Localization and quantification of Cd- and Cu-specific metallothionein isoform mRNA in cells and organs of the terrestrial gastropod Helix pomatia

Abstract

A quantitative assay based on real-time detection polymerase chain reaction (rtdPCR) was applied to analyze basal and metal-induced mRNA levels of two metallothionein (MT) isoforms (Cd–MT and Cu–MT) in organs of the terrestrial gastropod Helix pomatia. The results show that specific Cd–MT mRNA levels increase with Cd tissue burden, identifying hepatopancreas and gut as the main organs of Cd accumulation and, accordingly, the predominant organs of Cd-MT mRNA expression. In situ hybridization localized this isoform in epithelial cells of hepatopancreas, gut, and kidney. In contrast to the observed Cd-dependent inducibility of the Cd-binding MT isoform, gene expression of the Cu-binding MT could not be induced by either Cd or Cu exposure. Only very low mRNA amounts of the Cu–MT isoform were found in snail hepatopancreas and kidney, whereas the mantle exhibited high basal mRNA levels of this isoform. In situ localization revealed that the Cu-MT gene expression was restricted to one cell type, the so-called rhogocytes, which are present to various extents in the different organs examined. These results suggest a metal-specific sharing of functions between the two MT isoforms. The Cd–MT isoform apparently plays a crucial role in Cd detoxification, as demonstrated by the inducibility of this isoform, as well as its specific localization in the main metabolic and Cd storing organs. The predominant presence of Cu-MT in rhogocytes of snail mantle strengthens the hypothesis that this isoform may regulate Cu availability in hemocyanin synthesis.