Carboxylesterase activity in earthworm gut contents: Potential (eco)toxicological implications Juan C. Sanchez-Hernandez a, ⁎, Christophe Mazzia b , Yvan Capowiez c , Magali Rault b a Laboratory of Ecotoxicology, Faculty of Environmental Science, University of Castilla-La Mancha, Avda. Carlos III s/n, 45071, Toledo, Spain b Universite d'Avignon et des Pays de Vaucluse, UMR 406 Abeilles et Environnement, Site AGROPARC, F-84914, Avignon Cedex 09, France c INRA, Unite PSH, Site AGROPARC, F-84914 Avignon Cedex 09, France abstract article info Article history: Received 12 June 2009 Received in revised form 21 July 2009 Accepted 22 July 2009 Available online 3 August 2009 Keywords: Gut secretion Lumbricus terrestris Luminal carboxylesterases Organophosphorus pesticides Pesticide-detoxifying esterases Carboxylesterases (CbEs) are key enzymes in pesticide detoxification. These esterases are involved in the biochemical mechanism for pesticide resistance in some pest species, and further they are considered an efficient protective mechanism against acute toxicity by organophosphate (OP) pesticides in mammals. To gain knowledge on the role of CbEs in pesticide toxicity and natural tolerance in earthworms, we performed an enzyme kinetic analysis to investigate whether these annelids are able to secrete them into their gut lumen. We determined levels of CbE activity and isozyme abundance in the gut wall and ingested soil collected from different portions of the gastrointestinal tract of Lumbricus terrestris. Moreover, modulation of enzyme activity by selected substrates (α-naphthyl acetate [α-NA], 4-nitrophenyl valerate [4-NPV] and 4-nitrophenyl acetate [4-NPA]) and OP pesticides was examined to compare the response between tissue and soil CbEs. We found a high CbE activity in the ingested soil extracts from the crop/gizzard (α-NA-CbE = 8.43± 2.76 U mg -1 protein and 4-NPA- CbE = 5.98 ± 2.11 U mg -1 protein) compared to the gut wall. Three lines of evidences suggest that the gut epithelium is the main source of this luminal CbE activity. First, the effect of substrate concentrations on CbE activity from both the ingested soil extracts and gut tissues resulted in similar apparent K m and V max values. Second, native PAGE gels revealed that some of the CbE isozymes in the gut tissue were also present in the soil extracts. Third, tissue and soil CbEs showed the same sensitivity to inhibition by OPs. The concentrations of insecticide causing 50% of esterase inhibition (IC 50 ) was comparable between tissue (IC 50 s range = 4.01–9.67 nM dichlorvos and 8480–6880 nM paraoxon) and soil (IC 50 s range=6.01–11.5 nM dichlorvos and 8400–7260 nM paraoxon). Our results suggest a set of (eco)toxicological implications and environmental applications derived from the ability of earthworms to secrete these pesticide-detoxifying enzymes. © 2009 Elsevier Inc. All rights reserved. 1. Introduction Earthworms occupy a notable place in terrestrial ecotoxicology. They are common standard organisms in soil toxicity testing to assess either harmful effects of new registered chemicals or toxicity of historically contaminated soils (van Straalen and van Gestel, 1998). Moreover, these organisms are demonstrated to be excellent bioindicators in the field monitoring of soil pollution by metals (Spurgeon and Hopkin, 1999), although some earthworm species are able to tolerate highly metal- contaminated soils (Spurgeon and Hopkin, 2000). The induction of metal-sequestrating proteins is among the more plausible causes for earthworm survival in heavily metal-polluted soils (Vijver et al., 2004). Likewise, pesticide-detoxifying enzymes such as carboxylesterases (CbE, EC 3.1.1.1) can make the pesticide ineffective against individuals that naturally have high levels of these enzymes (Stenersen, 2004). These esterases detoxify organophosphate (OP) and carbamate (CB) pesticides and synthetic pyrethroids (SPs) by two main ways; hydrolysis of the ester bond (CBs, SPs and those OPs containing this type of bond such as malathion) and binding of the pesticide (OPs) to the active site of CbE (Maxwell 1992; Chambers et al., 1994; Wheelock et al., 2005; Crow et al., 2007). Inhibition of CbE activity by OPs involves nucleophilic attack of the catalytic serine residue in the enzyme active site on the electronic deficient oxidized phosphorous to form a stable enzyme–inhibitor complex (Satoh and Hosokawa, 1998). This inhibition is considered a stoichiometric mechanism for decreasing the OP concentration at the target site, i.e., acetylcholinesterase (Maxwell, 1992; Chanda et al., 1997). Carboxylesterases have been described in earthworms (Haites et al., 1972; Øien and Stenersen, 1984). The intestinal tract of Lumbricus terrestris presents a wide variety of enzyme; among them, CbE activity is predominant in the peripherical chloragog tissue (Prento, 1987). Recently, Sanchez-Hernandez and Wheelock (2009) reported up to twelve CbE isozymes in the gastrointestinal tract, body wall muscle and reproductive tissues of L. terrestris, being particularly abundant in the crop, gizzard and foregut. The abundance of CbEs in the gastrointestinal Comparative Biochemistry and Physiology, Part C 150 (2009) 503–511 ⁎ Corresponding author. Tel.: +34 925 268800; fax: +34 925 268840. E-mail address: juancarlos.sanchez@uclm.es (J.C. Sanchez-Hernandez). 1532-0456/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2009.07.009 Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part C journal homepage: www.elsevier.com/locate/cbpc