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Aquatic Toxicology 159 (2015) 225–232

Contents lists available at ScienceDirect

Aquatic Toxicology

j ourna l ho me pa ge: www.elsev ier .com/ locate /aquatox

-acetyl-�-d-glucosaminidase activity in feral Carcinus maenasxposed to cadmium

ofia Raquel Mesquitaa,b,∗, S eyda Fikirdes ici Ergenc, Aurélie Pinto Rodriguesa,b,. Teresa Oliva-Telesd, Cristina Delerue-Matosd, Laura Guimarãesa,∗

Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), University of Porto, Rua dos Bragas 289, P 4050-123 Porto, PortugalICBAS – Institute of Biomedical Sciences Abel Salazar, University of Porto, Rua Jorge Viterbo Ferreira 228, 4050-313 Porto, PortugalFaculty of Science, Ankara University, Department of Biology, 06100 Tandogan, Ankara, TurkeyREQUIMTE, School of Engineering, Polytechnic Institute of Porto, Rua Dr. António Bernardino de Almeida 431, 4200-072 Porto, Portugal

r t i c l e i n f o

rticle history:eceived 26 June 2014eceived in revised form3 November 2014ccepted 9 December 2014vailable online 17 December 2014

eywords:hore crabhitobiaselutathionenti-oxidant defencesnvironmental monitoring

a b s t r a c t

Cadmium is a priority hazardous substance, persistent in the aquatic environment, with the capacity tointerfere with crustacean moulting. Moulting is a vital process dictating crustacean growth, reproductionand metamorphosis. However, for many organisms, moult disruption is difficult to evaluate in the shortterm, what limits its inclusion in monitoring programmes. N-acetyl-�-d-glucosaminidase (NAGase) is anenzyme acting in the final steps of the endocrine-regulated moulting cascade, allowing for the cast off ofthe old exoskeleton, with potential interest as a biomarker of moult disruption. This study investigatedresponses to waterborne cadmium of NAGase activity of Carcinus maenas originating from estuaries withdifferent histories of anthropogenic contamination: a low impacted and a moderately polluted one. Crabsfrom both sites were individually exposed for seven days to cadmium concentrations ranging from 1.3 to2000 �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 cadmiummetabolism and tolerance were also assessed to better understand differential NAGase responses: activityof glutathione S-transferases (GST), glutathione peroxidase (GPx) glutathione reductase (GR), levels oftotal glutathiones (TG), lipid peroxidation (LPO), lactate dehydrogenase (LDH), and NADP+-dependentisocitrate dehydrogenase (IDH). Animals from the moderately polluted estuary had lower NAGase activityboth in the epidermis and digestive gland than in the low impacted site. NAGase activity in the epidermisand digestive gland of C. maenas from both estuaries was sensitive to cadmium exposure suggesting itsusefulness 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 possiblyconferring enhanced tolerance to these animals through its chelator action. Investigation of enhancedtolerance should thus be accounted for in monitoring programmes employing NAGase as biomarker toavoid data misinterpretation.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

In recent years efforts have been made to promote the sustain-ble use of water and to reduce pollutant inputs in orderto protect

∗ Corresponding author at: Interdisciplinary Centre of Marine and Environmentalesearch (CIIMAR/CIMAR), University of Porto, Rua dos Bragas 289, 4050-123 Porto,ortugal. Tel.: +351 223401800

E-mail addresses: smesquita@ciimar.up.pt (S.R. Mesquita),guimaraes@ciimar.up.pt (L. Guimarães).

ttp://dx.doi.org/10.1016/j.aquatox.2014.12.008166-445X/© 2014 Elsevier B.V. All rights reserved.

and improve the quality of the aquatic environment. The EU WaterFramework Directive (WFD) regulates important aspects, relatedto the prevention of aquatic pollution, wastewater treatment effi-ciency and impact assessment of regulated and non-regulatedsubstances, among others (Laane et al., 2012; Ruel et al., 2012).The metal cadmium has been identified as a priority hazardoussubstance by the European Union (Directive 2008/105/EC), repre-senting a risk to human and environmental health. It is commonly

found in aquatic systems, both from natural and anthropogenic ori-gins (Jensen and Bro-Rasmussen, 1992), and is persistent in theenvironment requiring relentless understanding and detection ofits detrimental effects.

2 c Toxicology 159 (2015) 225–232

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Table 1Physico-chemical parameters (mean ± SD) measured (in triplicate) in the local waterduring each sampling campaign. T, temperature (◦C); Sal, salinity (psu); DO, dis-solved oxygen (mg/L).

Minho Lima

T 14.1 ± 0.13 14.1 ± 0.16

26 S.R. Mesquita et al. / Aquati

Assessing the presence and effects of aquatic contaminantsequires long-term chemical and biological monitoring. In thisontext, it is particularly important to improve the range of cost-ffective parameters available to evaluate exposure and effects,nd complement the data obtained with chemical analysis, provid-ng an early detection of pollution problems (Hagger et al., 2008).iomarkers have been recognised as strategic tools in the assess-ent of environmental quality of coastal waters giving a significant

ontribution to the weight-of-evidence approaches involved inisk assessment and classification of ecosystems’ status recognisedy the WFD (Picado et al., 2007; Hagger et al., 2008). Biomarkeresponses reflect the impact of all the stressors to which therganisms may be exposed, providing warning signals about theealth status of species. The use of multibiomarker approaches

n invertebrates is also of marked importance. Not only do theyepresent more than 95% of the known animal species but alsohey supply keystone organisms for ecosystem function (Schulte-ehlmann et al., 2004). Moreover, several of them, includingrustaceans, have been used and are recommended to investigateontaminant effects and evaluate environmental quality (Morenot al., 2003; Zou, 2005; Picado et al., 2007; Mesquita et al., 2011;odrigues et al., 2013, 2014).

Crustacean moulting (ecdysis) is a periodic phase in therustacean life-cycle, essential for metamorphosis, growth, andeproduction, that involves the cast-off of the old exoskele-on exposing the new soft one formed beneath. Ecdysis isnder the control of a complex hormonal cascade. N-acetyl-�-d-lucosaminidase (NAGase), or chitobiase, is the last chitinolyticnzyme of that pathway. It is responsible for the degradation ofhitin oligosaccharides into monomers that will be re-absorbednto the new cuticle (Espie and Roff, 1995). Several studies havehown that crustacean moulting and/or NAGase activity may benhibited by exposure to endocrine disrupting compounds suchs polychlorinated biphenyls (PCBs), pesticides, 4-octylphenol,iethylphthalate, and several metals (Xie et al., 2004; Zou, 2005;eng and Zou, 2009; Zhang et al., 2010). Recently, the levels of epi-

ermal NAGase activity in Carcinus maenas were shown to varyn relation to the moult cycle (Mesquita et al., 2011). However,espite the ecological importance of C. maenas in estuarine andoastal food webs, and its sensitivity to chemical stress (Mesquitat al., 2011; Rodrigues et al., 2013, 2014; Rodrigues and Pardal,014), there is a lack of studies addressing the responses of itsAGase activity to priority contaminants. As a parameter measuredt the sub-individual level, of relatively simple and cost-effectiveetermination in tissues of exposed organisms, C. maenas NAGasectivity could provide an early diagnostic tool for identifying expo-ure to chemical stress and assess the status of this species inelation to its environment.

Earlier works reported adverse effects of cadmium on aquaticildlife, including the modulation of crustacean ecdysis andAGase activity. Reddy and Fingerman (1995) exposed the fiddlerrab Uca pugilator to cadmium and registered alterations in brainnd eyestalk neurosecretory tissues responsible for the regulationf ecdysis and other physiological processes. Moreno et al. (2003)bserved moult arrest in the estuarine crab Chasmagnathus granu-

ata triggered by cadmium exposure. Xie et al. (2004) showed thatAGase activity of the prawn Penaeus vannamei could be inhibited

n vitro by cadmium. Hence, the aim of the present work was tonvestigate the responses of C. maenas NAGase activity to cadmiumn relation to its tissue accumulation.

Since C. maenas from different areas may exhibit differentialensitivity to pollution (Rodrigues et al., 2013, 2014), labora-

ory experiments were carried out with crabs originating from aow impacted and a moderately contaminated estuary, providing

ider characterisation of NAGase interest for biomonitoring. More-ver, increased upregulation of detoxifying enzymes and molecules

pH 7.9 ± 0.2 7.7 ± 0.3Sal 8.2 ± 2.4 17.6 ± 3.6DO 9.5 ± 1.9 9 ± 2.9

involved in metal detoxification, often play a role in differentialsensitivity of species to contaminants (Knapen et al., 2004, Morganet al., 2007), possibly with associated metabolic costs. Therefore, tobetter understand NAGase responses in crabs from the two estu-aries and the consequent implications for biomonitoring, we alsoincluded in this study biomarkers involved in the biotransforma-tion process, anti-oxidant defences, oxidative stress and energyproduction, which are known to be involved in the response tocadmium 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), totalglutathione (TG), lactate dehydrogenase (LDH), NADP+-dependentisocitrate 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 Minhoand Lima rivers (Rodrigues et al., 2013) during autumn. The Minhoestuary is under low human pressure, with low historical levelsof metals and polycyclic aromatic hydrocarbons (PAHs) (Ferreiraet al., 2003; Reis et al., 2009). Cadmium concentrations in the samp-ling site ranged between 6.4 and 8.9 �g/g dw (Guimarães et al.,unpublished data). The Lima estuary has several industries locatednearby, and a harbour and a shipyard. It shows high populationdensity and receives wastewaters of industrial, soil leaching, live-stock and urban origin) (Ferreira et al., 2003). It is contaminated bymetals, with cadmium concentrations in the sampling site rangingbetween 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 eachsampling, using a multiparametric sea gauge WTW multi 340i withthe appropriate probes (pH SenTix 41 and Tetracon 325). The meanvalues, and corresponding standard deviation (SD), obtained forthese parameters were similar for the two sampling sites (Table 1).

The animals were immediately transported to the laboratoryin thermally insulated boxes and acclimated for at least 14 daysto laboratory conditions. During this period the crabs were keptindividually in 2 L capacity glass beakers filled with filtered seawa-ter (15 psu), in a temperature (16 ± 1 ◦C) and photoperiod (14:10 hlight:dark) controlled room. The recipients were covered and aer-ation was provided. Crabs were fed every other day, 1–2 h prior tomedium renewal.

2.2. Chemicals

Cadmium chloride (CdCl2, Cas no. 10108-64-2) was purchased

from Merck (Darmstadt, Germany), and the chemicals for enzy-matic analyses were purchased from Sigma–Aldrich Chemical(Steinheim, Germany). The Bradford reagent was purchased fromBio-Rad (Munich, Germany). For cadmium analysis nitric acid (65%,

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uprapur®, Merck, Darmstadt, Germany) was used; standard solu-ions were prepared daily by dilution of a cadmium stock solution1000 mg/L; Panreac, Barcelona, Spain) in 0.5% (v/v) aqueous nitriccid solution. Monobasic ammonium phosphate (Merck, Darm-tadt, Germany) was used as matrix modifier. The solutions wererepared with ultrapure water (resistivity = 18.2 M� cm) obtainedrom a Simplicity 185 water purification system (Millipore, Mol-heim, France).

.3. Exposure experiments

Organisms from both populations were exposed to five cad-ium concentrations (1.3, 8, 50, 320 and 2000 �g/L). Exposure

evels were chosen to include both environmentally relevant con-entrations and concentrations reported to cause accumulation in. maenas (Bjerregaard, 1990). Stock solutions of cadmium wererepared in ultra-pure water and diluted with filtered seawater15 psu) to prepare the test medium. Control groups prepared onlyith filtered seawater were included in the assays. Three replicates

f each treatment (control and cadmium) were prepared for eachopulation. In each replicate 4 crabs were individually exposed

n 2 L glass beakers, under temperature, photoperiod and aera-ion conditions previously described for the acclimation period.est solutions were renewed every other day. Water temperature,onductivity, salinity, pH, and dissolved oxygen (DO) were mon-tored during the assays in the old and the freshly prepared testolutions. No food was provided to the crabs during the assays. Sam-les of old (48 h) and freshly prepared test solutions of the highestxposure concentration were collected for determination of ionicadmium. At the end of the assays crabs were ice anaesthetised andamples of epidermis (beneath the dorsal carapace), muscle (firstair of locomotor appendices) and digestive gland were collectedor biomarker analysis. Each biomarker was determined in nineo twelve crabs per treatment. For each treatment, the remainingpidermis, muscle and digestive gland, together with the rest ofhe soft tissues of the crabs, hereafter referred to as whole-bodyoft tissues for simplicity, were collected, pooled, and used for theuantification of cadmium.

.4. Determination of cadmium in water samples andhole-body soft tissues

The cadmium quantifications were performed at 228.8018 nmith a High Resolution-Continuum Source Atomic Absorption

pectrophotometer (HR-CS-AAS, ContrAA 700, Analytik Jena, Jena,ermany) using electrothermal atomisation, after acid mineral-

sation by microwave-assisted digestion (MARS-X, CEM, Mathews,C, USA) in HP-500 Plus Teflon vessels (CEM, Mathews, NC, USA),ccording to APHA recommendations (APHA, 1992). Soft tissuesere lyophilised, ground and homogenised. The microwave acidigestion of soft tissue samples was performed using 130-mg por-ions and 10 mL of a 27% nitric acid solution (Reis and Almeida,008). For the digestion of the water samples 7-mL aliquots and

mL of a nitric acid solution (65%) were used (Reis and Almeida,008). The optimised microwave digestion temperature was 160 ◦C20 min) at a maximum pressure of 200 psi. The atomic adsorptionarameters were optimised and were based on the guidelines pro-ided by the equipment’s manufacturer. The external calibrationethod was used for cadmium quantification and the analyti-

al procedure was checked using samples spiked with knownmounts of cadmium. Satisfactory mean recoveries (90.5–114%)ere obtained. All samples were analysed in triplicate.

.5. Biomarkers

All biomarker determinations were performed as describedor C. maenas by Mesquita et al. (2011) and Rodrigues et al.

ology 159 (2015) 225–232 227

(2013). A portion of epidermis and digestive gland of each crabwas homogenised and centrifuged. The supernatant was recov-ered 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 thedetermination of LDH and IDH activities. Digestive gland washomogenised in K-phosphate buffer. An aliquot of homogenatewas reserved for determination of LPO levels. The remaininghomogenate was centrifuged and the post-mitochondrial super-natant recovered was used for the determination of GST, GPx andGR activities, and TG levels as well. Except for LPO, biomarkerswere expressed as a function of the concentration of protein in thesamples. Protein concentration was determined by the Bradfordmethod (Bradford, 1976). Bovine �-globuline was used as standard.Spectrophotometric readings were performed in a Bio-Tek PowerWave 340 microplate reader or a Jasco 6405 UV/VIS spectropho-tometer.

2.6. Data analysis

Results of cadmium concentrations in water are presented asmean ± SD (standard deviation). All other results are presented asmean ± SE (standard error of the mean). Significant differences inthe biomarker responses in relation to cadmium treatments andthe estuary of origin of the crabs were first sought by factorialtwo-way analysis of variance (ANOVA), to test for significance ofthe interaction term. When significance of this term was found,one-way ANOVA with planned comparisons was performed forthe interaction variable. ANOVA assumptions were tested usingthe Shapiro–Wilk and the Levene’s test. When appropriate, thelogarithmic transformation was applied to the data in order to ful-fil ANOVA assumptions. Significant differences were accepted forp < 0.05 in all tests performed. All statistical analyses were carriedout in SPSS IBM v19.0.

3. Results

3.1. Cadmium in water samples and whole-body soft tissues

Throughout the experiments water parameters were kept stablein all test treatments. Dissolved oxygen and pH ranged between9.0–9.2 mg/L and 7.7–7.8, respectively. Salinity varied between 15.0and 15.2 and temperature ranged between 16.0 and 16.3 ◦C.

In freshly prepared medium of the highest test treat-ment (2000 �g/L), measured dissolved cadmium was between95% and 104% of the nominal concentration, corresponding to1905 ± 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 the7-day experiments a significant concentration-dependent accumu-lation 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 theMinho and at 99.9 ± 17.5 �g/g dw in crabs from the Lima estuaryexposed to 2000 �g/L (Fig. 1).

3.2. Biomarkers

3.2.1. MoultingCrabs collected from the Lima estuary showed significantly

lower epidermal NAGase activity (ca. 65% on average) than thosecollected 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 decreasedNAGase activity (by 36% in Minho crabs and 45% in Lima crabs,p < 0.05).

228 S.R. Mesquita et al. / Aquatic Toxicology 159 (2015) 225–232

Fig. 1. Cadmium accumulation (mean ± SE) in whole body soft tissues of C. maenasf

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rom the Minho and the Lima estuaries after a 7-day exposure.

NAGase activity in the digestive gland was also significantlyower (about 40% on average) in crabs from the Lima than in thoserom the Minho estuary (Table 2, Fig. 2). Cadmium also caused

decrease of NAGase activity in Minho crabs, which was signifi-antly lower than controls at 2000 �g/L (−34%, p < 0.05). However,o significant differences among experimental groups were found

or Lima crabs.

able 2esults of the full-factorial two-way ANOVAs carried out to assess the effects ofadmium and the sampling site on moulting and detoxification biomarkers of C.aenas.

Parameter Source of variation df F p

MoultingNAGase Estuary 1, 106 68.04 <0.001Epidermis Cadmium 5, 106 2.90 0.017

Estuary × cadmium 5, 106 0.47 0.799NAGase Estuary 1, 122 136.88 <0.001Digestive gland Cadmium 5, 122 2.93 0.016

Estuary × cadmium 5, 122 3.08 0.012Biotransformation and anti-oxidant defences

GST

Estuary 1, 111 96.96 <0.001Cadmium 5, 111 2.25 0.049Estuary × cadmium 5, 111 2.38 0.043

GR

Estuary 1, 114 0.38 0.542Cadmium 5, 114 2.20 0.059Estuary × cadmium 5, 114 1.32 0.259

GPx

Estuary 1, 115 61.20 <0.001Cadmium 5, 115 1.22 0.306Estuary × cadmium 5, 115 0.48 0.791

TG

Estuary 1, 116 345.80 <0.001Cadmium 5, 116 5.37 <0.001Estuary × cadmium 5, 116 4.02 0.002

Oxidative damage

LPO

Estuary 1, 116 3.73 0.056Cadmium 5, 116 1.29 0.274Estuary × cadmium 5, 116 0.51 0.768

Energy production

IDH

Estuary 1, 122 0.75 0.388Cadmium 5, 122 2.87 0.018Estuary × cadmium 5, 122 0.23 0.950

LDH

Estuary 1, 122 6.53 0.012Cadmium 5, 122 0.39 0.854Estuary × cadmium 5, 122 0.48 0.787

Fig. 2. N-acetyl-�-d-glucosaminidase (NAGase) activity (mean ± SE) in the epider-mis and digestive gland of C. maenas, collected from the Minho and the Lima

estuaries, after 7-day exposure to cadmium. *Indicates significant differences inrelation to the control at p < 0.05. Different letters indicate significant differencesbetween sampling sites within each treatment level at p < 0.05.

3.2.2. Biotransformation, anti-oxidant defences and oxidativestress

GST activity in the digestive gland was strikingly higher (ca. 7.5folds, p < 0.05) in Minho crabs than in Lima crabs (Table 2, Fig. 3).Moreover, a different response pattern to cadmium exposure wasfound for the two study sites. In Minho crabs, induction of GSTincreased with the cadmium concentration, so that at 2000 �g/Lthe enzyme activity was 45% higher than that of the control group(p < 0.05). In Lima crabs, no significant differences among experi-mental groups could be depicted.

Opposite results were obtained for TG levels. The levels of glu-tathione were significantly higher in Lima than in Minho crabs(about 8 folds, p < 0.05) (Table 2, Fig. 3). In Lima crabs, TG levelsincreased with cadmium concentrations and were 50% higher inthe 2000 �g/L group, compared to the respective control group(p < 0.05). No significant differences among experimental groupswere found for Minho crabs.

Lima crabs additionally showed significantly lower GPx activity(ca. 55% on average) than Minho crabs (Table 2, Fig. 3). However, nodifferences among treatment groups were found in either site. Also,for GR activity and LPO levels there were no differences betweenestuaries or treatment groups (Table 2, Figs. 3 and 4).

3.2.3. Energy productionCrabs from the Minho and the Lima estuaries exhibited similar

levels of IDH activity and patterns of response to cadmium expo-sure. IDH activity increased with the cadmium concentration, in

S.R. Mesquita et al. / Aquatic Toxicology 159 (2015) 225–232 229

Fig. 3. Levels of total glutathiones (TG) and activity of the enzymes glutathione S-transferases (GST), glutathione reductase (GR) and glutathione peroxidase (GPx) in thedigestive gland of C. maenas, from the Minho and the Lima estuaries, after 7-day exposureDifferent letters indicate significant differences between sampling sites within each treat

Fig. 4. Lipid peroxidation (LPO) levels (mean ± SE) in the digestive gland of C. mae-nas, collected from the Minho and the Lima estuaries, after 7-day exposure tocadmium.

to cadmium. *Indicates significant differences in relation to the control at p < 0.05.ment level at p < 0.05.

crabs from both sites, with significant differences from the respec-tive control group found at >50 �g/L cadmium (Table 2, Fig. 5). Suchresults suggest an increase in the aerobic pathway of energy pro-duction to cope with the exposure. Compared to the control groups,at 2000 �g/L cadmium IDH was increased by 92% (p < 0.05) and 56%(p < 0.05) in the Minho and the Lima crabs, respectively.

Concerning LDH, differences observed were only related to theorigin of the crabs. Crabs from the Lima exhibited higher LDH activ-ity (ca. 44%, p < 0.05) than those from the Minho estuary.

4. Discussion

In the present study a concentration-dependent accumulationof cadmium in C. maenas tissues was found, which was linearly pro-portional to the cadmium level in the exposure media. Cadmiumconcentrations measured in whole body soft tissues of exposedorganisms were in accordance with previously reported accumu-lation for C. maenas exposed to similar levels for an equivalentperiod (Nørum et al., 2005 and references therein). The residuallevels detected in control animals (∼0.5 �g/L) from both sites were

within the range of background concentrations previously foundfor C. maenas males collected in uncontaminated areas (Bjerregaardet al., 2005). The data are also suggestive of similar cadmium uptakein crabs from the two studied sites. On this regard, it has been

230 S.R. Mesquita et al. / Aquatic Toxic

Fig. 5. Activity of the enzymes NADP+-dependent isocitrate dehydrogenase (IDH)and lactate dehydrogenase (LDH) in muscle of C. maenas, from the Minho and theLib

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ima estuaries, after 7-day exposure to cadmium. *Indicates significant differencesn relation to the control at p < 0.05. Different letters indicate significant differencesetween sampling sites within each treatment level at p < 0.05.

eported that uptake rates of cadmium did not show relevant sig-ificant differences between C. maenas from metal-rich and controlstuaries (Rainbow et al., 1999). According to these authors, thehronic exposure to raised trace metal availabilities was not suffi-ient to select for a reduction in dissolved trace metal uptake rateRainbow et al., 1999).

While moulting is a process that may be affected by environ-ental contaminants, its disruption is difficult to evaluate in an

xpeditious manner. The importance of this process for growth,eproduction and metamorphosis of aquatic crustaceans sets theeed for cost-effective environmental biomarkers of moulting dis-uption to be assessed in species recommended for use in pollutiononitoring, such as C. maenas (Picado et al., 2007). As an enzyme

cting in the final step of the endocrine cascade regulating moult-ng, the potential of NAGase activity as biomarker has been shownor some crustacean species (Zou, 2005). However, available infor-

ation on differential sensitivity of C. maenas NAGase activityo priority contaminants is scarce. The present study shows thenzyme activity may be inhibited by cadmium exposure, indicatingts potential for inclusion in monitoring programmes. Further, therevious history of exposure to contamination enhanced toleranceo cadmium, eliciting differential responses of NAGase enzyme,

robably through the involvement of glutathione.

The NAGase activity levels recorded here are in the same rangef those previously obtained for C. maenas epidermis and digestiveland (Mesquita et al., 2011). They were, however, clearly higher

ology 159 (2015) 225–232

than those observed for the fiddler crab (Zou and Fingerman, 1999).In addition to differences inherent to the species, a gender effectmay also account for these dissimilarities, as Zou and Fingerman(1999) determined the enzyme activity in females, whereas maleshave been used here. In the epidermis of C. maenas, NAGase activitywas significantly inhibited by 2000 �g/L cadmium, independentlyof the crabs’ site of origin, suggesting that upon prolonged expo-sure crabs may persist longer on intermoult stage or suffer moultarrest. The inhibition may result from cadmium interference withearly steps of the endocrine moulting cascade and/or from directeffect on the enzyme. Previous studies with eyestalk-ablated adultcrabs of the species C. granulata suggest that cadmium inhibitsmoulting by inhibiting the secretion of ecdysone (Moreno et al.,2003). Conversely, in vitro studies performed with purified NAGaseextracts from the prawn Litopenaeus vannamei have shown thatcadmium was able to inhibit this enzyme to some extent (Xieet al., 2004). Future research should address the mechanism bywhich inhibition may occur. NAGase activity in the digestive glandappeared to be more sensitive to cadmium than that of the epider-mis, although only in Minho crabs. In these organisms, inhibitionof the enzyme activity was observed in all cadmium treatments(1.3–2000 �g/L). The differences found between the epidermis anddigestive gland of Minho crabs may be related to the differentialdistribution and physiological relevance of organ-specific NAGaseisoforms (Mesquita et al., 2011; Peters et al., 1999). On the otherhand, the absence of NAGase inhibition noted in the digestive glandof Lima crabs suggests that these animals coped better with cad-mium exposure.

Interestingly, upon cadmium exposure Minho crabs exhibitedincreased GST activity (at >50 �g Cd/L), with no alterations ofTG levels. Lima crabs showed no changes in GST but significantincreases in TG levels (at 1.3 and 50–2000 �g/L). Glutathione is akey component of the redox cycle, and potent free radical scav-enger that also acts as cofactor of detoxification and antioxidantenzymes (e.g., GST, GR and GPx). The molecule deals with metalstress mainly by two mechanisms: by relieving oxidative stresseffects caused by metals through the formation of oxidised glu-tathione; and by forming complexes with metals (Hatcher et al.,1995). Other authors, working with cell lines, have reported thatelevation of cellular glutathione level is involved in cadmium resis-tance, rather than decreased cadmium uptake or enhanced cellularmetallothionein synthesis (Hatcher et al., 1995). They have alsoobserved that, despite the elevation of cellular glutathione level,the activities of glutathione redox cycle enzymes GPx and GR, andanti-oxidative enzymes catalase (CAT) and superoxide dismutase(SOD) remained unchanged (Hatcher et al., 1995). Hence, theseauthors suggested that enhanced expression of �-glutamylcysteinesynthetase, the rate-limiting enzyme for glutathione biosynthe-sis, was responsible for elevated cellular glutathione levels. In thepresent work, for both Minho and Lima crabs, no changes relativeto the respective controls were found in GPx or GR (the enzymeinvolved in glutathione recycling). Enhanced production of glu-tathione in Lima crabs appears, thus, to be an effective mechanismin preventing oxidative damage and cadmium NAGase inhibitionin the digestive gland. In contrast, GST induction in Minho crabs,appear to protect against lipid peroxidation, but were not able toprevent NAGase inhibition in the hepatopancreas upon cadmiumexposure. GSTs are multifunctional proteins, which in addition totheir intrinsic transferase activity, also have glutathione peroxi-dase activity towards ROS (Casalino et al., 2007). The activation ofGST enzymes triggered by cadmium exposure was also observedin several marine worms (Won et al., 2011 and references herein).

Moreover, according to previous studies GST may have a protectiverole against Cd toxicity, in terms of physiological alterations relatedto metabolism and cell protection (Wang and Wang, 2009; Wanget al., 2011; Won et al., 2011). Overall, the results from the present

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ork suggest that animals from Minho and Lima estuaries use dif-erent detoxifying mechanisms to deal with cadmium exposure,nd support the role of glutathione as chelating agent, conferringnhanced tolerance to this metal. GST induction may representn initial response to cadmium, as seen in Minho crabs, which innimals under chronic exposure to contamination, as Lima crabs,ay give way to glutathione chelation to deal with the chemical

hallenge and achieve a new physiological steady state, allowingomeostasis maintenance (Mouneyrac et al., 2011). Both mecha-isms appear to impose increased energetic burden in the aerobicathway of energy production as indicated by the significant induc-ion, relatively to controls, of IDH activity found in both Minho andima crabs. Mitochondrial IDH catalyses decarboxilation of isoci-rate into �-ketoglutarate with concurrent production of NADPHJo et al., 2001). It is also known to be induced by the presence ofeactive oxygen species (ROS), providing NADPH for the regener-tion of GSH in the mitochondria, which in turn protects againstxidative damage and cell death (Jo et al., 2001).

. Conclusions

Overall, C. maenas NAGase activity was sensitive to cadmiumxposure suggesting its usefulness for incorporation in monitoringrogrammes. Glutathione appears to confer enhanced tolerance toadmium, preventing NAGase inhibition, in animals with a previousistory of exposure to moderate contamination. Investigation ofhe enhanced tolerance should thus be accounted for in monitoringrogrammes employing NAGase as biomarker, to avoid data mis-

nterpretation. Tolerance to metal pollution has been considereds a trait that may facilitate adaptation to contaminated estuar-es (Dafforn et al., 2009), an important aspect given that C. maenass also a well known widespread non-indigenous species that hasolonised metal polluted areas (Cohen et al., 1995; Klassen andocke, 2007).

cknowledgements

The work was supported by European Regional Devel-pment Fund (ERDF) and Portuguese funds (FCT – Foun-ation for Science and Technology), within the scope ofroject CRABTHEMES (FCOMP-01-0124-FEDER-007383, COM-ETE Programme), and projects “PEst-C/MAR/LA0015/2013, PEst-/EQB/LA0006/2013 and ECORISK (reference NORTE-07-0124-EDER-000054, North Portugal Regional Operational Programme,N.2–O Novo Norte, National Strategic Reference Framework)”..P. Rodrigues was supported by a PhD fellowship from FCT

SFRH/BD/65456/2009).

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