Interactive effects of nickel and chlorpyrifos on Mediterranean mussel cAMP-mediated cell signaling and MXR-related gene expressions Silvia Franzellitti a, ⁎, Antonio Capuzzo b , Aldo Viarengo c , Elena Fabbri a a University of Bologna, Interdepartment Centre for Research in Environmental Sciences (CIRSA), via S. Alberto 163, 48123 Ravenna, Italy b University of Ferrara, Department of Biology and Evolution, via Borsari 46, 44100 Ferrara, Italy c University of Oriental Piedmont, Department of Science of Environment and Life (DISAV), via T. Michel 11, 15121 Alessandria, Italy abstract article info Article history: Received 11 May 2011 Received in revised form 13 July 2011 Accepted 14 July 2011 Available online 23 July 2011 Keywords: Mytilus galloprovincialis Chemical mixtures Nickel Chlorpyrifos Cyclic AMP PKA P-glycoprotein Major vault protein The aim of this study was to infer putative interactive effects of a binary mixture between nickel (Ni) and chlorpyrifos (CHP) on mussel cell signaling, and also to unravel downstream effects on transcriptional regulation mediating cytoprotective responses. Mussels were exposed for 4 days to Ni (0.77 mg/L), CHP (4.5 mg/L), or the mixture Ni/CHP (0.135 mg/L Ni and 0.61 mg/L CHP). Cyclic AMP content and PKA activity in gills were evaluated as biological endpoints related to cell signaling. Expression of the MgPgp (ABCB1) and MgMvp genes was also assessed as involved in the mussel MXR mechanism. Levels of cAMP and PKA activities were not modified in mussels exposed to Ni or CHP, whereas they significantly increased in organisms exposed to the mixture. Similar responses were also detected for MgPgp expression, which is thought to be under cAMP/PKA-mediated regulation. Expression of MgMvp was unaffected by CHP or Ni/CHP exposure, and increased by Ni. The differential regulation of MgPgp and MgMvp expressions could be ascribed to the different intracellular localization and function of the two transporters. On the whole, present data indicated that Ni and CHP elicited interactive effects on mussel physiology, both at the signal transduction and at the gene expression levels. © 2011 Elsevier Inc. All rights reserved. 1. Introduction Several anthropogenic activities lead to a gradual increase of aquatic contaminants, not only in quantity, but also in diversity. Thus, gaining relevant information about the mode of action of single compounds has been proven a challenging task. However, unpredict- able interactions of chemical mixtures can affect different biochemical pathways in natural environments. From a toxicological point of view, these interactions can strongly influence the overall impact of chemical stressors on living organisms. Although an increasing number of studies invested large efforts to understand mixture toxicity (Evrard et al., 2010; Banni et al., 2011; Dondero et al., 2011; Maria and Bebianno, 2011), the complex interplay of different toxicants among several biological pathways remains difficult to unravel. Pesticides and metals are largely used in several human activities, and are widely detected in the different aquatic compartments, including marine environments (Bayen et al., 2004; Labrada-Martagón et al., 2011). Among them, this study was focused on the metal nickel and the pesticide chlorpyrifos. Nickel (Ni) is found in coastal environments as a result of industrial discharges from electroplating, smelting, mining, and refining operations, as well as other industrial emissions (Brix et al., 2004). Chlorpyrifos (CHP) is a broad-spectrum organophosphate compound employed as the active ingredient in several insecticides, being among the most widely used insect control products (Lemus and Abdelghani, 2000). Both Ni and CHP are categorized as priority substances within the European Water Framework Directive for the protection of aquatic ecosystems (Directive, 2008/105/EC). Moreover, both compounds are detected in marine environments, where they tend to partition from the water column into benthic sediments due to their hydrophobicity and affinity for the organic matter. Therefore, sediments frequently become a long-term repository for such compounds, implying that benthic organisms including sessile bivalves can be particularly subjected to their putative joint toxicity. Nevertheless, scarce information is currently available about their putative sublethal effects and mode of action in marine species. Mytilus galloprovincialis and other marine mussels are extensively used as sentinel organisms in environmental monitoring programs due to their wide distribution, sedentary lifestyle, tolerance to a wide range of environmental conditions, and because they are filter feeders with a very low metabolism, which allows the bioaccumulation of many chemicals in their tissues (Viarengo et al., 2007). Recent studies indicated that mussels are suitable organisms to evaluate contaminant effects on physiological mechanisms involved in cell signaling and in Comparative Biochemistry and Physiology, Part C 154 (2011) 377–382 ⁎ Corresponding author at: University of Bologna, CIRSA, via S. Alberto 163, 48123 Ravenna, Italy. Tel.: + 39 0544937311; fax: + 39 0544937411. E-mail address: silvia.franzellitti@unibo.it (S. Franzellitti). 1532-0456/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2011.07.006 Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part C journal homepage: www.elsevier.com/locate/cbpc