Malachite green toxicity assessed on Asian catfish primary cultures of peripheral blood mononuclear cells by a proteomic analysis

Abstract

The potential genotoxic and carcinogenic properties reported for malachite green (MG) and the frequent detection of MG residues in fish and fish products, despite the ban of MG, have recently generated great concern. Additional toxicological data are required for a better understanding of the mechanism of action and a more comprehensive risk assessment for the exposure of fish to this fungicide. To date, the use of fish peripheral blood mononuclear cells (PBMCs) has not been exploited as a tool in the assessment of the toxicity of chemicals. However, PBMCs are exposed to toxicants and can be easily collected by blood sampling. The present study aims at better understanding the effects of MG by a proteomic analysis of primary cultured PBMC from the Asian catfish, Pangasianodon hypophthalmus, exposed to MG. The two lowest concentrations of 1 and 10 ppb were selected based on the MTS (water soluble tetrazolium salts) cytotoxicity test. Using a proteomic analysis (2D-DIGE), we showed that 109 proteins displayed significant changes in abundance in PBMC exposed during 48 h to MG. Most of these proteins were successfully identified by nano LC–MS/MS and validated through the Peptide and Protein Prophet of Scaffold™ software, but only 19 different proteins were considered corresponding to a single identification per spot. Our data suggest that low concentrations of MG could affect the mitochondrial metabolic functions, impair some signal transduction cascades and normal cell division, stimulate DNA repair and disorganize the cytoskeleton. Altogether, these results confirm that the mitochondrion is a target of MG toxicity. Further studies on the identified proteins are needed to better understand the mechanisms of MG toxicity in fish produced for human consumption.

Introduction

The triphenylmethane dye, malachite green (MG), used as an agent against bacterial, fungal and parasite infections in aquaculture has been banned for aquatic organisms intended for human consumption and as food additives since 2000 in the European Union (Sudova et al., 2007), since 1981 in the United States, and since 2003 in Japan (Schuetze et al., 2008). Despite this ban, MG is still used as confirmed by the frequent detection of its residues in fish and fish products (BfR, 2007). The quantities of MG residues in fish will depend on the origin of the exposure, either illegal applications in aquaculture or environmental residues. For instance, Scherpenisse and Bergwerff (2005) reported MG residue concentrations up to 24 μg/kg in trout and 7 μg/kg in Asian catfish from a local retailer in Utrecht (Netherlands). MG and LMG were detected with total concentrations up to 0.765 μg/kg fresh weight in tissues of eels caught from a canal (Teltowkanal) in Berlin, Germany (Schuetze et al., 2008) and up to 619 μg/kg measured in caviar of trout from Sweden (BfR, 2007). MG and, especially its reduced form, LMG, are persistent in edible fish tissues for extended periods of time (Jiang et al., 2009) depending on their fat content (Bauer et al., 1988). The major risk of contamination in fish flesh comes from the aquatic imported products from several Asian countries, especially Vietnam, where regulations about MG are less stringent (Arroyo et al., 2009, Love et al., 2011).

According to the reviews of Srivastava et al. (2004) and Sudova et al. (2007), MG exerts considerable toxic effects in fish. However, until now, these effects of MG have been investigated mostly in mammals, in part, due to the possible risk for human as consumers (Sudova et al., 2007). MG affects, at the transcriptional level, the cell cycle machinery in liver cells of rodents (Gupta et al., 2003). MG also leads to the inhibition of DNA synthesis in rat hepatocytes (Rao and Fernandes, 1996), to the generation of chromosomal damage in Syrian hamster embryo cells (Rao et al., 2001), to the reduction of capability of cells to proliferate and to impairment of mitochondrial activity in two human tumour cell lines (Stammati et al., 2005). Potential properties attached to MG, like genotoxic, teratogenic and carcinogenic properties, are still questioned. In this context, additional toxicological data are required for a more comprehensive risk assessment of the potential MG effects in farmed fish.

To advance our understanding of the mechanisms of action of MG, evaluation of cellular alterations after in vitro exposure is advisable to complement data acquired in fish at higher levels of biological organization. Isolated fish cells are recognized as valuable models to assess the impact of chemical substances on biological processes (Castaño et al., 2003, Davoren et al., 2005). Peripheral blood mononuclear cells (PBMCs) represent targets for chemicals in fish and as such they may be particularly suitable for mechanistically oriented studies on cell-specific toxicant action (Bols and Lee, 1991, Schirmer, 2006). Working on PBMC allows regular sampling in little invasive way and follows the new European Chemicals Legislation (REACH) asking for alternatives assays avoiding animal killing in ecotoxicology (Lilienblum et al., 2008, Rovida and Hartung, 2009). Blood is the perfect exchange medium between rearing water and the organism. It has been shown to be impacted by MG treatment. The fungicide is, in fact, reported to cause significant alterations in blood biochemical parameters, to affect haematological (Srivastava et al., 2004, Sudova et al., 2007) and immunological (Yonar and Yonar, 2010) variables in fish. Ding et al. (2009) recently demonstrated that MG can bind lysozyme.

Proteomic analysis, demonstrating changes in gene expression at the proteome level, is one of the possible strategies to provide insight into the underlying mechanisms of toxicity induced by xenobiotics. This approach has been recently applied in studies of environmental concern as for instance on gills of Chinese mitten crab (Eriocheir sinensis) during acclimation to cadmium (Silvestre et al., 2006) but also on zebrafish (Danio rerio) liver treated with tetrabromobisphenol-A (De Wit et al., 2008), on African clawed frogs (Xenopus laevis) exposed to polychlorinated biphenyls (PCBs) mixture Aroclor 1254 (Gillardin et al., 2009), on green and white sturgeon larvae (Acipenser medirostris and Acipenser transmontanus) exposed to heat stress and selenium (Silvestre et al., 2010a) and on the European bullhead (Cottus gobio) following exposure to cadmium sub-lethal concentrations (Dorts et al., 2011). To date, proteomic analyses are mostly applied on sequenced model species which may be, to some extent, poorly relevant from an ecotoxicological or aquaculture perspective (Forné et al., 2010). Silvestre et al. (2010b) carried out a proteomic analysis on the haemolymph of the giant tiger shrimp in Vietnamese pond systems. In the same context, our model species is the Asian catfish, Pangasianodon hypophthalmus, one of the most important farmed fish in view of its economic interest. The export of this species has sharply increased, in particular towards the major European, North-American and Asian markets (FAO, 2009). Fish are reared under intensive conditions with densities up to 150 fish m⁻³ (Rahman et al., 2006). Effective and relatively cheap antifungal agents like MG seem attractive for farmers to limit economic losses.

The present study aimed at identifying sensitive modifications of protein expression as markers for in vitro effects of MG on primary cultures of PBMC from the Asian catfish, P. hypophthalmus and at analysing through which mechanism MG and its derivatives could exert their potential adverse effects in fish.