Toxic effects of DDT and methyl mercury on the hepatocytes from Hoplias malabaricus
Introduction
Dichlorodiphenyltrichloroethane (DDT, an organochlorine pesticide) and the metal mercury (Hg), both from human sources, accumulate in tissues of fish from many rivers and lakes around the world (Table 1). While bioaccumulation is well-documented, the effects of the observed concentrations of DDT and Hg on fish liver cell function are poorly studied. This is an important issue since the impact on biochemical and cellular processes caused by these pollutants can affect fish physiology, behavior, reproduction and survival (Lam and Gray, 2003, Vasseur and Cossu-Leguille, 2003, Eggen et al., 2004, Moore et al., 2004).
Predatory fish typically have greater concentrations of DDT and Hg due to bioaccumulation along the aquatic food chain (Morel et al., 1998). Bioaccumulation of DDT and Hg in predatory fish is likely to cause similar adverse and toxic reactions in hepatocytes as those observed in mammals. These include damage to cellular biomolecules (Milaeva, 2006), uncoupling of oxidative phosphorylation and electron transport in mitochondria (Verity et al., 1994, Garg and Chang, 2006), intracellular ion imbalance and generation of reactive oxygen species (ROS) and free radicals (Barros et al., 1994, Shanker et al., 2005, Milaeva, 2006) and impairment of the antioxidant defense system (Cookson and Pentreath, 1996). Primary hepatocyte culture is a useful in vitro model for the investigation of contamination on fish liver function (Segner, 1998, Filipak Neto et al., 2007).
Both DDT and Hg are found in the waters of the Amazonian region, yet few studies have examined the impact those anthropogenic chemicals may have on native fish. Hoplias malabaricus (Bloch, 1794), known locally as the traíra, is a freshwater teleost predator abundant in the Amazon River system. This species is an interesting model to evaluate toxic effects of pollutants in Brazilian ecosystems because it has predator and voracious behavior bioaccumulating xenobiotics from food chain, has no migratory behavior, is widely distributed throughout South America and is easily maintained in experimental conditions (Mol et al., 2001, Porto et al., 2005, Rabitto et al., 2005, Souza Lima et al., 2005, Dorea et al., 2006, Oliveira Ribeiro et al., 2006, Olivero-Verbel et al., 2006, Alves Costa et al., 2007). For Brazilian tropical species, however, no studies have been developed to investigate the mechanisms of cell toxicity after in vitro exposure to DDT and MeHg exposure, despite the high occurrence of these pollutants in many species of fish (Table 1). This experimental system has recently been made available due to the establishment of primary hepatocyte cultures of H. malabaricus (Filipak Neto et al., 2006).
In this study, this method was used to evaluate whether the exposure to DDT, MeHg and their combination induces oxidative stress and cell death in hepatocytes of a native fish species under realistic concentrations. Also, we examined if the magnitude of the effect of co-exposure to DDT and MeHg (DDT*MeHg) was similar to the exposure to the higher concentration of MeHg, investigating the in vitro ROS production, antioxidant defense system, damage to biomolecules and cell survival. The concentrations of chemicals tested in the current work were similar to those found in liver and muscle of H. malabaricus collected in Amazon basin (Table 1). The use of environmentally relevant concentrations is a more realistic approach favoring the discussion about hepatotoxicity in wild species under chronic exposure.
Section snippets
Materials and methods
Hoplias malabaricus specimens (700–1100 g) were obtained from commercial farm (Curitiba, Paraná, Southern Brazil) and maintained in 80 l fiber-glass aquaria at room temperature (18–24 °C) and controlled photoperiod (12 h:12 h). Fishes were fed every 3 days on young, live carp Cyprinus carpio.
ROS
In order to verify the effects of DDT and MeHg on the production of ROS, intracellular concentrations of hydrogen peroxide (H2O2) and superoxide anion () were measured in H. malabaricus hepatocytes. H2O2 increased in MeHg I (14%), DDT*MeHg I (17%) and most in DDT (36%, Fig. 1A). was similar in all treatments, except for the decrease in MeHg II (18%, Fig. 1A).
Enzymatic activity of the CAT, SOD, GST, GR, G6PDH and δ-ALAd
Given the effects of DDT and MeHg on the production of ROS in H. malabaricus hepatocytes, we measured the activities of some
Discussion
Deregulation of redox intracellular milieu and increased hepatocyte death indicated that environmentally relevant concentrations of DDT and MeHg were very harmful xenobiotics for H. malabaricus hepatocytes. Here, we also indicated that oxidative stress was one important reason for hepatotoxicity of both xenobiotics.
Conflict of interest statement
The authors state that there are no conflicts of interest or relation between the data presented in this work and other data previously published.
Acknowledgements
The authors acknowledge Dr. Luis Cláudio Fernandes for scientific assistance, CAPES (Brazilian Agency for Science and Technology) for fellowship support and ARAUCARIA Foundation (Parana State Agency for Science and Technology) for reagents and equipment supply.
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