Differential metallothionein expression in earthworm ( Lumbricus rubellus) tissues

Abstract

This article describes the immunoperoxidase localization of metallothionein (MT) in the major organs and tissues of the earthworm Lumbricus rubellus sampled from a mine soil heavily polluted with Pb, Zn, and Cd. The polyclonal antiserum used was raised against the MT isoform (wMT2), the molecular characteristics and focal subcellular distribution of which indicate a primary role for it in the sequestration of certain nonessential metals, such as Cd. Intense MT immunostaining was detectable in chloragogenous tissue throughout the body: around the intestine, in the typhlosolar infolding, around blood vessels anterior and posterior to the crop/gizzard, and around the calciferous gland. Electron probe X-ray microanalysis of neutral red-labeled vesicular structures in the chloragogenous tissue indicated that this acidic compartment, probably lysosomal, yielded the strong Cd and S signals associated with Cd-MT. MT expression was also detected in the apical cytoplasm of intestinal epithelial cells; in coelomocytes contiguous with chloragocytes attached to the gut; within the narrow tubular region of nephridia, in the secretory epithelia of the calciferous gland, but not anywhere in the body wall. We concluded that (a) the main route of Cd uptake is probably via absorptive alimentary surfaces, and not across the external epidermal layer; (b) nephridia are involved with Cd excretion and/or are a major target of Cd-induced pathological damage; (c) tentatively, a combination of immunohistochemistry and proton-induced X-ray emission analysis indicated that the calciferous gland is probably not a major “heavy metal” excretory route.