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(41-03) Cd accumulation and in situ localisation of a Cd-inducible metallothionein isoform in organs of the terrestrial gastropod Helix pomatia. Chabicovsky, Monika*,1, Dallinger, Reinhard1, 1 Institut of Zoology and Limnology, University of Innsbruck, Innsbruck, Austria ABSTRACT- Metallothioneins (MTs) are cystein rich proteins, representing a superfamily of widely differentiated proteins, binding essential and non-essential heavy metals. In terrestrial pulmonates, cadmium can induce synthesis of specific Cd-MT isoforms, which therefore have been linked to Cd detoxification in a wide range of phylogenetic orders. To better understand the biological function, as well as kinetic and dynamic features of the Cd specific isoform, Cd-MT RNA was localised in relevant organs of H. pomatia. To define metal burden, Cd content of the tissues was measured by atomic absorption spectrophotometry. In control animals Cd was predominantly accumulated in 3 organs: hindgut, foregut and midgut gland, while only traces of Cd could be measured in mantle, foot and kidney. Cd treatment for 3 weeks induced an at least 2fold Cd accumulation in all organs, however, the elevation of the Cd concentration was more than 14fold in midgut gland and foregut. In order to detect even very low levels of the Cd specific MT isoform RT-PCR was applied as a very sensitive method, using specific primers for the Cd-MT cDNA. Results suggest a nearly ubiquitous distribution of the Cd-MT mRNA at least at very low levels, in both control as well as metal treated animals. For in situ localisation of the mRNA a specific digoxigenin-labeled probe was used in in situ hybridisation method (ISH). The most intensive signal for hybridized Cd-MT mRNA was observed in the midgut gland of Cd-treated animals, consistently with its suggested role in Cd detoxification. If staining reaction time was extended kidney, foot, fore- and hindgut of Cd-treated individuals also showed organ and cell specific hybridisation. While in midgut gland, kidney and foot all types of cells contained the RNA, the gene expression of Cd-MT was cell-specific in the gut. In untreated animals Cd-MT mRNA was only detected in the surrounding tissue of the kindey. The mantle was the only organ in which Cd-MT mRNA could not be detected by ISH (expecting a higher detection limit compared with PCR). Concluding, ISH with Cd-MT-specific probes demonstrates the presence of this isoform in the major organs involved in absorption (fore- and hindgut), primary detoxification (midgut gland, analogous to vertebrate liver) and accumulation / excretion (kidney, analogous to vertebrate kidney) of cadmium. Key words: metallothionein, isoform, gastropod, distribution |
