Behaviour and biomarkers of oxidative stress in Gambusia holbrooki after acute exposure to widely used pharmaceuticals and a detergent
Introduction
Biomarkers of oxidative stress have been largely used for the assessment of effects induced by several classes of chemical contaminants on organisms. The assessment of alterations on key enzymatic activities of sentinel species following exposure to contaminated waters has been one of the major uses of biomarkers in environmental studies. A review by Livingstone (2001) reports the role of biomarkers of oxidative stress in Ecotoxicology, showing the main perspectives for the future evolution and purpose of this special class of biomarkers. Oxidative stress biomarkers comply with basic requirements, such as: responsiveness; low cost; simple procedures; applicability under varied testing conditions; and sensitivity to a high number of environmental contaminants. Several authors used oxidative stress parameters as non-specific end-points to assess the effects of single chemicals and complex mixtures of aquatic environmental contaminants. For example, they have been used in tissues of the fish Wallaga attu (Pandey et al., 2003) and Catostomus commersoni (Oakes and Van Der Kraak, 2003) to evaluate the effects of complex mixtures, in tissues of Nile tilapia, Oreochromis niloticus, to evaluate the effects of cadmium (Almeida et al., 2002) and to evaluate the effects of mercury on Salmo salar (Berntssen et al., 2003).
A large number of bioindicators and test-organisms have been suggested for the evaluation of ecotoxicity of anthropogenic compounds on aquatic ecosystems. Among them, the majority are freshwater species and considerably less studies have been performed with saltwater species despite their importance to assess the possible impact of chemicals in estuaries and brackish water environments. The use of Gambusia holbrooki in Ecotoxicology is due to its intrinsic characteristics, as well as additional features related to laboratory conditions of maintenance and testing: G. holbrooki, commonly known as mosquitofish, is an euryhaline organism that is able to easily adapt to varied environmental conditions. Therefore, it is widely distributed in both freshwater systems and estuaries of temperate regions, including Portuguese rivers and estuaries. Besides its wide geographical distribution, this fish is characterized by high fecundity and can be considered a secondary consumer in aquatic food webs. Furthermore, this species is not only abundant but is also easy to capture. In addition, keeping this species under laboratory-controlled conditions is relatively easy. A large number of fish can be reared in small aquaria or tanks, due to the small body size (male size between 2 and 2.5 cm long).
Therapeutic agents and personal care products, including detergents used in several formulations, are intensively and continuously used. Some of them are resistant to metabolic degradation and are lipophilic. These two factors contribute for the presence of these substances in the environment and in animal tissues after their use. These compounds may show biological activity in the environment (Jones et al., 2002; Miao et al., 2002; Daughton and Ternes, 1999; Halling-Sørensen et al., 1998) and induce harmful effects on wild organisms even at very low concentrations. In addition, synergistic effects may occur as a result of the simultaneous exposure to several different compounds (Cleuvers, 2003). Therefore, pharmaceutical and personal care products can be considered as environmental contaminants of particular concern.
Diazepam is a benzodiazepine, currently used in human and veterinary therapeutics. This compound has anxiolytic, sedative and muscle relaxing effects. These pharmacological activities result from the enhancement of GABAergic transmission at benzodiazepine-sensitive GABAA-receptors (Mohler et al., 1996). However, diazepam has also been shown to influence cellular redox systems (Musavi and Kakkar, 1998, Musavi and Kakkar, 2003), an effect not fully understood. The presence of diazepam at concentrations up to 0.04 μg l−1 has been reported in effluents from a large number of German sewage treatment plants (Ternes, 1998) and at concentrations ranging from 0.7 to 1.2 ng l−1 in River Po, Italy (Zuccato et al., 2000).
The fibrate clofibrate was widely used in many countries as a blood lipid regulator before being withdrawn from human therapeutics in most countries of Western Europe. In spite of having being withdrawn, it is still being detected in effluents from sewage treatment plants in concentrations up to 0.8 μg l−1 (Andreozzi et al., 2003). The pharmacological activity of clofibrate and related compounds occurs through activation of the nuclear peroxisome proliferator activated receptors (PPARs). This activation leads to the transcriptional activation of the genes encoding for the peroxisomal oxidation system and cytochrome P450 CYP4A isoforms (mainly CYP4A1 and CYP4A3). Hyperproduction of hydrogen peroxide is favoured by the hepatic expression of hydrogen peroxide-generating peroxisomal fatty acyl-CoA oxidase, followed by a compensatory mechanism of disproportionate increase in the activity of hydrogen peroxide-degrading enzymes, such as catalase. The toxic effects of hydrogen peroxide are consequent to: (a) the mentioned overproduction and to (b) a simultaneous decrease in the activity of glutathione peroxidase (GPx) (Yeldandi et al., 2000). Clofibrate has been found to cause oxidative stress in experimental animal models, such as rodents (Cai et al., 1995; Qu et al., 2001), probably due to an exacerbated hydrogen peroxide production. Clofibrate exposure was also related with significantly elevated levels of hydrogen peroxide in the red algae Kappaphycus alvarezii and in the medium surrounding the exposed algae (Barros et al., 2003).
Clofibric acid is the main pharmacological active derivative of clofibrate and several other fibrates. This compound has an estimated persistence of 21 years in the environment (Buser et al., 1998). Besides the above-referred effects attributed to clofibrate, clofibric acid is also considered a potential endocrine disrupter, since it interferes with the synthesis of cholesterol (Pfluger and Dietrich, 2001). The low efficacy of sewage treatment plants in removing clofibric acid allows its entrance in the aquatic environment. In fact, during a normal passage through a sewage treatment plant, only 50% of the initial amount of clofibric acid is effectively removed (Ternes, 1998). Therefore, clofibric acid has been found at concentrations of 270 ng l−1 in tap water (Heberer, 2002), of 0.55 μg l−1 in surface waters of Swiss lakes (Buser et al., 1998), of 103 ng l−1 in Detroit River water (Boyd et al., 2003), of 18 ng l−1 in the estuary of the River Elba, from 0.28 to 1.35 ng l−1 in North Sea water (Weigel et al., 2002), of 1.6 μg l−1 in the majority of German sewage treatment plants (Ternes, 1998), and of 5 ng l−1 in effluents of Greek sewage treatment plants (Koutsouba et al., 2003).
Sodium dodecylsulphate (SDS) is a widely used detergent, contained for example in household products, industrial mixtures and cosmetic and toiletry products, including cleansing creams, liquid soaps and shampoos, bubble baths, bath and shower gels, and tooth pastes (Sirisattha et al., 2004). Its continuous input into the environment can be responsible for the high concentrations that have been found in specific areas. For example, concentrations between 0.2 and 10 mg l−1 of SDS were reported in irrigation fields contaminated with wastewater (Dizer, 1990). This compound has been shown to increase surface tension of phosphatidylcholine monolayers (Cserháti et al., 2002). Therefore, it is of utmost importance to ascertain its effects on cellular processes involving phospholipids.
The aim of the present study was to investigate the effects of diazepam, clofibrate, acid clofibric, and SDS on oxidative stress parameters of G. holbrooki. Oxidative stress enzymatic biomarkers were measured in hepatic and gill tissues, in order to investigate patterns of response in these tissues and to quantify the extent of alterations caused by the mentioned compounds. Lipid peroxidation was also quantified in both tissues, as well as the oxidative status of the intracellular glutathione pool. The choice of using liver for analytical procedures is justified by the fact that liver is the major organ involved in detoxification in vertebrates. Effects on gills were also investigated, due their role as a primary barrier against the entrance of xenobiotics into the body and probably also as a first line of detoxification and elimination of harmful agents. Considering that diazepam is a well-known central nervous system depressant, swimming behaviour was also evaluated in the experiments with this xenobiotic.
Section snippets
Fish 96-h exposure
G. holbrooki males were captured in the estuary of the Minho River (North of Portugal), at mean salinity of 6 g l−1. This estuary has been used as a reference estuary in previous studies of our working group, since it is considered a low impacted one (Ferreira et al., 2003; Antelo et al., 1996). Fish were captured using hand nets, immediately transported to the laboratory, and kept in ASTM hard water medium (ASTM, 1980) supplemented with 6 g l−1 of sodium chloride. Males were separated from
Results
To simplify the analysis of the results, for local (tissues), L and G denote liver and gills, respectively.
Diazepam
For enzyme e, local l, and concentration c, the model iswith residuals ε∼N(0,Σ), which is equivalent towhere ε′=eε∼LN(0,Σ). The covariance matrix Σ considered different standard deviations for (the residuals corresponding to) some enzymes: all enzymes were grouped except GRed and Cu–ZnSOD, which were left alone.
Estimates for the parameters in (1) are: a(L)=−0.70±0.141 (P=0.000), a(G)=−0.64±0.141 (P=0.000), b(GRed)=0, b
Clofibrate
For enzyme e, local l, and concentration c, the model iswith residuals ε∼N(0,Σ), which is equivalent towhere ε′=eε∼LN(0,Σ). The covariance matrix Σ considered: (a) different standard deviations according to the enzyme: all enzymes were grouped except GSSG, which was left alone; (b) the same correlation coefficient for each 2×2 matrix of individual/enzyme.
Estimates for the parameters in (3) are: a(GRed)=4.75±1.609
Clofibric acid
For enzyme e, local l, and concentration c, the model iswith residuals ε∼N(0,Σ), which is equivalent towhere ε′=eε∼LN(0,Σ). The covariance matrix Σ considered: (a) different standard deviations according to the enzyme: all enzymes were grouped except GRed and Cu–ZnSOD, which were left alone; (b) the same correlation coefficient for each 2×2 matrix of individual/enzyme.
Estimates for the parameters in (5) are: a
SDS
For enzyme e, local l, and concentration c, the model iswith residuals ε∼N(0,Σ), which is equivalent towhere ε′=eε∼LN(0,Σ). The covariance matrix Σ considered different standard deviations according to the enzyme: all enzymes were grouped except GSSG and Cu–ZnSOD, which were coupled together.
Estimates for the parameters in (7) are: a(L)=−0.21±0.081 (P=0.009), a(G)=−0.23±0.081 (P=0.005), b(L)=0.05±0.020 (P
Diazepam
Our study showed a significant increase in the activity of GRed with the rise in diazepam concentration, both in gill and liver tissue suggesting an oxidative stress-related adaptative response. Similarly, the selenium-dependent fraction of GPx was also shown to be significantly increased in both gills and liver of exposed organisms. Manganese SOD was also elevated, but only in hepatic tissues of G. holbrooki.
Musavi and Kakkar (1998) assessed the GRed levels in the brains of rats to which were
Conclusion
Our conclusions point to an overall diminished oxidative-related stress response caused by SDS and diazepam. However, males of G. holbrooki exhibited a highly responsive abnormal behaviour following diazepam exposure. Oxidative-based alterations were observed for clofibric acid, and, mainly, clofibrate, with modifications at several biomarkers. In spite of the significant responses, their ecological relevance may be low, since actual concentrations in the ecosystem are in a much lower order of
Acknowledgments
The present work was partially funded by “Fundação para a Ciência e a Tecnologia” (B. Nunes Ph.D. Grant SFRH/BD/866/2000), by Project “CONTROL” (POCTI/MAR/MAR/15266/1999) and by the European Fund FEDER.
We would like to gratefully acknowledge Miss Muriel Harkes for her dedicated effort and valuable contributions during the preparation and revision of the present paper.
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