Acute and chronic effects of clofibrate and clofibric acid on the enzymes acetylcholinesterase, lactate dehydrogenase and catalase of the mosquitofish, Gambusia holbrooki
Introduction
A large number of therapeutic agents are found unchanged in the aquatic environment, after its elimination from the patient’s body (Daughton and Ternes, 1999). An even larger number of chemical compounds are found in the environment as the result of biological degradation by the organisms present in ecosystems. These components are of particular concern since they have important biological activity even at low concentrations. Furthermore, some of them are bioaccumulated and/or biomagnified in food webs. Besides the pharmaceutical agents per se, their metabolites may also possess biological activity, which can be similar or entirely different from their parent compound. In addition, the input of pharmaceuticals into the environment must be considered as a continuous process, in which very small amounts of a huge variety of substances are systematically delivered. The inefficiency of sewage treatment plants concerning the degradation of pharmaceuticals contributes to this situation (Petrović et al., 2003). The presence of several compounds in effluents of sewage treatment plants has been reported (Ternes, 1998, Miao et al., 2002), thus contributing for the presence of these chemical agents in the environment (Winkler et al., 2001). From a dynamic standpoint, changes in populations might be induced as a result of decades of subtle alterations, which could be wrongly attributed to evolutionary trends (Daughton and Ternes, 1999).
Clofibrate is an exclusive human use drug, designed to activate peroxisome proliferator activating receptors, in order to enhance lipid metabolism (lipid regulation), therefore lowering the lipid content of the body. This therapeutic activity allows it to be used in weight control. Earlier data involving this chemical and its effect over the rat, a classical animal model, showed a high prevalence of peroxisome proliferation (even as an acute effect), indicating the possibility of occurrence of oxidative stress, which may lead to irreversible damage by lipid peroxidation, and to the establishment of carcinogenetic processes (Qu et al., 2001). The increase of liver weight and volume has been also described (Latruffe et al., 2001). Due to these adverse effects, clofibrate was withdrawn from commercial circuits in most European countries.
Clofibrate has been detected in effluents from sewage treatment plants (Andreozzi et al., 2003) in concentrations up to 0.8 μg l−1. Its main metabolite, clofibric acid, possesses biological intracellular activity and has a high persistence in the environment (Daughton and Ternes, 1999), with an estimated persistence of 21 years (Buser et al., 1998) and holding endocrine disruption activity through interference with cholesterol synthesis (Pfluger and Dietrich, 2001). During passage through a sewage treatment plant, a loss of only 50% of the initial amount of clofibric acid was detected (Ternes, 1998). Due to these properties, clofibric acid may be found at concentrations of 270 ng l−1 in tap water (Heberer, 2002), 0.55 μg l−1 in surface waters of Swiss lakes (Buser et al., 1998), 1.6 μg l−1 (Ternes, 1998) in the majority of German sewage treatment plants, 103 ng l−1 in Detroit River water (Boyd et al., 2003), 5 ng l−1 in effluents of Greek sewage treatment plants (Koutsouba et al., 2003) 18 ng l−1 in the estuary of the River Elba, and 0.28–1.35 ng l−1 in North Sea water (Weigel et al., 2002).
The mosquitofish Gambusia holbrooki is an euryhaline organism widely distributed in both freshwater systems and estuaries of temperate regions. This fish possesses high fecundity and has been considered as a representative secondary consumer in aquatic ecosystems. These characteristics lead this species to be considered as a suitable animal model in Ecotoxicology.
The present study intended to evaluate both acute and chronic effects induced by exposure to clofibrate and clofibric acid on the enzymes acetylcholinesterase (AChE), lactate dehydrogenase (LDH) and catalase (CAT) of G. holbrooki. These enzymes were selected for use as putative biomarkers of effect in this study since they are key enzymes in biological processes determinant for the survival of the individuals: neurotransmission, anaerobic metabolism and peroxisome proliferation, respectively. In addition, several organ weight/total body weight ratios were used as end-points. Since total body weight was expected to change after clofibrate and clofibric acid exposure, the ratios liver weight/head weight and liver weight/gills weight were also determined. In chronic studies, males and females were independently exposed to evaluate potential sexual differences.
Section snippets
General procedures
Fish were collected in the estuary of Minho river, located in the northern region of Portugal. This estuary was chosen due to its characteristics of low urban, industrial and agricultural contamination (Ferreira et al., 2003), and has been used as a reference estuary in previous studies by our group. Fish were temporarily (4–5 days) kept in 30 l maintenance plastic tanks, filled with water from the sampling site. Females were separated from males. A first phase of laboratory maintenance involved
Acute clofibrate exposure
Exposure to clofibrate did not induce significant alterations on head AChE activity of mosquitofish (F = 3.05; d.f. = 6, 40; p > 0.05) (Fig. 1a). The hepatic CAT activity was significantly decreased following clofibrate exposure, and a plateau was observed for all tested concentrations (Fig. 1b) (F = 8.31; d.f. = 6, 40; p ⩽ 0.05). Muscular LDH activity was not significantly altered (F = 0.42; d.f. = 6, 36; p > 0.05) (Fig. 1c). No significant differences were found between exposed and non-exposed animals in what
Discussion
Neither body weight reduction nor hepatomegally were observed after acute exposure of male mosquitofish to clofibrate, thus suggesting that both toxicants did not exert mobilization of fat tissue/hypolipidemic effects. Hepatomegally, when referring to mammals, occurs due to simultaneous hepatothrophy and hyperplasia, as stated by Hildebrand et al. (1999). These effects do not appear to have occurred in G. holbrooki under the tested conditions. Chronic exposure of male mosquitofish to clofibrate
Acknowledgements
The present work was partially funded by “Fundação para a Ciência e a Tecnologia” (B. Nunes Ph.D. grant SFRH/BD/866/2000) and by Project “CONTROL” (POCTI/MAR/MAR/15266/1999).
Bruno André Nunes, B.Sc, Researcher as Ph.D. student in the Group of Ecotoxicology of the Centre of Marine and Environmental Research (CIIMAR). Involved in research on Ecotoxicology (biomarkers, contamination of estuarine environments by pharmaceutical residues, development of methodologies of toxicity evaluation; aquatic toxicology involving crustaceans, algal species and fish).
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Bruno André Nunes, B.Sc, Researcher as Ph.D. student in the Group of Ecotoxicology of the Centre of Marine and Environmental Research (CIIMAR). Involved in research on Ecotoxicology (biomarkers, contamination of estuarine environments by pharmaceutical residues, development of methodologies of toxicity evaluation; aquatic toxicology involving crustaceans, algal species and fish).
Félix Dias Carvalho, Ph.D., Auxiliar Professor at the Faculty of Pharmacy, University of Porto, where he is a lecturer of Ecotoxicology since 1998. His research is related with the evaluation of molecular mechanisms, both in basic toxicology and in ecotoxicology. He has published over 60 full papers related to these areas.
Lúcia Maria das Candeias Guilhermino, Ph.D., Associate Professor in the Institute of Biomedical Sciences of Abel Salazar, University of Porto, Portugal. Head of the Group of Ecotoxicology of the Centre of Marine and Environmental Research (CIIMAR). Involved in research on Ecotoxicology (biomarkers, aquatic ecotoxicology, methods of evaluation of toxicity; biomonitoring).