Biochemical biomarkers in Oreochromis niloticus exposed to mixtures of benzo[a]pyrene and diazinon.

Abstract

Biochemical biomarkers (the activities of acetylcholinesterase, 7-ethoxyresorufin-O-deetilase, carboxylesterase, catalase, glutathione peroxidase and glutathione S-transferase) were evaluated in Nile tilapia (Oreochromis niloticus) that had been exposed to benzo[a]pyrene (BaP) and the organophosphate pesticide diazinon (DZ), at 0.5 mg/L. The animals were pre-exposed to BaP for three days, and DZ was then added to both non-exposed and pre-exposed groups, being exposed for 2 and 7 additional days. The level of BaP was also measured in the bile. BaP caused the induction of phase I and II enzymes, and DZ caused carboxylesterase inhibition in gills but not in liver. AChE activity was unchanged. No significant modulation was observed in antioxidant enzymes. When in combination with BaP, DZ caused a significant decrease of EROD and GST induction. Levels of BaP in the bile were also increased in fish exposed to BaP combined with DZ, indicating an interference of DZ in responses activated by BaP.

Introduction

The measurement of induction of ethoxyresorufin-O-Deethylase (EROD, indicative of cytochrome P450 1A) in fish has been extensively used as a contamination biomarker to identify the exposure to organic xenobiotics, such as polycyclic aromatic hydrocarbons (PAHs), dioxins and polychlorobyphenyls (Flammarion et al., 1996; Whyte et al., 2000). The inhibition of esterases by organophosphate or carbamate pesticides (OPs and CMs) is also used as exposure and effect biomarkers for these compounds in environmental monitoring (Vioque-Fernández et al., 2007a, Vioque-Fernández et al., 2007b, Vioque-Fernández et al., 2009). However, data on the effects of complex mixtures of these contaminants in fish biomarkers are scarce.

It has been known that phosphorothionate pesticides are poor cholinesterase inhibitors, and must undergo activation to the oxon form by cytochrome P450 isoforms in order to inhibit esterases (Straus et al., 2000). Nevertheless, it has been also demonstrated that metabolites generated during P450-mediated conversion of thionates to the oxon forms are capable of inhibiting cytochrome P450 themselves (Tang et al., 2002). It was proposed that CYP-mediated desulfuration of chlorpyrifos in rats produces chlorpyrifos-oxon (Fukuto, 1990) in the process releasing the sulfur ion, which can then suppress CYP activity through binding onto the heme group (Tang et al., 2002). Suppression of CYP1A by OPs has been also described for fish (Flammarion et al., 1998). Similarly, Wheelock et al. (2005) observed a 30% decrease in CYP1A levels in Chinook salmon (Oncorhynchus tshawytscha) that had been exposed to chlorpyrifos.

Apart from desulfuration activity, OPs can also induce CYP isoforms involved in their detoxification (dearylation) (Neal and Halpert, 1982). Therefore, it is evident that other compounds that induce P450s could alter both the toxicity of the parent insecticide as well as the timing of esterase inhibition via the production of the oxon-derivative insecticide. Strauss et al. (2000) demonstrated that P450 isoforms induced by Aloclor 1254 are not able to increase desulfuration or dearylation of the OP chlorpyrifos, but this idea was not tested for other P450 inducers, such as the PAH benzo[a]pyrene (BaP).

Depending on the isoform profile of P450s and the substrate specificities of the induced isoform(s), induction can lead to either increases in the production of activated metabolites with enhanced toxicity or increased detoxication with enhanced protection from toxicity (Binder et al., 1984; Stegeman and Hahn, 1994; Straus et al., 2000). Moreover, if cytochrome P450 isoforms are inhibited in the activation process, the inhibition can impair the phase I defense mechanisms involved in detoxification of most contaminants that could be present in combination with OPs in aquatic environments that have been contaminated by other contaminants.

Diazinon (DZ) is a moderately persistent organophosphorothionate pesticide largely used in agriculture (Larkin and Tjeerdema, 2000). Its toxicity to animals is due to classical inhibition of cholinesterases, which poses risks to non-target organisms that inhabit natural environments close to agricultural fields. However, there are no data concerning DZs toxic effects when in combination with CYP inducers. It is possible that, in combination with other chemicals, DZ could exert a different toxic response in aquatic organisms, and this remains to be studied.

Exposure to PAHs and OPs has been related to oxidative stress generation in aquatic animals due to the production of reactive oxygen species (ROS) during their metabolism in cells (Winston and Di Giulio, 1991; Bagchi et al., 1995; Gultekin et al., 2000, Gultekin et al., 2001; Valavanidis et al., 2006, van der Oost et al., 2003). In healthy organisms, there is a balance between production and elimination of ROS. When the production of ROS is greater than its removal, an oxidative stress condition can be established. To deal with oxidative stress, the cells possess antioxidant defenses, such as the enzymes catalase (CAT), glutathione peroxidase (GPx) and also GST, a phase II biotransformation enzyme with peroxidase activity (Torres et al., 2002; Almeida et al., 2005, Almeida et al., 2007; Limón-Pacheco and Gonsebatt, 2009).

It has been shown that BaP exposure can significantly induce phase I and II biotransformation enzymes and oxidative stress. However, the main effect of DZ in organisms is esterase inhibition, even though oxidative stress has also been involved in the toxicology of both compounds. Even so, there are no data on the effect of these contaminants together in aquatic organisms. Thus, the goal of this study was to evaluate various biochemical responses of tilapias after controlled exposure to BaP and DZ alone or in combination. DZ is a thion OP that should be converted to the oxon form by cytochrome P450 to inhibit esterases. Therefore, we were interested to know whether P450 isoforms induced after BaP pre-exposure are able to either increase or decrease esterase inhibition after DZ exposure. We were further interested in knowing whether DZ in combination with BaP is able to decrease the EROD induction caused by BaP, and if there is any other influence of one of these compounds in the oxidative stress responses activated by the other contaminant after exposure to mixtures of both compounds. Levels of BaP in the bile of the tilapias were also monitored during the experiment.

Tilapia, a wide-spread fish species in tropical countries, is the most cultivated freshwater fish in Brazil, with increasing occurrence in Brazilian natural environments. Nile tilapia, which is a hardy fish, is an omnivorous feeder and feed by frequent foraging in the whole water column with occasional contact with sediments (Pathiratne et al., 2009). These characteristics make Nile tilapia a suitable indicator species for bio-monitoring of aquatic pollution in tropical environments (Rodríguez-Fuentes and Gold-Bouchot, 2004; Gold-Bouchot et al. 2006).