Aquatic Toxicology 159 (2015) 225–232
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Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox
N-acetyl--d-glucosaminidase activity in feral Carcinus maenas exposed to cadmium Sofia Raquel Mesquita a,b,∗ , S¸eyda Fikirdes¸ici Ergen c , Aurélie Pinto Rodrigues a,b , M. Teresa Oliva-Teles d , Cristina Delerue-Matos d , Laura Guimarães a,∗ a
Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), University of Porto, Rua dos Bragas 289, P 4050-123 Porto, Portugal ICBAS – Institute of Biomedical Sciences Abel Salazar, University of Porto, Rua Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal c Faculty of Science, Ankara University, Department of Biology, 06100 Tandogan, Ankara, Turkey d REQUIMTE, School of Engineering, Polytechnic Institute of Porto, Rua Dr. António Bernardino de Almeida 431, 4200-072 Porto, Portugal b
a r t i c l e
i n f o
Article history: Received 26 June 2014 Received in revised form 13 November 2014 Accepted 9 December 2014 Available online 17 December 2014 Keywords: Shore crab Chitobiase Glutathione Anti-oxidant defences Environmental monitoring
a b s t r a c t Cadmium is a priority hazardous substance, persistent in the aquatic environment, with the capacity to interfere with crustacean moulting. Moulting is a vital process dictating crustacean growth, reproduction and metamorphosis. However, for many organisms, moult disruption is difficult to evaluate in the short term, what limits its inclusion in monitoring programmes. N-acetyl--d-glucosaminidase (NAGase) is an enzyme acting in the final steps of the endocrine-regulated moulting cascade, allowing for the cast off of the old exoskeleton, with potential interest as a biomarker of moult disruption. This study investigated responses to waterborne cadmium of NAGase activity of Carcinus maenas originating from estuaries with different histories of anthropogenic contamination: a low impacted and a moderately polluted one. Crabs from both sites were individually exposed for seven days to cadmium concentrations ranging from 1.3 to 2000 g/L. At the end of the assays, NAGase activity was assessed in the epidermis and digestive gland. Detoxification, antioxidant, energy production, and oxidative stress biomarkers implicated in cadmium metabolism and tolerance were also assessed to better understand differential NAGase responses: activity of glutathione S-transferases (GST), glutathione peroxidase (GPx) glutathione reductase (GR), levels of total glutathiones (TG), lipid peroxidation (LPO), lactate dehydrogenase (LDH), and NADP+ -dependent isocitrate dehydrogenase (IDH). Animals from the moderately polluted estuary had lower NAGase activity both in the epidermis and digestive gland than in the low impacted site. NAGase activity in the epidermis and digestive gland of C. maenas from both estuaries was sensitive to cadmium exposure suggesting its usefulness for inclusion in monitoring programmes. However, in the digestive gland NAGase inhibition was found in crabs from the less impacted site but not in those from the moderately contaminated one. Altered glutathione levels were observed in cadmium-treated crabs from the contaminated site possibly conferring enhanced tolerance to these animals through its chelator action. Investigation of enhanced tolerance should thus be accounted for in monitoring programmes employing NAGase as biomarker to avoid data misinterpretation. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In recent years efforts have been made to promote the sustainable use of water and to reduce pollutant inputs in orderto protect
∗ Corresponding author at: Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), University of Porto, Rua dos Bragas 289, 4050-123 Porto, Portugal. Tel.: +351 223401800 E-mail addresses:
[email protected] (S.R. Mesquita),
[email protected] (L. Guimarães). http://dx.doi.org/10.1016/j.aquatox.2014.12.008 0166-445X/© 2014 Elsevier B.V. All rights reserved.
and improve the quality of the aquatic environment. The EU Water Framework Directive (WFD) regulates important aspects, related to the prevention of aquatic pollution, wastewater treatment efficiency and impact assessment of regulated and non-regulated substances, among others (Laane et al., 2012; Ruel et al., 2012). The metal cadmium has been identified as a priority hazardous substance by the European Union (Directive 2008/105/EC), representing a risk to human and environmental health. It is commonly found in aquatic systems, both from natural and anthropogenic origins (Jensen and Bro-Rasmussen, 1992), and is persistent in the environment requiring relentless understanding and detection of its detrimental effects.
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Assessing the presence and effects of aquatic contaminants requires long-term chemical and biological monitoring. In this context, it is particularly important to improve the range of costeffective parameters available to evaluate exposure and effects, and complement the data obtained with chemical analysis, providing an early detection of pollution problems (Hagger et al., 2008). Biomarkers have been recognised as strategic tools in the assessment of environmental quality of coastal waters giving a significant contribution to the weight-of-evidence approaches involved in risk assessment and classification of ecosystems’ status recognised by the WFD (Picado et al., 2007; Hagger et al., 2008). Biomarker responses reflect the impact of all the stressors to which the organisms may be exposed, providing warning signals about the health status of species. The use of multibiomarker approaches in invertebrates is also of marked importance. Not only do they represent more than 95% of the known animal species but also they supply keystone organisms for ecosystem function (SchulteOehlmann et al., 2004). Moreover, several of them, including crustaceans, have been used and are recommended to investigate contaminant effects and evaluate environmental quality (Moreno et al., 2003; Zou, 2005; Picado et al., 2007; Mesquita et al., 2011; Rodrigues et al., 2013, 2014). Crustacean moulting (ecdysis) is a periodic phase in the crustacean life-cycle, essential for metamorphosis, growth, and reproduction, that involves the cast-off of the old exoskeleton exposing the new soft one formed beneath. Ecdysis is under the control of a complex hormonal cascade. N-acetyl--dglucosaminidase (NAGase), or chitobiase, is the last chitinolytic enzyme of that pathway. It is responsible for the degradation of chitin oligosaccharides into monomers that will be re-absorbed into the new cuticle (Espie and Roff, 1995). Several studies have shown that crustacean moulting and/or NAGase activity may be inhibited by exposure to endocrine disrupting compounds such as polychlorinated biphenyls (PCBs), pesticides, 4-octylphenol, diethylphthalate, and several metals (Xie et al., 2004; Zou, 2005; Meng and Zou, 2009; Zhang et al., 2010). Recently, the levels of epidermal NAGase activity in Carcinus maenas were shown to vary in relation to the moult cycle (Mesquita et al., 2011). However, despite the ecological importance of C. maenas in estuarine and coastal food webs, and its sensitivity to chemical stress (Mesquita et al., 2011; Rodrigues et al., 2013, 2014; Rodrigues and Pardal, 2014), there is a lack of studies addressing the responses of its NAGase activity to priority contaminants. As a parameter measured at the sub-individual level, of relatively simple and cost-effective determination in tissues of exposed organisms, C. maenas NAGase activity could provide an early diagnostic tool for identifying exposure to chemical stress and assess the status of this species in relation to its environment. Earlier works reported adverse effects of cadmium on aquatic wildlife, including the modulation of crustacean ecdysis and NAGase activity. Reddy and Fingerman (1995) exposed the fiddler crab Uca pugilator to cadmium and registered alterations in brain and eyestalk neurosecretory tissues responsible for the regulation of ecdysis and other physiological processes. Moreno et al. (2003) observed moult arrest in the estuarine crab Chasmagnathus granulata triggered by cadmium exposure. Xie et al. (2004) showed that NAGase activity of the prawn Penaeus vannamei could be inhibited in vitro by cadmium. Hence, the aim of the present work was to investigate the responses of C. maenas NAGase activity to cadmium in relation to its tissue accumulation. Since C. maenas from different areas may exhibit differential sensitivity to pollution (Rodrigues et al., 2013, 2014), laboratory experiments were carried out with crabs originating from a low impacted and a moderately contaminated estuary, providing wider characterisation of NAGase interest for biomonitoring. Moreover, increased upregulation of detoxifying enzymes and molecules
Table 1 Physico-chemical parameters (mean ± SD) measured (in triplicate) in the local water during each sampling campaign. T, temperature (◦ C); Sal, salinity (psu); DO, dissolved oxygen (mg/L). Minho T pH Sal DO
14.1 7.9 8.2 9.5
± ± ± ±
Lima 0.13 0.2 2.4 1.9
14.1 7.7 17.6 9
± ± ± ±
0.16 0.3 3.6 2.9
involved in metal detoxification, often play a role in differential sensitivity of species to contaminants (Knapen et al., 2004, Morgan et al., 2007), possibly with associated metabolic costs. Therefore, to better understand NAGase responses in crabs from the two estuaries and the consequent implications for biomonitoring, we also included in this study biomarkers involved in the biotransformation process, anti-oxidant defences, oxidative stress and energy production, which are known to be involved in the response to cadmium exposure (Perrin and Watt, 1971; Manca et al., 1991; Hatcher et al., 1995; Stohs et al., 2000; Wang and Wang, 2009). These were the activities of enzymes glutathione peroxidase (GPx), glutathione S-transferases (GST), glutathione reductase (GR), total glutathione (TG), lactate dehydrogenase (LDH), NADP+ -dependent isocitrate dehydrogenase (IDH) activities, and lipid peroxidation (LPO) levels. 2. Materials and methods 2.1. Crab sampling and maintenance in the laboratory Crabs were collected at the mouth of the estuaries of the Minho and Lima rivers (Rodrigues et al., 2013) during autumn. The Minho estuary is under low human pressure, with low historical levels of metals and polycyclic aromatic hydrocarbons (PAHs) (Ferreira et al., 2003; Reis et al., 2009). Cadmium concentrations in the sampling site ranged between 6.4 and 8.9 g/g dw (Guimarães et al., unpublished data). The Lima estuary has several industries located nearby, and a harbour and a shipyard. It shows high population density and receives wastewaters of industrial, soil leaching, livestock and urban origin) (Ferreira et al., 2003). It is contaminated by metals, with cadmium concentrations in the sampling site ranging between 27 and 91.5 g/g dw (Guimarães et al., unpublished data). Male crabs (41.6 ± 0.97 mm, cephalothorax width; mean ± SD) in intermoult stage (C4) (Drach, 1939; O’Halloran and O’Dor, 1988) were captured using baited hand-nets. Water temperature, salinity, dissolved oxygen, and pH were measured, in triplicate, during each sampling, using a multiparametric sea gauge WTW multi 340i with the appropriate probes (pH SenTix 41 and Tetracon 325). The mean values, and corresponding standard deviation (SD), obtained for these parameters were similar for the two sampling sites (Table 1). The animals were immediately transported to the laboratory in thermally insulated boxes and acclimated for at least 14 days to laboratory conditions. During this period the crabs were kept individually in 2 L capacity glass beakers filled with filtered seawater (15 psu), in a temperature (16 ± 1 ◦ C) and photoperiod (14:10 h light:dark) controlled room. The recipients were covered and aeration was provided. Crabs were fed every other day, 1–2 h prior to medium renewal. 2.2. Chemicals Cadmium chloride (CdCl2 , Cas no. 10108-64-2) was purchased from Merck (Darmstadt, Germany), and the chemicals for enzymatic analyses were purchased from Sigma–Aldrich Chemical (Steinheim, Germany). The Bradford reagent was purchased from Bio-Rad (Munich, Germany). For cadmium analysis nitric acid (65%,
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Suprapur® , Merck, Darmstadt, Germany) was used; standard solutions were prepared daily by dilution of a cadmium stock solution (1000 mg/L; Panreac, Barcelona, Spain) in 0.5% (v/v) aqueous nitric acid solution. Monobasic ammonium phosphate (Merck, Darmstadt, Germany) was used as matrix modifier. The solutions were prepared with ultrapure water (resistivity = 18.2 M cm) obtained from a Simplicity 185 water purification system (Millipore, Molsheim, France). 2.3. Exposure experiments Organisms from both populations were exposed to five cadmium concentrations (1.3, 8, 50, 320 and 2000 g/L). Exposure levels were chosen to include both environmentally relevant concentrations and concentrations reported to cause accumulation in C. maenas (Bjerregaard, 1990). Stock solutions of cadmium were prepared in ultra-pure water and diluted with filtered seawater (15 psu) to prepare the test medium. Control groups prepared only with filtered seawater were included in the assays. Three replicates of each treatment (control and cadmium) were prepared for each population. In each replicate 4 crabs were individually exposed in 2 L glass beakers, under temperature, photoperiod and aeration conditions previously described for the acclimation period. Test solutions were renewed every other day. Water temperature, conductivity, salinity, pH, and dissolved oxygen (DO) were monitored during the assays in the old and the freshly prepared test solutions. No food was provided to the crabs during the assays. Samples of old (48 h) and freshly prepared test solutions of the highest exposure concentration were collected for determination of ionic cadmium. At the end of the assays crabs were ice anaesthetised and samples of epidermis (beneath the dorsal carapace), muscle (first pair of locomotor appendices) and digestive gland were collected for biomarker analysis. Each biomarker was determined in nine to twelve crabs per treatment. For each treatment, the remaining epidermis, muscle and digestive gland, together with the rest of the soft tissues of the crabs, hereafter referred to as whole-body soft tissues for simplicity, were collected, pooled, and used for the quantification of cadmium.
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(2013). A portion of epidermis and digestive gland of each crab was homogenised and centrifuged. The supernatant was recovered for the determination of NAGase activity (Mesquita et al., 2011). Muscle samples were homogenised in the respective buffers, centrifuged, and the supernatants recovered were used for the determination of LDH and IDH activities. Digestive gland was homogenised in K-phosphate buffer. An aliquot of homogenate was reserved for determination of LPO levels. The remaining homogenate was centrifuged and the post-mitochondrial supernatant recovered was used for the determination of GST, GPx and GR activities, and TG levels as well. Except for LPO, biomarkers were expressed as a function of the concentration of protein in the samples. Protein concentration was determined by the Bradford method (Bradford, 1976). Bovine ␥-globuline was used as standard. Spectrophotometric readings were performed in a Bio-Tek Power Wave 340 microplate reader or a Jasco 6405 UV/VIS spectrophotometer. 2.6. Data analysis Results of cadmium concentrations in water are presented as mean ± SD (standard deviation). All other results are presented as mean ± SE (standard error of the mean). Significant differences in the biomarker responses in relation to cadmium treatments and the estuary of origin of the crabs were first sought by factorial two-way analysis of variance (ANOVA), to test for significance of the interaction term. When significance of this term was found, one-way ANOVA with planned comparisons was performed for the interaction variable. ANOVA assumptions were tested using the Shapiro–Wilk and the Levene’s test. When appropriate, the logarithmic transformation was applied to the data in order to fulfil ANOVA assumptions. Significant differences were accepted for p < 0.05 in all tests performed. All statistical analyses were carried out in SPSS IBM v19.0. 3. Results 3.1. Cadmium in water samples and whole-body soft tissues
2.4. Determination of cadmium in water samples and whole-body soft tissues The cadmium quantifications were performed at 228.8018 nm with a High Resolution-Continuum Source Atomic Absorption Spectrophotometer (HR-CS-AAS, ContrAA 700, Analytik Jena, Jena, Germany) using electrothermal atomisation, after acid mineralisation by microwave-assisted digestion (MARS-X, CEM, Mathews, NC, USA) in HP-500 Plus Teflon vessels (CEM, Mathews, NC, USA), according to APHA recommendations (APHA, 1992). Soft tissues were lyophilised, ground and homogenised. The microwave acid digestion of soft tissue samples was performed using 130-mg portions and 10 mL of a 27% nitric acid solution (Reis and Almeida, 2008). For the digestion of the water samples 7-mL aliquots and 3 mL of a nitric acid solution (65%) were used (Reis and Almeida, 2008). The optimised microwave digestion temperature was 160 ◦ C (20 min) at a maximum pressure of 200 psi. The atomic adsorption parameters were optimised and were based on the guidelines provided by the equipment’s manufacturer. The external calibration method was used for cadmium quantification and the analytical procedure was checked using samples spiked with known amounts of cadmium. Satisfactory mean recoveries (90.5–114%) were obtained. All samples were analysed in triplicate. 2.5. Biomarkers All biomarker determinations were performed as described for C. maenas by Mesquita et al. (2011) and Rodrigues et al.
Throughout the experiments water parameters were kept stable in all test treatments. Dissolved oxygen and pH ranged between 9.0–9.2 mg/L and 7.7–7.8, respectively. Salinity varied between 15.0 and 15.2 and temperature ranged between 16.0 and 16.3 ◦ C. In freshly prepared medium of the highest test treatment (2000 g/L), measured dissolved cadmium was between 95% and 104% of the nominal concentration, corresponding to 1905 ± 50.2 g/L (mean ± SD) and 2076 ± 27.6 g/L, respectively. In the old exposure medium measured dissolved cadmium was 4% to 15% lower than in the freshly prepared one. At the end of the 7-day experiments a significant concentration-dependent accumulation of cadmium in the whole-body soft tissues was observed, which peaked at 89.6 ± 14.7 g/g dw (mean ± SD) in crabs from the Minho and at 99.9 ± 17.5 g/g dw in crabs from the Lima estuary exposed to 2000 g/L (Fig. 1). 3.2. Biomarkers 3.2.1. Moulting Crabs collected from the Lima estuary showed significantly lower epidermal NAGase activity (ca. 65% on average) than those collected from the Minho estuary (Table 2, Fig. 2). However, the response pattern of crabs from both estuaries was similar. High exposure concentrations (2000 g/L) significantly decreased NAGase activity (by 36% in Minho crabs and 45% in Lima crabs, p < 0.05).
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Fig. 1. Cadmium accumulation (mean ± SE) in whole body soft tissues of C. maenas from the Minho and the Lima estuaries after a 7-day exposure.
NAGase activity in the digestive gland was also significantly lower (about 40% on average) in crabs from the Lima than in those from the Minho estuary (Table 2, Fig. 2). Cadmium also caused a decrease of NAGase activity in Minho crabs, which was significantly lower than controls at 2000 g/L (−34%, p < 0.05). However, no significant differences among experimental groups were found for Lima crabs.
Table 2 Results of the full-factorial two-way ANOVAs carried out to assess the effects of cadmium and the sampling site on moulting and detoxification biomarkers of C. maenas. Parameter
Source of variation
Moulting NAGase Epidermis
Estuary Cadmium Estuary × cadmium Estuary NAGase Digestive gland Cadmium Estuary × cadmium Biotransformation and anti-oxidant defences Estuary Cadmium GST Estuary × cadmium
df
F
p
1, 106 5, 106 5, 106 1, 122 5, 122 5, 122
68.04 2.90 0.47 136.88 2.93 3.08
