Cd/Zn exposure interactions on metallothionein response in Eisenia fetida (Annelida, Oligochaeta) S. Demuynck ⁎ ,1 , F. Grumiaux ⁎ ,1 , V. Mottier, D. Schikorski, S. Lemière, A. Leprêtre Laboratoire d'Ecologie Numérique et d'Ecotoxicologie, UPRES EA 3570, FR 1818 CNRS, Université des Sciences et Technologies de Lille, F-59655 Villeneuve d'Ascq Cedex, France Received 20 November 2006; received in revised form 27 February 2007; accepted 1 March 2007 Available online 12 March 2007 Abstract We studied metallothionein (MT) response in the manure worm Eisenia fetida after exposures to cadmium (Cd), zinc (Zn) or cadmium and zinc spiked media. MT was studied both at the protein level by Dot Immunobinding Assay, (DIA) and at the expression level by Northern blotting. Cd was highly accumulated by worms whereas Zn body concentration was regulated. In addition, Zn would limit Cd accumulation in worms exposed to low Cd concentrations (1 and 8 mg Cd kg - 1 of dry soil). Exposure to a mixture of Cd and Zn at high concentrations increased cytosolic MT levels. This increase would allow worms to regulate body Zn concentrations and also to limit Cd toxicity. Cd exposures increased gene expression of Cd-binding MT isoform (MT 2A) whereas Zn did not. However, when both metals were at high concentrations in the exposure medium, this expression was further increased. Several hypotheses are proposed to explain the results and the best approach to estimate metal exposure of this earthworm species is given. Further experiments have now to be performed to evaluate the usefulness of these MT responses for field contaminated soils toxicity assessment using this earthworm species. © 2007 Elsevier Inc. All rights reserved. Keywords: Eisenia fetida; Earthworm; Metallothionein; Cadmium–zinc interactions; Northern blotting; Dot Immunobinding Assay; Gene expression 1. Introduction Oligochaetes are susceptible to metal pollution more than many other groups of soil invertebrates (Bengtsson et al., 1992) and since they are preyed upon by numerous animals, they may represent a potential vector of secondary poisoning in polluted environments (Getz et al., 1977; Ireland, 1983). Indeed, metal accumulation turns out to be critical for survival and fitness of worm predators (e.g., birds or small mammals) (Cooke et al., 1992; Abdul Rida and Bouché, 1994). Species of choice is the compost-dwelling brandling worm, Eisenia fetida (Annelida Oligochaeta) recommended as a toxicity test species (OECD, 1984) and currently used to determine the effect of pollutants on soil biological quality. Metallothioneins (MTs) constitute a super-family of low- molecular-mass thermoresistant proteins (6–8 kDa), with high cysteine content and a high affinity with various metals (Kägi, 1991). MTs of terrestrial invertebrates, including earthworms, were proposed as biomarkers (Stürzenbaum et al., 1998; Morgan et al., 1999a) to assess the impact of metallic pollution on the environment (Dallinger et al., 2000). Indeed, MTs participate in homeostasis of essential metals, such as zinc and copper, and to detoxification of trace metals like cadmium or mercury (e.g., Dabrio et al., 2002). Thus, MTs are widely considered as biochemical environmental indicators of metal contamination (George and Olsson, 1994; Amiard and Cosson, 1997; Langston et al., 1998; Raspor et al., 1999). Moreover, the MT-mRNA inductibility by trace metals has been proposed as a biomarker (Chan, 1995, Tom et al., 1999). Indeed, Spurgeon et al. (2004) found a negative correlation between mt-2 expression and reproduction in Lumbricus rubellus, so impacts at low level of biological organisation seems to lead to detrimental consequences on population dynamics. Previous works performed on Cd-exposed E. fetida, suggested that “MT responses” (MT levels or MT gene expression levels) could be considered as sensitive potential biomarkers of Cd-exposure (Brulle et al., 2006; Demuynck Comparative Biochemistry and Physiology, Part C 145 (2007) 658 – 668 www.elsevier.com/locate/cbpc ⁎ Corresponding authors. Tel.: +33 3 20436531; fax: +33 3 20436732. E-mail address: sylvain.demuynck@univ-lille1.fr (S. Demuynck). 1 S. Demuynck and F. Grumiaux contributed equally to this work. 1532-0456/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2007.03.001