doi:10.1111/jfd.12362
Journal of Fish Diseases 2016, 39, 295–308
Histological and hormonal changes in the European eel (Anguilla anguilla) after exposure to environmental cocaine concentration F Gay1,2, I Ferrandino1, A Monaco1, M Cerulo3, G Capasso1 and A Capaldo1 1 Department of Biology, University of Naples Federico II, Naples, Italy 2 Department of Chemistry and Biology, University of Salerno, Salerno, Italy 3 Department of Translational Medical Sciences, Pediatric Surgery, University Federico II, Naples, Naples, Italy
Abstract
The aim of this study was the assessment of histological and hormonal changes induced in the European eel from environmental concentrations of cocaine. Silver eels were exposed to 20 ng L 1 of cocaine during 50 days; at the same time, control, vehicle control and two post-exposure recovery groups (3 and 10 days) were made. The general morphology of the skin and the intestine, and the plasma levels of prolactin, cortisol and dopamine were evaluated. In the skin, cocaine decreased the number and size of mucous cells, increased the thickness of the epidermis and altered the club cells and the basal lamina. In the intestine, cocaine increased the thickness of the epithelium and the number of mucous cells and reactivated the structure of the intestine and of the intestinal musculature. Moreover, cocaine increased plasma prolactin, cortisol and dopamine levels. These results suggest that cocaine induced histological changes, directly and/or through the hormonal changes observed. Considering the complex life cycle of the eel, the changes induced by cocaine in the skin, the intestine and the endocrine system could threaten the ability of the eel to successfully migrate and reproduce. Keywords: cocaine and eel histology, cocaine and eel hormones, cocaine and fish, eel skin and Correspondence A Capaldo, Department of Biology, University Federico II, Via Mezzocannone 8, 80134 Naples, Italy (e-mails: [email protected] and [email protected]) Ó 2015 John Wiley & Sons Ltd
295
intestine, environmental cocaine, environmental illicit drugs.
Introduction
There is broad evidence that the major drugs of abuse enter the sewage network contaminating the receiving surface waters (Zuccato et al. 2005, 2008; Postigo, de Alda & Barcelo 2010; Daughton 2011; Pal et al. 2013; Rosi-Marshall et al. 2014). Although their strong pharmacological activities (Rehm, Taylor & Room 2006; Pal et al. 2013) let foresee toxic effects to the aquatic organisms, only few studies have been performed to date, and the only data concern the cytotoxic and genotoxic (Binelli et al. 2012), and sublethal (Parolini et al. 2013) effects induced in the freshwater mussel Dreissena polymorpha by environmental cocaine, and cocaine metabolite benzoylecgonine, respectively. As data about the effects of environmental illicit drugs on fish were lacking, a study was started to evaluate and compare the effects on fish of the most frequent drugs in surface waters. In this perspective, the different metabolites, generally widespread in the aquatic environment, were not evaluated. The first studies showed that the European eels (Anguilla anguilla) chronically exposed to environmental cocaine concentrations bioaccumulate cocaine in their tissues (Capaldo et al. 2012) and undergo endocrine alterations (Gay et al. 2013). These results let foresee histological changes as well; therefore, in this study,
F Gay et al. Eel changes induced by cocaine
Journal of Fish Diseases 2016, 39, 295–308
the epidermis and the intestine of the eels were examined. Histological changes have been widely used as biomarkers in the evaluation of the health of fish exposed to contaminants (Teh, Adams & Hinton 1997; Pacheco & Santos 2002; Thophon et al. 2003; Camargo & Martinez 2007; Capkin, Birinciogl & Altinok 2009; Ebrahimi & Taherianfard 2011). Moreover, the histological biomarkers allow examining specific organs, playing key role in the organism physiology, are easy to identify and serve as warning signs of damage to animal health (Fanta et al. 2003; Camargo & Martinez 2007). The epidermis and the intestine were chosen because they bioaccumulate cocaine after a chronic exposure (Capaldo et al. 2012). Together with gills, the epidermis and the intestine are the first interfaces of the organism exposed to the aquatic environment and therefore a primary target for the action of environmental pollutants on fish. The epidermis is the major protective barrier of the organism to its external environment, enabling fish to move on or between hard substrata, or to dig into soft bottoms with minimal abrasion to their body (Fishelson 1996; Burkhardt-Holm, Wahli & Meier 2000). The intestine plays a key role in feeding (Tesch 2003) and osmoregulation, especially in sea water (Evan 1993; Loretz 1995; Bentley 2002; Lionetto et al. 2005; Marshall & Grosell 2006; Kim et al. 2008). Moreover, both the epidermis and the intestinal epithelium produce mucus having predominantly protective functions (Domeneghini et al. 2005) and are very sensitive to environmental contaminants (Braunbeck & Appelbaum 1999; Burkhardt-Holm et al. 2000; Rosety et al. 2001; Pacheco & Santos 2002; Capkin et al. 2009; Geeraerts & Belpaire 2010; Jeheshadevi et al. 2014). In teleosts, prolactin (PRL) plays an important role in osmotic regulation in fresh water. It regulates ion and water movements in intestine, skin, gills, kidney and urinary bladder and stimulates mucus secretion by skin, intestine and gill (Norris 2007; Breves, McCormick & Karlstrom 2014). Moreover, in fish, PRL is involved in epithelial cell proliferation (Sakamoto & McCormick 2006; Takahashi, Takahashi & Sakamoto 2006; Takahashi et al. 2013), together with cortisol, the stress hormone involved in both epithelial cell proliferation and apoptosis (Iger et al. 1995; Van der Salm, Nolan & Wendeelar Bonga 2002; Sakamoto & McCormick 2006; Takahashi et al. 2006). For this reason, plasma PRL and cortisol levels Ó 2015 John Wiley & Sons Ltd
296
were evaluated; moreover, as in teleosts dopamine (DA), the main mediator of cocaine effects (Nestler & Malenka 2004), inhibits PRL release (Norris 2007), DA levels were evaluated too.
Materials and methods
Chemicals Cocaine free-base was purchased from SigmaAldrich Inc. Ethyl 3-aminobenzoate, methanesulfonic acid salt 98% (MS-222) was purchased from Aldrich Chemical Corporation Inc. Animals Adult specimens of European eel (Anguilla anguilla) (38.85 0.39 cm; 85.38 1.60 g; Mean SD) (silver eel stage) were obtained from a local fish dealer. They were acclimated to the laboratory for 1 month, as previously described (Capaldo et al. 2012), and kept in 300-L glass aquaria under a natural photoperiod, in dechlorinated, well-aerated tap water, with the following physicochemical conditions: salinity 0, ammonia
Journal of Fish Diseases 2016, 39, 295–308
Histological and hormonal changes in the European eel (Anguilla anguilla) after exposure to environmental cocaine concentration F Gay1,2, I Ferrandino1, A Monaco1, M Cerulo3, G Capasso1 and A Capaldo1 1 Department of Biology, University of Naples Federico II, Naples, Italy 2 Department of Chemistry and Biology, University of Salerno, Salerno, Italy 3 Department of Translational Medical Sciences, Pediatric Surgery, University Federico II, Naples, Naples, Italy
Abstract
The aim of this study was the assessment of histological and hormonal changes induced in the European eel from environmental concentrations of cocaine. Silver eels were exposed to 20 ng L 1 of cocaine during 50 days; at the same time, control, vehicle control and two post-exposure recovery groups (3 and 10 days) were made. The general morphology of the skin and the intestine, and the plasma levels of prolactin, cortisol and dopamine were evaluated. In the skin, cocaine decreased the number and size of mucous cells, increased the thickness of the epidermis and altered the club cells and the basal lamina. In the intestine, cocaine increased the thickness of the epithelium and the number of mucous cells and reactivated the structure of the intestine and of the intestinal musculature. Moreover, cocaine increased plasma prolactin, cortisol and dopamine levels. These results suggest that cocaine induced histological changes, directly and/or through the hormonal changes observed. Considering the complex life cycle of the eel, the changes induced by cocaine in the skin, the intestine and the endocrine system could threaten the ability of the eel to successfully migrate and reproduce. Keywords: cocaine and eel histology, cocaine and eel hormones, cocaine and fish, eel skin and Correspondence A Capaldo, Department of Biology, University Federico II, Via Mezzocannone 8, 80134 Naples, Italy (e-mails: [email protected] and [email protected]) Ó 2015 John Wiley & Sons Ltd
295
intestine, environmental cocaine, environmental illicit drugs.
Introduction
There is broad evidence that the major drugs of abuse enter the sewage network contaminating the receiving surface waters (Zuccato et al. 2005, 2008; Postigo, de Alda & Barcelo 2010; Daughton 2011; Pal et al. 2013; Rosi-Marshall et al. 2014). Although their strong pharmacological activities (Rehm, Taylor & Room 2006; Pal et al. 2013) let foresee toxic effects to the aquatic organisms, only few studies have been performed to date, and the only data concern the cytotoxic and genotoxic (Binelli et al. 2012), and sublethal (Parolini et al. 2013) effects induced in the freshwater mussel Dreissena polymorpha by environmental cocaine, and cocaine metabolite benzoylecgonine, respectively. As data about the effects of environmental illicit drugs on fish were lacking, a study was started to evaluate and compare the effects on fish of the most frequent drugs in surface waters. In this perspective, the different metabolites, generally widespread in the aquatic environment, were not evaluated. The first studies showed that the European eels (Anguilla anguilla) chronically exposed to environmental cocaine concentrations bioaccumulate cocaine in their tissues (Capaldo et al. 2012) and undergo endocrine alterations (Gay et al. 2013). These results let foresee histological changes as well; therefore, in this study,
F Gay et al. Eel changes induced by cocaine
Journal of Fish Diseases 2016, 39, 295–308
the epidermis and the intestine of the eels were examined. Histological changes have been widely used as biomarkers in the evaluation of the health of fish exposed to contaminants (Teh, Adams & Hinton 1997; Pacheco & Santos 2002; Thophon et al. 2003; Camargo & Martinez 2007; Capkin, Birinciogl & Altinok 2009; Ebrahimi & Taherianfard 2011). Moreover, the histological biomarkers allow examining specific organs, playing key role in the organism physiology, are easy to identify and serve as warning signs of damage to animal health (Fanta et al. 2003; Camargo & Martinez 2007). The epidermis and the intestine were chosen because they bioaccumulate cocaine after a chronic exposure (Capaldo et al. 2012). Together with gills, the epidermis and the intestine are the first interfaces of the organism exposed to the aquatic environment and therefore a primary target for the action of environmental pollutants on fish. The epidermis is the major protective barrier of the organism to its external environment, enabling fish to move on or between hard substrata, or to dig into soft bottoms with minimal abrasion to their body (Fishelson 1996; Burkhardt-Holm, Wahli & Meier 2000). The intestine plays a key role in feeding (Tesch 2003) and osmoregulation, especially in sea water (Evan 1993; Loretz 1995; Bentley 2002; Lionetto et al. 2005; Marshall & Grosell 2006; Kim et al. 2008). Moreover, both the epidermis and the intestinal epithelium produce mucus having predominantly protective functions (Domeneghini et al. 2005) and are very sensitive to environmental contaminants (Braunbeck & Appelbaum 1999; Burkhardt-Holm et al. 2000; Rosety et al. 2001; Pacheco & Santos 2002; Capkin et al. 2009; Geeraerts & Belpaire 2010; Jeheshadevi et al. 2014). In teleosts, prolactin (PRL) plays an important role in osmotic regulation in fresh water. It regulates ion and water movements in intestine, skin, gills, kidney and urinary bladder and stimulates mucus secretion by skin, intestine and gill (Norris 2007; Breves, McCormick & Karlstrom 2014). Moreover, in fish, PRL is involved in epithelial cell proliferation (Sakamoto & McCormick 2006; Takahashi, Takahashi & Sakamoto 2006; Takahashi et al. 2013), together with cortisol, the stress hormone involved in both epithelial cell proliferation and apoptosis (Iger et al. 1995; Van der Salm, Nolan & Wendeelar Bonga 2002; Sakamoto & McCormick 2006; Takahashi et al. 2006). For this reason, plasma PRL and cortisol levels Ó 2015 John Wiley & Sons Ltd
296
were evaluated; moreover, as in teleosts dopamine (DA), the main mediator of cocaine effects (Nestler & Malenka 2004), inhibits PRL release (Norris 2007), DA levels were evaluated too.
Materials and methods
Chemicals Cocaine free-base was purchased from SigmaAldrich Inc. Ethyl 3-aminobenzoate, methanesulfonic acid salt 98% (MS-222) was purchased from Aldrich Chemical Corporation Inc. Animals Adult specimens of European eel (Anguilla anguilla) (38.85 0.39 cm; 85.38 1.60 g; Mean SD) (silver eel stage) were obtained from a local fish dealer. They were acclimated to the laboratory for 1 month, as previously described (Capaldo et al. 2012), and kept in 300-L glass aquaria under a natural photoperiod, in dechlorinated, well-aerated tap water, with the following physicochemical conditions: salinity 0, ammonia
