTmRNA concentrations, which resulted significantly higher in hepatopancreas of molluscs from Porto Garibaldi.

Expression of metallothionein cDNA in a freshwater crab, Sinopotamon yangtsekiense, exposed to cadmium

This paper aims at evaluating the induction of metallothionein (MT) synthesis in gills, heart and hepatopancreas of a freshwater crab, Sinopotamon yangtsekiense. Crabs were exposed to different cadmium concentrations (from 0 to 5 mg/L) from 24 to 96 h. The amount of cadmium (Cd) accumulated in gills, heart and hepatopancreas of the crab showed an increase with an increase in the treatment concentration. Furthermore, the amount of Cd accumulated in these tissues showed an increase with an increase in duration. On the other hand, MT and β-actin genes were amplified using reverse transcription PCR (RT-PCR). Metallothionein gene expression induced by Cd was analyzed using real-time quantitative polymerase chain reaction. Its expression levels were correlated to duration time and increased with cadmium concentrations in water, whilst β-actin was expressed at a relatively constant level. The MT transcript levels in gills and hepatopancreas were higher than that in heart. Furthermore, the MT mRNA expression was not time dependent, and no relationship with Cd accumulation followed the time course of treatment at the same dose Cd treatment. But the clear dose response of MT to cadmium exposure of S. yangtsekiense is a proof of the feasibility of MT as a biomarker for testing cadmium pollution.

Response of metallothionein gene-1 to laboratory exposure to heavy metals and thermal stress in the freshwater prawn Macrobrachium rosenbergii

Metallothioneins, metal-inducible proteins, are being characterized from different organisms and shown as potential biomarkers of exposure to pollution by certain heavy metals. Here we report the identification of a new metallothionein cDNA (433 bp) from the shrimp Macrobrachium rosenbergii, putatively encoding a 61 residue polypeptide. Tissue specific analysis indicated that Mar-MT-I ( M. rosenbergii Metallothionein Gene-1) is expressed with the highest levels in the hepatopancreas and lowest in the thoracic ganglia, and none in the gills or muscles. In addition, our data showed that Mar-MT-I is differentially regulated in the hepatopancreas by certain heavy metals and thermal stress: Cd and Cu produce somewhat similar expression profile patterns, Zn has a reductional effect and thermal stress alone entirely stops its expression. These results show that Mar-MT-I mRNA levels can potentially be used as biomarkers for Cd, Cu or Zn pollution individually. However, in the case of combined metal treatment, different combinations of these metals have quite different effect on Mar-MT-I expression. Therefore, factors of such differential behaviors should be kept as a priority for further biomonitring studies.

Cloning and characterization of cDNA probes for the analysis of metallothionein gene expression in the Mediterranean bivalves: Ruditapes decussatus and Cerastoderma glaucum

cDNA probes have been developed for subsequent use in monitoring the cadmium exposure of the clam Ruditapes decussatus and the cockle Cerastoderma glaucum using metallothionein (MT) gene expression in different tissues of these species. Two partial MT cDNAs were isolated from Ruditapes decussatus and Cerastoderma glaucum. The identification of the nucleotide sequences showed that the cDNAs consist of 480 bp coding 72 amino acid proteins containing 21 cysteine residues organized in Cys-X-Cys motifs as classically described for MTs. The induction of MT gene expression in CdCl(2) treated bivalves was confirmed by dot blot analysis and suggests a potential specific tissue expression rate.

Metallothionein genes from hydrothermal crabs (Bythograeidae, Decapoda): Characterization, sequence analysis, gene expression and comparison with coastal crabs

Hydrothermal vent conditions can alter DNA and hydrothermal organisms may develop detoxification mechanisms and/or genetic adaptations. Hydrothermal vent animals notably synthesize a high quantity of metallothioneins (MT). Recent studies have revealed that the levels of MT within hydrothermal crustacean tissues are higher than those found in other vent animals. To improve our understanding of the environmental impacts exerted on the vent organisms, we characterized the metallothioneins (cDNA and Mt genes) of several members of the Bythograeidae ( Bythograea thermydron, Cyanagraea praedator and Segonzacia mesatlantica) which is the only endemic hydrothermal crab family. In comparison, the isolation of metallothionein cDNA was also carried out in several coastal crab families. The results showed that the hydrothermal crabs possess Mt composed of three exons and two introns presenting conserved splicing signals. The cDNA sequences isolated from distinct crabs showed multiple substitutions. In spite of the unique environmental conditions, the protein sequence analysis revealed no specific amino acid residue for the MT of the three hydrothermal crabs. However, gene expression analysis performed by real-time PCR based on S. mesatlantica (hydrothermal crab) compared to Pachygrapsus marmoratus (coastal crab) confirmed the higher metallothionein induction in hydrothermal crabs suggested by others authors.

Cloning of a river pufferfish (Takifugu obscurus) metallothionein cDNA and study of its induction profile in cadmium-exposed fish

We report here the full-length cDNA sequence of metallothionein (MT) gene from an anadromous river pufferfish, Takifugu obscurus (order: Tetradotiformes; family: Tetradontidae). Phylogenetic relationship analysis revealed that the identified MT has high sequence similarity with many Perciformes fish species. The tissue distribution and concentration- and time-dependent expression of MT mRNA were studied in fish exposed to cadmium. Liver showed the highest level of MT gene expression followed by other tissues (brain, gill and kidney) in response to cadmium exposure. Muscle showed a weak expression response of MT gene. Time-course study revealed highest early phase (at 6h) expression in the brain and late persistence of induction in the intestine. MT mRNA expression showed a concentration-dependent expression in all the tissues. However, induction in brain and liver occurred at much lower concentrations as compared to other tissues. Our results demonstrate that MT in T. obscurus is induced by cadmium exposure which indicates that it plays a functionally conserved function of metal detoxification.

Cloning and characterization ofTsMT3, a type 3 metallothionein gene from salt cress (Thellungiella salsuginea)

A full-length type 3 plant metallothionein cDNA was isolated from 200 mM NaCl stressed shoots of the salt cress (Thellungiella salsuginea). The 447 bp TsMT3 cDNA sequence has a 207 bp open reading frame (ORF) and encodes a deduced 69 residue peptide of molecular weight 7.52 kDa. Southern blot analysis indicates that, there is only one copy of TsMT3 in the T. salsuginea genome. The accumulation of TsMT3 mRNA is enhanced by the stress imposed by PEG6000, 200 mM NaCl, 50 microM ABA, 4 degrees C, 40 microM CuSO(4) or 25 microM CdCl2. The expression vector pET28-TsMT3 was heterologously expressed in Escherichia coli to define the contribution of TsMT3 to heavy metal tolerance. In the presence of 2 mM CuSO4, 0.3 mM Pb(NO3)2 or 0.4 mM CdCl2, TsMT3 expressing cells exhibited enhanced metal tolerance and accumulated more metal than the controls. We believe that TsMT3 is probably involved in the processes of metal homeostasis, tolerance, and reactive oxygen species (ROS) scavenging.

Physiological responses and expression of metallothionein (MT) and superoxide dismutase (SOD) mRNAs in olive flounder, Paralichthys olivaceus exposed to benzo[a]pyrene

We cloned complementary DNA (cDNA) encoding metallothionein (MT) and superoxide dismutase (SOD) from the liver of olive flounder, Paralichthys olivaceus. The full-length MT cDNA consists of 183 base pairs (bp) and encodes a protein of 60 amino acids; partial SOD cDNA consists of 326 bp and encodes a protein of 109 amino acids. We investigated the dose- and time-related effects of the polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) on MT and SOD mRNA using quantitative polymerase chain reaction (QPCR). The expression levels of MT mRNA were highest at 24 h (about five times) in 10 microg/L BaP, and at 6 h (about twelve times) in 30 microg/L BaP. The expression levels of SOD mRNA were highest at 12 h (about three times) in 10 microg/L BaP, and at 6 h (about six times) in 30 microg/L BaP, and then decreased toward the end of the experiment. We also measured plasma glucose and cortisol, all of which increased with BaP exposure. These results suggest that MT and SOD play an important role in the detoxification of reactive oxygen species (ROS) caused by BaP exposure, and thus may be indicators of oxidative stress responses.

Cloning, expression and characterization of metallothionein from the Antarctic clam Laternula elliptica

The genes for two apparent subtypes of metallothionein (MT) isoform were isolated from the Antarctic clam Laternula elliptica. Determination of the nucleotide sequence showed that the gene consists of 222 bp that code a 73-amino acid protein. The comparison between MT cDNA sequences of L. elliptica and other bivalves showed strong homologies on positions of cysteine residues, which are important for their metal binding abilities. The gene for the MT was inserted into a pET vector and overexpressed as a carboxyl terminal extension of glutathionein- S-transferase (GST) in Escherichia coli. After the GST fusion proteins had been purified by glutathione–Sepharose affinity chromatography column and digested with enterokinase, the MT was purified with gel filtration and analyzed for its biochemical properties. Recombinant MTs were reconstituted with Cd, Cu, and Zn, and kinetic studies of the reactions with electrophilic disulphide, DTNB, were investigated to explore their metal binding ability. It is revealed that the Cd–MT and Zn–MT react with DTNB biphasically, and that Zn–MT reacts with DTNB more rapidly, and with a significantly greater pseudo-first-order rate constant. Cu–MT reacts monophasically and releases metal slowly from MT.

Shark (Scyliorhinus torazame) metallothionein: cDNA cloning, genomic sequence, and expression analysis

Novel metallothionein (MT) complementary DNA and genomic sequences were isolated from a cartilaginous shark species, Scyliorhinus torazame. The full-length open reading frame (ORF) of shark MT cDNA encoded 68 amino acids with a high cysteine content (29%). The genomic ORF sequence (932 bp) of shark MT isolated by polymerase chain reaction (PCR) comprised 3 exons with 2 interventing introns. Shark MT sequence shared many conserved features with other vertebrate MTs: overall amino acid identities of shark MT ranged from 47% to 57% with fish MTs, and 41% to 62% with mammalian MTs. However, in addition to these conserved characteristics, shark MT sequence exhibited some unique characteristics. It contained 4 extra amino acids (Lys-Ala-Gly-Arg) at the end of the beta-domain, which have not been reported in any other vertebrate MTs. The last amino acid residue at the C-terminus was Ser, which also has not been reported in fish and mammalian MTs. The MT messenger RNA levels in shark liver and kidney, assessed by semiquantitative reverse transcriptase PCR and RNA blot hybridization, were significantly affected by experimental exposures to heavy metals (cadmium, copper, and zinc). Generally, the transcriptional activation of shark MT gene was dependent on the dose (0-10 mg/kg body weight for injection and 0-20 microM for immersion) and duration (1-10 days); zinc was a more potent inducer than copper and cadmium.