Biochemical alterations in caged Nile tilapia Oreochromis niloticus☆
Introduction
In developing countries the intense industrial activity, associated to limited availability of coordinated environmental initiatives, increases the risk to natural water resources. Joinville, the largest city in Santa Catarina state, southern Brazil, is home to more than 200 industrial plants from which effluents are discharged without proper treatment into rivers crossing the industrial area, as evidenced by preliminary toxicological studies (Knie, 2002). The lack of continuous monitoring, or restriction to a few variables, is a major drawback for the effective evaluation of contamination levels.
Regarding protocols of aquatic contamination studies, acetylcholinesterase inhibition and cytochrome P450 (CYP) induction are classical biomarkers, wherein oxidative stress-related parameters, such as antioxidant enzymes, glutathione status and oxidative damage to lipids are complementary biomarkers (Livingstone, 1990, Livingstone, 2001). Among phase II enzymes, the glutathione S-transferase (GST) induction is an important factor in determining the sensitivity of cells to a broad spectrum of toxic chemicals (Gadagbui and James, 2000).
Different studies showed that fish from impacted areas have increased levels of CYP-dependent activity (Payne et al., 1984; Spies, 1989; Collier et al., 1996; Kirby et al., 1999; Lewis et al., 2006). The CYP1A expression has been assessed as an exposure biomarker for polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyl (PCBs) including livers of fish caged at contaminated areas in Brazil (Bainy et al., 1999; Ventura et al., 2002). CYP2B expression is induced in rodents when exposed to PAHs such as non-planar PCBs, polybrominated biphenyl, dichloro-diphenyl-trichloroethane, dieldrin, heptachlor and chlordane (Lewis and Lake, 1997). Although most known forms of CYP2B are expressed at low constitutive levels, some examples in fish demonstrate induction of CYP2B (Bainy et al., 1999; Schlezinger and Stegeman, 2001). The CYP3 isoforms make up the major part of the hepatic and small intestine CYP content in both mammalian and fish species. They play a dominant role in drug and xenobiotic metabolism, which are typically induced by rifampicin, phenobarbital and dexamethasone (Hegelund and Celander, 2003; Li et al., 2008). In fish the induction pattern is more heterogeneous (Okey, 1990; Bresolin et al., 2005; Li et al., 2008).
Detoxifying processes, leading to amplification in antioxidant defense systems, are required to prevent their deleterious effects (Winston and DiGiulio, 1991). When the cellular capacity to counteract ROS is overcome an oxidative stress condition is established (Sies, 1999). The classical adaptive response following mild to moderate oxidative stress leads to an increase in antioxidant protection, e.g. the induction of antioxidant enzymes (e.g. Correia et al., 2007; Hansen et al., 2007). However, toxic oxidative burdens can impair such a response and initiate cell damage (Prieto et al., 2007).
The cellular thiol status, along with antioxidant enzymes, can be an important marker of oxidative attack and may be useful in biomonitoring programs (Livingstone, 2001). Glutathione (GSH) is the most important non-protein thiol group participating in conjugation reactions and antioxidant defenses, as well as in signaling pathways (Sies, 1999). An experimental approach to identify whether the cells are under oxidative stress is to measure oxidized glutathione (GSSG) formation, which is accumulated under oxidative stress and reflects in an increased GSH/GSSG ratio (Sies, 1999). Conjugation reactions or oxidative burdens can lead to decreased GSH levels (Dickinson and Forman, 2002), frequently associated to end products of lipid peroxides, like thiobarbituric acid reactive substances (TBARS), damage to DNA and to proteins (Prieto et al., 2007; Alink et al., 2007).
Mitogen-activated protein kinases (MAPKs) are a family of evolutionary conserved enzymes that play critical regulatory roles in cell physiology. These proteins may respond to chemical and physical environmental stresses, thereby controlling cell survival and adaptation (Cowan and Storey, 2003). Such proteins have been identified in aquatic organisms playing key roles in cell physiology (MacDougal et al., 1999).
Caspase-3 is an important component in the cascade leading to the apoptotic mode of cell death. In zebra fish, overexpression of caspase-3 induces extensive apoptosis and several developmental abnormalities, in association to an elevated sensitivity to environmental stress. This stress sensitivity can be ameliorated by knocking down caspase-3 activity (Yamashita et al., 2008), implying caspase-3 in physiological processes in fish. The fish caspases pathway are similar to the known mammalian apoptotic machinery (Yabu et al., 2001), supporting the use of caspase-3 activation as an apoptotic index in the Nile tilapia brain.
The industrial area of the Joinville city was chosen and the field experiments were made in the river named “Rio do Braço”, the major source of water to Joinville city. Aiming to establish the feasibility of an in situ short term exposure protocol, caged tilapia was used as potential biomonitoring species. To test the usefulness of oxidative stress-related parameters, cell signaling proteins as biomarkers and to validate protocols to be used in environmental assessment programs, tilapias were caged for one week in two different sites of Braço river. Antioxidant enzymes, glutathione status and lipid peroxidation end products were analyzed in parallel to other markers of contamination, such as cytochrome P450A1, 2B-like and 3A immunocontent, glutathione S-transferase and cholinesterase activity. Caspase-3 activation, ERK 1/2 and p38MAPK phosphorylation, along with HSP70 expression, were evaluated in the brain as potential indicators of contamination.
Section snippets
Area of study
Joinville, is the largest city in Santa Catarina state, Brazil and is one of the most important industrial areas of southern Brazil (Fig. 1).
According to the available data, site S1 has low levels of oxygen and values of cyanides, phosphorous, nitrite, nitrate and thermotolerant fecal coliforms above allowance levels (Oliveira et al., 2009). Point S1 receives water carrying industrial effluents, mainly from the metal/mechanic industry, and possibly loaded with metals (Knie, 2002). Contamination
Oxidative markers
Liver GSH-t levels were significantly decreased in animals caged at site S2, but not at S1, while GSSG was increased in site S1 (Fig. 2). As shown in Fig. 2C, there was a decrease in GSH/GSSG ratio in the liver of the animals exposed to contaminated waters in both the sites, when compared to the reference animals (REF). Blood GSH-t was decreased in animals exposed in site S2 (Fig. 3A), similar to the response found in the liver. Plasma-SH levels (Fig. 3B) were not altered, while Hb content
Discussion
The GSH-t levels in both blood and liver were decreased in site S2 while GSSG was elevated in the site S1. Owing to these alterations in the glutathione levels, the rate GSH/GSSG was significantly decreased in both the polluted sites. The rate of GSH/GSSG is used as an index of oxidative stress (Sies, 1999). The second pro-oxidant parameter analyzed was the end product of lipid peroxidation, TBARS levels, that was increased in sites S2, but not in S1. The TBARS levels have been frequently used
Conclusions
In conclusion, data presented here indicate: (a) a possible occurrence of oxidative stress in animals from both sites, associated to an induction of antioxidant enzymes activity GPx, catalase and GR in site S2. (b) The presence of agonists of CYP1A and CYP2B-like in site S1. (c) The sign of pesticide contamination, as indicated by the lower cholinesterase activity in site S1. (d) Occurrence of apoptosis in the fish brain, based on the increased activation of caspase-3 in both sites. (e)
Acknowledgments
We would like to acknowledge Fundação Municipal 25 de Julho, Joinville, SC, for supplying the fish used in this study and for technical support in the field experiments and Dr. John J. Stegeman for giving CYP1A and CYP2B-like antibodies. Funding sources: International Foundation for Science (IFS Research Grant Agreement Number A/3636) and Ministry of Science and Technology of Brazil research grant in the program: MCT/CNPq/CT-Agro/CT-Hidro/MAPA-SDC-SPAE 44/2008, Proc. Nr. 577253/2008-5.
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Experimental procedures were approved by the local ethical committee and conducted in accordance with national and institutional guidelines for the protection of animal welfare, as acknowledged in the M&M section. CEUA protocol number: PP00039.
- 1
Present address: Universidade Federal do Pampa, Rua Antônio Trilha 1847, 97300-000 São Gabriel, RS, Brasil.
- 2
These authors contributed equally to this work.
- 3
Present address: Instituto de Física de São Carlos, Universidade de São Paulo, 13560-590 São Carlos, SP, Brazil.