A molecular approach to the study of metallothionein induction in oysters

Abstract

The objective of this study was to provide basic information about molluscan metallothionein (MT) induction at the molecular level in order to increase our understanding of the response of molluscs to metal exposure and metal tolerance. This required the development of tools and methods to analyze MT mRNA in oysters and examination of basic exposure parameters. This work will provide significant basic structural information for use in comparative studies and evaluate a possible mechanism of short-term tolerance in an aquatic invertebrate. The cDNA for oyster MT was isolated by cloning the product of a reverse transcription-polymerase chain reaction amplification. The cDNA was characteristic of MT sequences and confirmed the previous classification of Crassostrea virginica MT as a class I MT. The cDNA was used to develop methods to quantify MT mRNA in oyster gill tissue. Ribonuclease protection-based assays were necessary to quantify MT mRNA in oysters not exposed to metals. These methods were applied to examine the time- and exposure-concentration dependence of oyster gill MT mRNA during Cd exposure and the effect of sublethal Cd challenge on gill MT mRNA accumulation in oysters preexposed to a range of low Cd concentrations. Initial treatments of control (0.0003 {dollar}\mu{dollar}M) and 0.0044, 0.044, and 0.44 {dollar}\mu{dollar}M Cd exposure for 21 d were followed by challenge with 0.44 {dollar}\mu{dollar}M Cd. MT mRNA accumulation during preexposure exhibited concentration- and time-dependence, increasing at all preexposure concentrations. During the challenge, MT mRNA increased in all samples and the concentration-dependence of MT mRNA in relation to preexposure concentrations was maintained. MT mRNA concentrations during the challenge were significantly increased in oysters preexposed to the two highest Cd concentrations in comparison with the controls. Since MT mRNA levels are correlated with the rate of MT translation in previous studies, these data implicate new MT synthesis in acquired tolerance to Cd toxicity. The results of this study complement ongoing research at the protein level and will be part of a foundation upon which the effects of metal pollution on natural populations of organisms can be evaluated.