ABCB1 and ABCC4 efflux transporters are involved in methyl parathion detoxification in ZFL cells Bruna Félix Nornberg, Carolina Reyes Batista, Daniela Volcan Almeida, Gilma Santos Trindade, Luis Fernando Marins ⇑ Instituto de Ciências Biológicas, Universidade Federal do Rio Grande – FURG, Brazil article info Article history: Received 2 July 2014 Accepted 18 September 2014 Available online 5 October 2014 Keywords: ABC transporters Methyl parathion Multixenobiotic resistance (MXR) Verapamil abstract The multi-xenobiotics resistance (MXR) mechanisms are the first line of defense against toxic substances in aquatic organisms and present great importance in the adaptation related to contaminated environ- ments. Methyl parathion (MP) is a widely used organophosphate pesticide, which has been associated to various toxic effects in organisms. In the present work, we studied the main genes related to efflux transporters in zebrafish liver (ZFL) cells exposed to MP with and without an inhibitor of ABC transporters (verapamil). The results concerning transporters activity showed that the MXR mechanism is activated to detoxify from methyl parathion. The toxic effects of MP on ZFL cells were increased in the presence of the efflux transporter inhibitor, once cell viability was significantly decreased in co-exposure experiments. The combined exposure to MP and the inhibitor caused an increase in gene expression of P-gp1 (Abcb1) and MRP4 (Abcc4), suggesting that these transporters isoforms are associated with MP efflux. In general, the expression of genes related to the antioxidant defense system (ADS) was significantly increased in ZFL cells co-exposed to MP and verapamil. These data provide useful insights for better understanding of MP detoxification mechanism in fish hepatocytes. Ó 2014 Published by Elsevier Ltd. 1. Introduction Aquatic organisms are constantly exposed to many pollutants dissolved in the water and great part of these organisms can toler- ate exposure to these compounds. Kurelec (1992) has shown that this tolerance capacity is related to the multi-xenobiotics mecha- nism (MXR), which is considered the first line of defense against toxic compounds (Epel, 1998). MXR is widely distributed in biolog- ical systems, from unicellular organisms to vertebrates (Annilo et al., 2006). It is mediated by the transmembrane transporters proteins from ATP-binding cassette (ABC) family, which are responsible for the excretion of many xeno/endobiotics from the cell by ATP hydrolysis (Bard, 2000). ABC transporters constitute a highly conserved superfamily of proteins among vertebrates and exert many functions in the organisms. In zebrafish, the ABC trans- porter genes are organized in 8 sub-families, which are designated from ABCA to ABCH (Dean and Annilo, 2005). Some ABC transport- ers from ABCB, ABCC and ABCG subfamilies have been reported with functions related to MXR (Buss and Callaghan, 2008; Zucchi et al., 2010; Faria et al., 2011). The ABCB1 (P-gp, MDR1), members of the ABCC (MRP) subfam- ily and breast cancer resistance protein (BCRP, ABCG2) are toxico- logically the most relevant proteins in efflux of xenobiotics in mammals (Doyle and Ross, 2003). P-gp may act eliminating hydroxylated xenobiotics metabolites after its modification by phase I enzymes (cytochromes P450), or leading non-metabolized xenobiotics out of the cell (Bard, 2000; Xu et al., 2005). MRPs have been reported to promote resistance against heavy metals in some mammals and fish species (Zucchi et al., 2010; Long et al., 2011). Besides, MRP and P-gp present significant overlap in substrate specificities, particularly evincing affinity for compounds conju- gated with glutathione, glucuronate or sulfate (Cole et al., 1994; Buss and Callaghan, 2008). ABCG2 is a widely studied transporter in humans due to its capacity to grant resistance to chemothera- pics used against breast cancer. Concerning fish species, Abcg2 expression was detected in liver of rainbow trout Oncorhynchus mykiss (Zaja et al., 2008a). In zebrafish, four paralogs of Abcg2 gene were already identified (Annilo et al., 2006). ABCB1 and ABCG2 transporters are located in the apical membrane of cells, facilitating the movement of substances and promoting the excretion of xenobiotics. ABCB1 substrates are http://dx.doi.org/10.1016/j.tiv.2014.09.010 0887-2333/Ó 2014 Published by Elsevier Ltd. ⇑ Corresponding author at: Instituto de Ciências Biológicas, Universidade Federal do Rio Grande – FURG, Av. Itália km 8, 96203-900 Rio Grande, RS, Brazil. E-mail address: dqmluf@furg.br (L.F. Marins). Toxicology in Vitro 29 (2015) 204–210 Contents lists available at ScienceDirect Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit