Elsevier

Toxicology in Vitro

Volume 22, Issue 7, October 2008, Pages 1705-1713
Toxicology in Vitro

Toxic effects of DDT and methyl mercury on the hepatocytes from Hoplias malabaricus

https://doi.org/10.1016/j.tiv.2008.07.006Get rights and content

Abstract

Here, we examined the impact of dichlorodiphenyltrichloroethane (DDT) and monomethyl mercury (MeHg) on the redox milieu and survival of hepatocytes from Hoplias malabaricus (traíra). After isolation and attachment of cells, we established one control and four treatments: DDT (50 nM of DDT), MeHg I (0.25 μM of MeHg), MeHg II (2.5 μM of MeHg) and DDT * MeHg I (combination of 50 nM of DDT and 0.25 μM of MeHg). After four days the exposed hepatocytes presented significantly increased damage in lipids (all treatments), proteins (DDT * MeHg I and MeHg II) and reduced cell viability (all treatments). Also the antioxidant enzymes catalase, glucose-6-phosphate dehydrogenase (G6PDH), glutathione reductase and superoxide dismutase were affected. The current data showed that despite of some protective responses, the increased disturbs on membrane lipids and proteins, increased hydrogen peroxide levels, and decreased glutathione concentration and cell viability strongly indicate oxidative stress as the reason of hepatotoxicity due to DDT and MeHg exposure. In addition, DDT and MeHg together had greater effect than alone when G6PDH and glutathione-S-transferase activities and lipids damage were considered. These findings are indicative of hepatotoxicity occurring at realistic concentrations of DDT and MeHg found in Amazonian fish tissues.

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 (O2) were measured in H. malabaricus hepatocytes. H2O2 increased in MeHg I (14%), DDT*MeHg I (17%) and most in DDT (36%, Fig. 1A). O2 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.

References (62)

  • J.G. Dorea et al.

    Fish mercury bioaccumulation as a function of feeding behavior and hydrological cycles of the Rio Negro, Amazon. Comparative biochemistry and physiology

    Toxicology and Pharmacology

    (2006)
  • M.S. Evans et al.

    Elevated mercury concentrations in fish in lakes in the Mackenzie River Basin: the role of physical, chemical, and biological factors

    The Science of the Total Environment

    (2005)
  • M. Ferreira et al.

    Organochlorine contaminants in flounder (Platichthys flesus) and mullet (Mugil cephalus) from Douro estuary, and their use as sentinel species for environmental monitoring

    Aquatic Toxicology

    (2004)
  • F. Filipak Neto et al.

    Use of hepatocytes from Hoplias malabaricus to characterize the toxicity of a complex mixture of lipophilic halogenated compounds

    Toxicology in Vitro

    (2007)
  • T.K. Garg et al.

    Methylmercury causes oxidative stress and cytotoxicity in microglia: attenuation by 15-deoxy-delta 12,14-prostaglandin J2

    Journal of Neuroimmunology

    (2006)
  • L. Holsbeek et al.

    Mercury speciation and accumulation in Bangladesh freshwater and anadromous fish

    The Science of the Total Environment

    (1997)
  • Z.-Y. Jiang et al.

    Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low density lipoprotein

    Analytical Biochemistry

    (1992)
  • J.H. Keen et al.

    Mechanism for several activities of the glutathione-S-transferases

    The Journal of Biological Chemistry

    (1976)
  • J.P. Kim

    Methylmercury in rainbow trout (Oncorhynchus mykiss) from Lakes Okareka, Okaro, Rotomahana, Rotorua and Tarawera, North Island, New Zealand

    The Science of the Total Environment

    (1995)
  • P.K. Lam et al.

    The use of biomarkers in environmental monitoring programmes

    Marine Pollution Bulletin

    (2003)
  • R.L. Levine et al.

    Carbonyl assays for determination of oxidatively modified proteins

    Methods in Enzymology

    (1994)
  • M. Mela et al.

    Effects of dietary methylmercury on liver and kidney histology in the neotropical fish Hoplias malabaricus

    Ecotoxicology and Environmental Safety

    (2007)
  • E.R. Milaeva

    The role of radical reactions in organomercurials impact on lipid peroxidation

    Journal of Inorganic Biochemistry

    (2006)
  • J.H. Mol et al.

    Mercury contamination in freshwater, estuarine, and marine fishes in relation to small-scale gold mining in Suriname, South America

    Environmental Research

    (2001)
  • M.N. Moore et al.

    An integrated biomarker-based strategy for ecotoxicological evaluation of risk in environmental management

    Mutation Research

    (2004)
  • C.A. Oliveira Ribeiro et al.

    Hematological findings in neotropical fish Hoplias malabaricus exposed to subchronic and dietary doses of methylmercury, inorganic lead, and tributyltin chloride

    Environmental Research

    (2006)
  • J. Olivero-Verbel et al.

    Contracaecum sp. infection in Hoplias malabaricus (moncholo) from rivers and marshes of Colombia

    Veterinary Parasitology

    (2006)
  • G.G. Pandit et al.

    Distribution and fate of persistent organochlorine pesticides in coastal marine environment of Mumbai

    Environment International

    (2006)
  • A. Pastore et al.

    Analysis of glutathione: implication in redox and detoxification

    Clinica Chimica Acta

    (2003)
  • J.I.R. Porto et al.

    Mutagenic effects of mercury pollution as revealed by micronucleus test on three Amazonian fish species

    Environmental Research

    (2005)
  • I.S. Rabitto et al.

    Effects of dietary Pb(II) and tributyltin on neotropical fish, Hoplias malabaricus: histopathological and biochemical findings

    Ecotoxicology and Environmental Safety

    (2005)
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