Chronic effects of realistic concentrations of non-essential and essential metals (Lead and Zinc) on oxidative stress biomarkers of the mosquitofish, Gambusia holbrooki

Metallic contamination is widespread, particularly in areas impacted by human activities. Human activities result in high loads of metals being discarded into the aquatic compartment, reinforcing the need to evaluate their toxic effects especially on exposed fish. The purpose of this study was to determine the toxic response (namely, antioxidant levels and lipoperoxidative damage) in both liver and gills of the freshwater fish species Gambusia holbrooki, exposed to lead and zinc. Fish were exposed for 28 days (chronic exposure) to ecologically relevant concentrations of the selected compounds. The following oxidative stress/damage biomarkers were evaluated: glutathione-S-transferases (GSTs), glutathione reductase (GR), and thiobarbituric acid reactive substances (TBARS). The results indicate that lead caused a significant oxidative response, with significant increase of the enzymatic antioxidant defense (GSTs activity in hepatic tissue, and GR activity in branchial tissue) of exposed organisms. On the other hand, zinc caused a significant inhibition of G. holbrooki hepatic GR, a biological response that may be related to the antioxidant activity exhibited by this metal. The obtained results are of high importance, especially if one considers that the obtained toxic responses occurred at low, albeit ecologically relevant, levels of exposure. Metal-containing waste (from domestic, agricultural and industrial sources) that is continuously released can have significant impacts on the environment, particularly aquatic ecosystems (López-Galindo et al. 2010). In recent years, the effects of metallic pollution in fish were demonstrated, showing that these organisms are able to bioaccumulate these pollutants (Greco et al. 2010). In addition, metals can induce severe alterations, including an oxidative stress response in various cell types, and also significant alterations of the elimination profile of foreign molecules from the body (Wang et al. 2009; Johnston et al. 2010). Thus, oxidative stress biomarkers, and others (especially those implicated in the metabolism of toxic molecules), have become an important assessment tool in aquatic toxicology (Livingstone 2001; Wang et al. 2009; Jin et al. 2010; Johnston et al. 2010) to monitor the occurrence of pollutants, and deleterious effects potentially exerted in aquatic organisms. The use of molecular oxygen in normal respiratory processes in mitochondria results in the physiological production and release of reactive oxygen species (ROS; Chance et al. 1979; Wallace 1999); exposure to xenobiotics (including metals) can increase the production of ROS through several mechanisms, such as interference in the electron transport within the mitochondrial membrane and subsequent accumulation of reactive intermediates (Herrero et al. 2008). This may result in cellular damage, namely by inactivation of antioxidant enzymes, depletion of nonenzymatic antioxidants, and membrane lipid peroxidation (Modesto and Martinez 2010). The review by Franco et al. (2009) referenced that some of the most common environmental contaminants, including several metallic species (e.g., iron, copper, chromium, cobalt, vanadium, cadmium, arsenic, nickel), could trigger apoptosis through the interference with regulating cellular mechanisms. Metals are known to interfere at several subcellular levels, such as the mitochondria (oxidation of mitochondrial RNA, and activation of the intrinsic apoptotic pathway), the endoplasmatic reticulum, and nuclear DNA. Moreover, established oxidative imbalance can cause irreversible oxidative damage in DNA and other macromolecules, or even death of organisms (Jin et al. 2010; Li et al. 2010; Modesto and Martinez 2010). ROS are removed or inactivated by antioxidant defenses, and the balance between these radicals and the antioxidant defense of living organisms is fundamental for their protection against oxidative stress and its deleterious consequences (Li et al. 2010; Modesto and Martinez 2010). Nevertheless, the exposure to chemical pollutants also may contribute to oxidative stress, by altering this equilibrium, and consequently inducing a decrease in the antioxidant defense system efficiency (Solé et al. 1996; Livingstone 2001). Fish cope with the harmful effects of oxidative stress through adaptive responses, namely by increased activity of enzymes involved in the biotransformation and metabolism of a wide range of environmental contaminants and their metabolites (Ognjanovic et al. 2008; Modesto and Martinez 2010). The antioxidant defense system of the majority of organisms is composed by a multitude of enzymes, among which it is possible to identify glutathione reductase (GR), and glutathione-S-transferases (GSTs). These are the most frequently studied biomarkers of oxidative stress in fish (Jin et al. 2010; Modesto and Martinez 2010; Pereira et al. 2010). Lipoperoxidation (LPO) estimation also has been found to have a high value as a biomarker of toxic effects, because this parameter reflects the onset of cellular damage, as a result of oxidation of membrane lipids (Ognjanovic et al. 2008; Pereira et al. 2010). Amongst all compounds present in the environment, in growing amounts and with evident human origin, metals are particularly important. Metals are widely dispersed in the aquatic environment, being released by anthropogenic activities (mining, release of domestic products into sewage; plumbing degradation; emissions from nuclear plants, from smelters and from burning fossil fuels) but also by natural sources (e.g., volcanoes) (Hozhina et al. 2001; Thompson et al. 2005; Rose and Shea 2007; Connan and Tack 2010; Aktar et al. 2011). Metallic species are fundamental for life, being part of macromolecules and enzymes (Liu and Thiele 1997). However, metals also are prone to establish redox cycles if in the presence of molecular oxygen, giving rise to the production of ROS (Herrero et al. 2008). Consequently, metals are dual in their effects: albeit vital, they are eminently toxic (Liu and Thiele 1997). Metals (especially transition metals) are toxic and capable of exerting important deleterious effects of oxidative nature in exposed organisms, as reviewed by Valavanidis et al. (2006). Metals, such as arsenic, cadmium, lead, mercury, chromium, nickel, manganese, and iron, especially in their waterborne form, can indeed increase the production of reactive oxygen species (Jadhav et al. 2007). However, exposure to metals conducing to oxidative stress is commonly followed by a set of immediate physiological adaptations (e.g., through antioxidant defenses) to prevent their adverse effects. For example, the study conducted by Grinevicius et al. (2009) on textile effluents rich in metallic species evidenced antioxidant responses in the freshwater fish Danio rerio. Despite the activation of antioxidant mechanisms following chemical insults by metals, damage (e.g., lipid peroxidation) is likely to occur, as reported by Siddique et al. (2008) after exposing Drosophila melanogaster to metals present in tannery effluents. Mining effluents also were proven to induce oxidative stress responses in fish (Kelly and Janz 2009), anurans (Marques et al. 2011), and mammals (Reglero et al. 2009). The purpose of the present study was to evaluate the chronic effects induced by exposure to environmentally realistic concentrations of two metals (nonessential lead and essential zinc) on oxidative stress parameters of the freshwater fish Gambusia holbrooki. Being distinct in nature and biological effects, these two metals are dispersed widely in Portuguese estuaries (Mucha et al. 2003; Fernandes et al. 2008). However, it is important to know in detail their toxic effects when in ecologically relevant levels to ascertain about the putative common toxic mechanisms and to know the biological responses elicited by fish to cope with these two different compounds. The oxidative stress parameters GSTs, GR, and thiobarbituric acid reactive substances (TBARS) were measured in hepatic and gill tissues to serve as putative biomarkers of effect in this study, provided their role in key biological processes determinant for the survival of the individuals: the enzymes are involved in detoxification by phase II metabolism (conjugation with glutathione) and antioxidant activity, and TBARS are indicative of lipoperoxidative damage (Nunes et al. 2008, 2015a, b). As target organs, we chose liver and gills because the liver is the main organ of xenobiotic metabolism in fish, and the gills are the primary barrier against the entrance of xenobiotics into the body and also are the first line of detoxification and elimination of deleterious compounds (Wood and Soivio 1991; Evans et al. 2005).