Functional Analysis of Avian Metallothionein Isoforms: An Ecotoxicological Approach for Assessing Potential Tolerability to Element Exposure

Abstract

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, TI, 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 microM), 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.