Functional Analysis of Avian Metallothionein Isoforms: An

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Environ. Sci. Technol. 2008, 42, 9391–9396

Functional Analysis of Avian Metallothionein Isoforms: An Ecotoxicological Approach for Assessing Potential Tolerability to Element Exposure DONG-HA NAM, EUN-YOUNG KIM,* AND HISATO IWATA Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan

Received May 19, 2008. Revised manuscript received September 28, 2008. Accepted October 6, 2008.

To assess the inducibility of avian metallothionein (MT) genes and potential tolerability of their protiens to element exposure, we investigated the transcriptional inducibilities of cormorant (Phalacrocorax carbo) and mallard (Anas platyrhynchos) MT genes in CV-1 cells by elements and detoxification potencies of their in vitro synthesized MT proteins. About 1.3 kb of 5′-upstream regions were sequenced for cormorant MT1 and 2 genes, where two metal-responsive elements were identified. Both cormorant MT promoters were dose dependently activated by Cd, Zn, Cu, and CH3Hg, whereas no transactivation was detected by Pb, Tl, Ag, inorganic Hg, Cr(VI), Cr(III), As(V), As(III), Ni, Co, Rb, and Bi, suggesting a shared transactivational mechanism of both MTs for specific elements. These findings support our previous results, where Cu and Zn concentrations were shown to be positively correlated with hepatic MT1/2 mRNA levels in wild cormorants. Comparison of EC50 and LOEL (lowest observed effect level) for each element revealed that Cd was the most potent inducer of MT1/2 promoters, followed by CH3Hg, Zn, and Cu. Since LOELs of CH3Hg for both MT promoters were higher than the hepatic levels in wild cormorants, hepatic CH3Hg concentration may not be high enough to induce MT mRNA in the wild population. Although LOELs of Cd were much lower than the hepatic concentrations detected in wild cormorants, no significant correlation was observed between hepatic Cd levels and MT mRNAs. This may be due to the masking effect of multiple elements, probably by Zn and/or Cu that were highly accumulated in wild cormorants. Cotreatment of Cd with Zn supported a possible suppression of Cd-induced MT expression by Zn in wild cormorants. MT1 and 2 proteins of cormorant and mallard endowed Escherichia coli with significantly higher growth rate than control to Cd exposure (500-1000 µM), implying that avian MTs could be involved in the detoxification of intracellular Cd. This study provides the first evidence on the inducibility of avian MT isoforms by specific elements and functional significance of each avian MT isoform in detoxifying intracellular heavy metals. Our in vitro approaches demonstrate their validity in predicting the response of MTs to element exposure in a wild avian population. * Corresponding author phone/fax: +81-89-927-8172; e-mail: [email protected]. 10.1021/es801332p CCC: $40.75

Published on Web 11/07/2008

 2008 American Chemical Society

Introduction Induction of metallothionein (MT), a cystein-rich metalbinding protein, is most likely a compensatory response for detoxification of metals through their binding to the induced MT proteins (1-3). In terms of sensitivity to metal stress, understanding the regulatory mechanism and the molecular characterization of MTs are necessary to assess the risk of metal exposure to the target species. The metal-induced MT expression is primarily regulated at the transcriptional level through the metal-regulatory transcriptional factor (MTF1). The core heptanucleotide metal-regulatory element (MRE; TGCRCNC), which is a target DNA sequence of MTF-1, has been identified in the 5′-flanking regions of MT genes (2, 4). The cis-acting MREs are highly conserved in all mammalian MT genes, although the number of copies and distribution of MREs are different among species (5). In avian species, MREs have been identified in the promoter region of chicken MT2, indicating that MTF-1 is essential for the transcriptional activation of chicken MT2 by metals (6, 7). Except for the sequences of chicken MT2 gene promoter, however, nothing is known about the structure and function of avian MT promoters. Unlike mammals, in which multiple forms of MTs (MT1 to MT4) were characterized, a single avian MT gene and its exclusive expression have been alleged to be present in avian species (6). As for other avian MT forms, Villarreal et al. (8) found a second MT gene (MT1) in silico in the chicken genome. However, they neither isolated gene/cDNA nor investigated the expression and function of the second avian MT. In addition, no information on the second avian MT protein synthesis and its functional role(s) is available, although the authors investigated metal-binding ability of the first chicken MT protein (8). Recently, we identified two distinct MT isoforms in three avian species, MT1 and MT2 cDNAs that were isolated from chicken, common cormorant, and mallard (9). Messenger RNAs of MT1 and MT2 isoforms were ubiquitously expressed in various tissues/organs of cormorant and mallard. Thus, our findings clearly indicate that the expression of two MT isoforms is a general feature in avian species. Our previous study (9) showed that the expression levels of both hepatic MT isoforms MT1 and MT2 in a population of wild cormorants were positively correlated with the residue levels of Cu and Zn, suggesting both isoforms are induced by these metals and there is a sharing transcriptional mechanism between MT1 and MT2 isoforms. In contrast, in the liver of mallard, only MT2 expression levels showed a positive correlation with Cu concentrations, and no correlation was found between MT1 expression and metal concentrations, indicating different transcriptional regulations of MT isoforms in response to different elements. In mouse, Zn and Cd simultaneously elevate the transcription rates of both major MT isoforms MT1 and MT2 (10, 11), whereas human MT homologues are regulated by metal ions in different ways (5, 12). These earlier reports indicate that there are interspecies and isoform specific differences in the transcriptional regulation by promoter/enhancer regions of MTs (13, 14). Thus, more information on the inducibility and function of avian MT isoforms in relation to metal exposure may provide insight into the detoxification potential of metals in avian species. Toward a comprehensive understanding of the inducibility of avian MT genes and potential tolerability of their proteins to element exposure, we investigated transactivation potenVOL. 42, NO. 24, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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cies of cormorant MT1 and MT2 promoter/enhancer regions and detoxification potential of their proteins by element exposure.

Materials and Methods Sequencing of the 5′-Flanking Regions of ccMT1 and ccMT2. Promoter/enhancer regions of common cormorant MT1 (ccMT1) and MT2 (ccMT2) were cloned and sequenced using genomic DNAs in cormorant liver sample. Detailed procedures are shown in the Supporting Information. Construction of ccMT1 and ccMT2 Promoter/Enhancer Reporter Plasmids. The 5′-flanking regions of ccMT1 (1030 bp) and ccMT2 (1271 bp) were subcloned into pGL4.10[luc2] Basic Vector. Detailed procedures are shown in the Supporting Information. Cell Culture, Transient Transfection, Ligand Treatment, and Reporter Gene Assay. Detailed procedures are shown in the Supporting Information. Construction of Expression Vectors of MTs from Cormorant and Mallard. The entry clone of each avian MT isoform was recombined with a destination vector pETDEST42 to construct high-level inducible expression vectors. Detailed procedures are shown in the Supporting Information. Heterologous Expression and Immunochemical Detection of Avian MTs. A constructed expression plasmid, pETDEST42, was used to transform BL21 (DE3) Escherichia coli strains. Detailed procedures are shown in the Supporting Information. Growth Rates of E.coli Harboring Each Avian MT. Detailed procedures of the experiment are shown in the Supporting Information. Statistical Analysis. Each experiment was performed in triplicate, and the results were presented as mean ( SD. ANOVA followed by Sheffe’s posthoc test was performed with SPSS package, version 12.0 (SPSS Japan Inc., Tokyo, Japan). P < 0.05 was regarded as statistically significant. EC50 (50% of effective concentration) and LOEL (lowest observed effect level) for the transactivation of MT promoter/enhancer regions by individual elements were calculated using SigmaPlot, version 9.0 (Systat Inc., Richmond, CA).

Results and Discussion Nucleotide Sequences of ccMT1 Promoter/Enhancer Regions. We have succeeded in isolating about 1230 bp and 1278 bp of the 5′-flanking regions of ccMT1 and ccMT2 genes with respect to putative transcription initiation site, respectively (Figures S1 and S2). The 5′-flanking region (572 bp) of the chicken MT1 gene was obtained from the chicken genome database (Figure S3). Sequential analysis of these regions revealed that ccMT promoters lacked a canonical TATAAA motif, but the motif was replaced by GATAAA in ccMT1 and CATAAA in ccMT2. The chicken MT2 TATA box consists of the canonical element TATAAA motif (6), whereas chicken MT1 promoter identified from chicken genomic database has a variant GATAAA sequence. Two potential MREs and a single GC box are present within the promoter/enhancer regions of ccMT1. ccMT2 gene is preceded by two MREs, a single antioxidant responsive element (ARE), four GC boxes, and two E boxes. The contiguous DNA that flanks the 5′ end of the chicken MT1 gene contains three MREs, a single ARE, and five GC motifs. Comparison of the 5′-flanking regions of avian MTs revealed that the identities of DNA sequences between MT1 and MT2 were 29% for cormorant and 32% for chicken. MT orthologs between cormorant and chicken showed relatively high sequence identities (34% for MT1 and 35% for MT2). Transactivation Potencies of ccMT1 Promoter/Enhancer Regions by Exposure of a Single Element. We constructed reporter plasmids containing promoters of ccMT1 (975 bp of 9392

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the 5′-flanking sequences including a putative cap site (+1) and 55 bp of untranslated sequences) and ccMT2 (1247 bp of 5′-flanking sequences including a putative cap site (+1) and 24 bp of untranslated sequences). Using these reporter plasmids, we investigated ligand-dependent transactivation potencies of ccMT1 and ccMT2 promoter/enhancer regions by an in vitro reporter gene assay. Both ccMT1 and ccMT2 promoter/enhancer regions were dose dependently activated by treatment with Cd, methyl Hg, Cu, and Zn (Figure 1). The comparison of LOELs for individual elements revealed that Cd was the most potent activator for ccMT1 (0.10 µM) and ccMT2 (0.10 µM) promoters, followed by methyl Hg (10 µM for ccMT1 and 5.0 µM for ccMT2), Zn (50 µM for both ccMTs), and Cu (200 µM for ccMT1 and 50 µM for ccMT2) (Figure 2). The in vitro transactivation potency for ccMT2 by each element was greater than that for ccMT1. On the other hand, no transcriptional activation by all the other 12 elements (Pb, Tl, Ag, inorganic Hg, Cr(VI), Cr(III), As(V), As(III), Ni, Co, Rb, and Bi) tested in this study was observed (Figure 1). It is known that transactivation potencies of the chicken MT2 promoter by metals were similar between the homologous (primary chicken embryo fibroblasts) and heterologous (monkey kidney) cells (6). These results indicate that the basic metal regulatory mechanism is well-conserved between vertebrates, and heterologous MT expression has little effect on the transactivation potency. Similar responses of ccMT1 and ccMT2 promoters by Cd, Zn, Cu, and methyl Hg in the present study are consistent with our previous findings in which the two MT mRNAs showed significant positive correlations with Zn and Cu levels in the liver of wild cormorants (9). Whereas the structures of MT gene enhancer/promoter regions are species- and isoform-specific, MT gene generally contains multiple copies of MREs that confer metal-induced transcriptional activity (5, 15). Assuming that expression of cormorant MT isoforms is regulated by metal-binding regulatory proteins including MTF-1, similar responses of the two MT promoters to Cd, Zn, Cu, and methyl Hg may be due to a sharing mechanism of transcription (e.g., same regulatory protein(s) with MREs and/or other responsive elements in the promoter/enhancer regions) (10, 11), even if there are variations in the distribution of MRE and other elements in the two promoter/enhancer regions (Figures S1 and S2). Although LOELs for the transactivation of ccMT1 and ccMT2 induced by Cd, Zn, Cu, and methyl Hg exposure were similar, transactivation potency of ccMT2 promoter was about 1.5 to 3.0-fold more prominent than that of ccMT1 promoter. Expression profiles of ccMT mRNAs in various tissues (liver, kidney, muscle, heart, lung, spleen, pancreas, and gonad) of wild cormorants also showed that the mRNA expression ratios of ccMT2 to ccMT1 ranged from 2- to 14-fold (9), reflecting the higher transcriptional potential of ccMT2 promoter by these elements. Since hepatic Ag and Rb levels exhibited significant correlations with ccMT2 mRNA in the liver of wild cormorants (9), we investigated whether both elements can induce transcriptional activation of cormorant MT promoters in our in vitro assay system. The results exhibited no transactivation potency of cormorant MT1 and MT2 promoters within the ranges of Ag and Rb levels detected in the wild cormorant population (Figure 1). Thus, the significant correlations of Ag and Rb with MT2 mRNA expression may be explained as due to the involvement of MT2 proteins induced by other elements (e.g., Zn and/or Cu), and also the hepatic MT2 proteins may play a role in the retention of Ag and Rb. The well-known mechanism of Tl toxicity is related to the interference of potassium-dependent processes in the (Na+/ K+)-ATPase activity, as well as the high affinity to sulfhydryl groups from proteins leading to the depletion of GSHpx (glutathione peroxidase) activity (16). The MT induction associated with Tl exposure has not yet been investigated.

FIGURE 1. Transcriptional activities of cormorant MT1 and MT2 promoter/enhancer regions by various elements. The luciferase activity in CV-1 cells transfected with the pGL4.10[luc2]-ccMT1 promoter (gray-shaded box) and pGL4.10[luc2]-ccMT2 promoter (9) are shown as relative (as a fold induction) to the activity in control cells transfected with pGL4.10[luc2] basic vector (0). Asterisks show statistical differences from control cells (*, p < 0.05; **, p < 0.01; ***, p < 0.001). The results observed in this study indicate that Tl is not a potent inducer of the transactivation of ccMT1 and ccMT2 promoters. In the present study, no apparent transactivation of ccMT1 and ccMT2 promoters by inorganic Hg was observed, whereas both ccMT promoters were significantly activated by methyl Hg exposure (Figure 1). It is known that the transactivation potencies of arsenic, chromium, and mercuric compounds are dependent on their chemical forms in mammalian MTs (17-19). Our results also suggest that ccMT inducibility may vary with the valence state of the elements. Interspecies Comparison of MT Promoter/Enhancer Regions. Previously, we compared the coding sequences of avian MTs with mammalian MT genes to provide insight into the evolutionary history of these genes (9). Since avian MTs were phylogenetically segregated based on their isoforms but not on species, we presume that avian MT gene duplication may precede species differentiation. When the 5′-flanking regions of avian MTs and mammalian MTs for which DNA sequences are available (500-1200 bp) were compared, avian MTs showed about 30% homology to one another, and 14-19% sequence identities with different mammalian MTs (22-26% with MT1s, 25-29% with MT2s, 22-29% with MT3s, and 14-25% with MT4s). We also compared the 5′-flanking regions of the avian MT genes to those of mammalian MT genes (especially mouse MT1 and human MT2A genes), which have been characterized by deletion mapping and electrophoretic mobility shift assay (5, 15, 20) to reveal conserved sequences which might play a role in regulating MT gene transactivation. Avian MT1s and human MT2A genes contained a 15 bp sequence MRE at approximate positions -20 and -60 relative to TATAAA motif (Figure S4). Avian MT2s and mouse MT1 genes had a conserved sequence of 15 bp MRE at -20 to -40 (Figure S4). The proximal sequences (over 300 bp) including MRE

distribution of 5′- promoter/enhancer regions between avian and mammalian MTs are likely to be conserved, implying a similar inducibility among vertebrate MTs. Comparison of proximal DNA sequences of avian MT genes (over 300 bp from TATAAA motif), where most MREs are located in vertebrates MT genes, showed slightly high identities to mouse MT1 (31-36%) and human MT2A (35-41%) when compared to those fo mammalian MT3/4 (23-30%). Although there is no conclusive evidence which mammalian MT isoform is orthologous to avian MTs, it seems plausible that avian MTs are closer to mammalian MT1 and 2 than to MT3 and 4 with respect to proximal DNA sequences including MREs in the promoter/enhancer regions. Elements vary in their effectiveness as inducers of different MT isoforms in mammals. Human MT1A and MT2A are induced by heavy metals in different manners (5, 12), whereas rodent MT homologues are similarly regulated by Zn, Cd, and/or Cu (10, 11, 21). When the expression vector for MTF-1 was cotransfected into cells along with a reporter vector containing mouse MT1 promoter, metals including Cd, Zn, Cu, Ag, Bi, Hg, Ni, and Co induced reporter gene activities via MTF-1 that recognizes MRE in the promoter regions (13). Induction of MTs in the rat liver by various metals including Pb has also been reported (22-24). The transactivation potencies of ccMT1 and ccMT2 promoters by Zn, Cd, and Cu appeared to be similar to those of rodent MT1s and MT2s, but the responses by other elements including inorganic Hg, Ag, Bi, Ni, Co, and Pb were different between cormorant and rodent MTs. Such dissimilar responses may be associated with their different structures of promoter/enhancer regions. Hepatic Concentration vs LOEL for in Vitro ccMT Transactivation. To examine whether the relationships between MT mRNA expression levels and element accumulation observed in a wild cormorant population (9) VOL. 42, NO. 24, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Transcriptional activities of cormorant MT1 and MT2 promoter/enhancer regions by elements. (A) CV-1 cells were transfected with the reporter plasmids (pGL4.10[luc2]-ccMT1 promoter (gray-shaded box), pGL4.10[luc2]-ccMT2 promoter (9), and pGL4.10[luc2] basic vector (0)) that were left untreated or treated with Zn2+, Cd2+, or Zn2+ and Cd2+ for 5 h. (B) CV-1 cells transfected with reporter plasmids were either left untreated or treated with Cr6+ and/or Cd2+ before harvest for 5 h. Luciferase activity was determined as relative (as a fold induction) to the activity in control cells transfected with the pGL4.10[luc2] basic vector (0). Asterisks show statistical differences from control cells (*, p < 0.05). could be reproduced by our in vitro reporter gene assay, we compared element concentrations in the liver of wild cormorants with their LOELs for transactivation of ccMT1 and ccMT2 promoters (Figure S5). The comparison revealed that Cd was the most potent inducer of ccMT1 and ccMT2 promoters, followed by methyl Hg, Zn, and Cu. As the LOELs of ccMT promoters by Zn and Cu were lower than the levels in liver of wild cormorant populations, the significant positive correlations of MT1 and MT2 mRNA levels with Zn and Cu concentrations found in the wild population appeared to be rational. Since LOELs of methyl Hg for the transactivation of both MT promoters were higher than the levels in the cormorant livers, methyl Hg concentration in the wild population may be considered as not high enough to induce MT mRNA expression. Whereas LOEL for Cd was much lower than Cd levels in the wild cormorants, no significant correlation of MT mRNAs with Cd levels was observed in the wild population (9). Because ccMT1 and ccMT2 promoters are transactivated principally by Zn and/or Cu, this could be due to the masking effect of multiple element exposures, especially by Zn in the wild population. Transactivation Potencies of ccMT Promoter/Enhancer Regions by Coexposure of Two Elements. To investigate whether Zn2+ could affect Cd-induced transactivation of MT genes, cells were cotreated with Cd2+ (2 µM) and Zn2+ (100-300 µM), and the results were compared with those of a single injection of Zn2+ (200 µM) or Cd2+ (2 µM) (Figure 2A). After a single injection of 2 µM Cd2+ (that corresponds to an 9394

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average level in the liver of wild cormorant population) or 200 µM Zn2+ (that is a less than average level (700 µM) that can induce cellular toxicity in CV-1 cells) into the cells for 5 h, transactivation of ccMT1 and ccMT2 promoters significantly increased when compared to that in nontreated cells. By treatment with a mixture of Cd (2 µM) and Zn (100, 200, or 300 µM), no alteration of transactivation was detected in comparison with that of Zn (200 µM) alone. Cotreatment with Cd (2 µM) and 200 or 300 µM Zn inhibited the transactivational activity of MT1 and MT2 by 40%, when compared to the result of Cd (2 µM) alone. These results are consistent with previous findings that the expression of MT was not additive by cotreatment with Cd (5.5 µM) and Zn (308 µM), whereas each metal was found to induce MT expression in rat liver (25). However, the mechanism of suppression of transactivation by the mixture of Cd and Zn remains unclear. Hexavalent chromium (100 µM) preferentially suppresses the induction of mouse MT1 and human MT2A in HepG2 cells by Cd (30 µM) or Zn (100 µM), although Cr(VI) alone does not activate an expression of those MTs (26). Since it is apparent that the inhibitory effect by Cr(VI) occurs at the transcriptional level, following the DNA binding of MTF-1, Cr(VI) might have inhibited the interaction of the MTF-1 transactivation domains with other cofactors (26). To examine whether Cr(VI) affects Cd-induced transactivation of ccMT1 and ccMT2 promoters, we treated the ccMT promotertransfected cells with Cd2+ (2 µM) and Cr6+ (0.2, 2, or 20 µM) (Figure 2B). Hexavalent chromium alone was found to be unable to induce MT gene transactivation at concentrations ranging from 0.01 to 10 µM (levels detected in the liver of wild cormorants). No suppressive effect of Cr6+ (0.2, 2, or 20 µM) on transactivation by Cd2+ was observed. Therefore, it is likely that Zn and/or other elements, but not Cr6+, could conceal Cd-induced MT expression in the wild cormorant population. Effects of Heavy Metals on Growth Rate of E. coli Harboring Avian MT isoform. For physiological and functional analysis of MTs, E. coli has been used as a host for the heterologous expression of mammalian MTs (27-29). Although the first chicken MT (MT2) was also heterologously synthesized in E.coli for characterizing its metal-binding ability (8), no information on the second avian MT (MT1) protein synthesis and its functional role(s) is available. In this regard, cormorant and mallard MT1 and MT2 isoforms were individually expressed in E. coli. By Western blot analysis, a single cross-reactive band with a molecular weight of 10.3-10.5 kDa for each avian MT isoform was detected in the E. coli supernatant fractions, where the cells were inoculated for 0.5 h (Figure S6A) and for 5 h (Figure S6B) after adding IPTG. These results suggest that avian MTs were efficiently inducible under the current experimental condition. Each avian MT protein in E. coli was quantified after 5 h of inoculation, indicating a similar production efficiency of recombinant proteins for the two MT isoforms both in cormorant and mallard. Figure 3 shows a growth rate of E. coli after administration of Cd, Ag, Zn, and Cu. Up to 100 µM Cd concentrations had no apparent effect on the overall growth rate of the E. coli harboring control and individual avian MT expression vector. However, at 500 and 1000 µM Cd, E. coli transfected with each MT showed a significantly higher growth rate than the control vector, but no isoform- and species-dependent growth rate was observed among transformed MTs. It is generally accepted that the basic function of MT is to protect cells from the toxic effects of heavy metals. The expression of mouse MT1 endowed resistance in E. coli up to 830 µM Cd (27). Other investigations showed that in E. coli (JM105) expressing human MT2 after Cd exposure (893 µM) about 30% of Cd in the cells was present in the form of Cd-binding

FIGURE 3. Effects of heavy metals (Cd, Ag, Zn, and Cu) on growth of E. coli harboring with (ccMT1/MT2, Mallard MT1/2) or without (control) MT genes. Asterisks show statistical difference from control cells (*, p < 0.05; **, p < 0.01). MT, indicating a greater tolerance of E. coli transformed with human MT2 rather than those with control vector (28). Expression of MT1 and MT2 from cormorant and mallard also endowed E. coli with resistance to high levels of Cd (500 and 1000 µM), suggesting that avian MTs could be involved in detoxification of intracellular Cd. In contrast, no significant difference in growth rates between the cells transformed with control and each avian MT expression vector was observed by treatment with Ag (0-500 µM), Zn (0-2000 µM), and Cu (0-2000 µM) (Figure 3). In previous studies, E. coli expressing human MT2 that were incubated in the medium containing Zn (0-4600 µM) and Cu (0-4600 µM) showed no resistance (28), whereas the expression of mouse MT1 in E. coli showed a detoxification potential to those heavy metals (27). The difference in the ability of resistance between mouse MT1 and human MT2 in E. coli is unclear. It has been reported that E.coli showed resistance to Zn, Cu, or Ag by their intrinsic heavy metal regulatory machinery (30-32). In this study, E. coli carrying control vector showed similar resistance to Zn, Cu, or Ag, compared with cells expressing each avian MT isoform. It is possible that an innate control system in E.coli participated primarily in the regulation of Zn, Cu, or Ag; thus, the detoxification ability of the avian MT heterologously synthesized in E. coli might be overwhelmed by their indigenous control system. The physiological role and metal-binding mechanism of each avian MT isoform deserve further investigation. The present study provides the first evidence on the inducibility of avian MT isoforms by specific elements and functional significance of each avian MT isoform to detoxify intracellular heavy metals. Our in vitro approaches clearly demonstrate the validity in predicting the response of MTs to elemental exposure in wild avian population. Therefore, data from these in vitro experiments may be useful for

understanding the species-specific sensitivity to elemental exposure, and further for assessing the potential risk of elements in a variety of avian species. Further studies are necessary to explore physiological consequences associated with MT induction by elemental exposure.

Acknowledgments This study was supported by Grants-in-Aid for “21st Century COE Program” and “Global COE Program” from the Ministry of Education, Culture, Sport, Science and Technology, Japan. The award of the JSPS (Japan Society for the Promotion of Science) Postdoctoral Fellowship for Foreign Researchers in Japan to Dong-Ha Nam (ID No. 06058) is acknowledged.

Supporting Information Available Experimental procedures and figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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