Marine Environmental Research xxx (2013) 1e7
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Effects of thermal stress and nickel exposure on biomarkers responses in Mytilus galloprovincialis (Lam) Hajer Attig a, Naouel Kamel a, Susanna Sforzini b, Alessandro Dagnino b, Jebali Jamel a, Hamadi Boussetta a, Aldo Viarengo b, Mohamed Banni a, b, * a b
Laboratory of Biochemistry and Environmental Toxicology, ISA, Chott-Mariem, 4042 Sousse, Tunisia Department of Environmental and Life Sciences, University of Piemonte Orientale Amedeo Avogadro, Via Bellini 25 G, 15100 Alessandria, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 October 2013 Received in revised form 7 December 2013 Accepted 9 December 2013
The present work aimed to assess the Mytilus galloprovincialis digestive gland biomarkers responses to nickel (Ni) exposure along with a heat stress gradient. Mussels were exposed to a sublethal dose of nickel (13 mM) along with a temperature gradient (18 C, 20 C, 22 C, 24 C and 26 C) for 4 days. Metallothionein (MTs) content was assessed as specific response to metals. Catalase (CAT), glutathione Stransferase (GST) activities and malondialdehyde (MDA) were measured as biomarkers of oxidative stress and lipid peroxidation. The cholinergic system was monitored using the acetylcholinesterase activity (AChE). Moreover, Ni uptakes along with the exposure temperatures were assessed. A correlation matrix (CM) between the investigated biomarkers and the exposure temperatures and a Principal Component Analysis (PCA) were achieved. Our data showed a negative effect of temperature increase on mussel’s antioxidant and detoxification response to Ni exposure being more pronounced in animals exposed to the 24 C and 26 C. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Heat stress Nickel Global warming Biomarker response Oxidative stress Mussels
1. Introduction Human activity results in the deposition of a large variety of xenobiotics into the environment on a continual basis, which effectively ensures wildlife exposure to complex mixtures of environmental stressors (Narbonne et al., 1999). Marine environment and especially intertidal zones receive chemical input from many diverse sources of contamination, such as wastewater and industrial discharge, agricultural and urban runoff (Banni et al., 2005; Tlili et al., 2010). Moreover, one consequence of the increase of CO2 levels, recognized as a major environmental problem at the global level, is climate change; continuous temperature increases may represent an important risk to marine ecosystems, especially coastal areas (Scholze et al., 2006). In recent years, a growing attention on the ecological and ecotoxicological effects of nickel (Ni) contamination has been considered (Kienle et al., 2009; Banni et al., 2011; Dondero et al., 2011). Indeed, as the result of accelerated consumption of nickelcontaining products nickel compounds are released to the
* Corresponding author. Laboratory of Biochemistry and Environmental Toxicology, Higher Institute of Agronomy, Chott Mariem, Sousse 4042, Tunisia. Tel.: þ216 73 327 544; fax: þ216 73 327591. E-mail address: [email protected] (M. Banni).
environment at all stages of production and utilization. Their accumulation in the environment may represent a serious hazard to wild life and human health (Papachristou et al., 1993; Kienle et al., 2009; Vandenbrouck., 2009; Attig et al., 2010). Ni is a metal of high environmental relevance that has been shown to exert longterm toxic effects to aquatic organisms including bivalves and fish (Pane et al., 2003; Attig et al., 2010; Banni et al., 2011; Dondero et al., 2011). In addition to the direct production of free radicals, Ni was suspected to cause depletion of the antioxidant enzymes system (Denkhaus and Salnikov, 2002) and therefore it should be considered a pro-oxidant agent. Although scientists generally have a good understanding of the toxicity of individual environmental stressors, there is a great need to bridge the gap between our understanding of the toxic effects of exposure to individual stressor and those effects from exposure to mixtures of these stressors. The study of the biological responses of organisms to different environmental conditions and the quantitative evaluation of their physiological status are being considered as a successful approach for the assessment of environmental quality (Banni et al., 2009, 2010; Viarengo et al., 2007). Mussels, Mytilus galloprovincialis are filter feeding organisms, often used in monitoring programs to evaluate the accumulation of contaminants in their tissues and the consequent effects on biological processes (Banni et al., 2011; Canesi et al., 2011; Viarengo et al., 2007). The digestive gland of mussels has been used as a model system for studying the
0141-1136/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.marenvres.2013.12.006
Please cite this article in press as: Attig, H., et al., Effects of thermal stress and nickel exposure on biomarkers responses in Mytilus galloprovincialis (Lam), Marine Environmental Research (2013), http://dx.doi.org/10.1016/j.marenvres.2013.12.006
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biomarkers response to several environmental stressors and chemicals loads in this organism (Banni et al., 2007; Viarengo et al., 2007; Dondero et al., 2011; Banni et al., 2011). Indeed, Biomarkers, considered as the changes that may occur, from the molecular to the organism level, are often employed to evaluate the physiological status of organisms exposed to environmental stressors (Viarengo et al., 2007). The present work sought to identify the effects of simultaneous changes in temperature and Ni supply on conventional biomarker of exposure in the digestive gland tissues of mussels. Le selected biomarkers were; metallothionein content, catalase, glutathione Stransferase (GST) and acetylcholinesterase activities. Moreover, the levels of malondialdehyde (MDA) were determined. We also investigated the Ni loads in digestive gland tissues along with the temperature gradient. 2. Material and methods 2.1. Animals and treatments Specimens of M. galloprovincialis (Lam) 4e5 cm shell length were purchased from an aquaculture farm in Bizerta Lagoon (Tunisia) and further acclimatised to aerated and daily renewed clean sea water in an aquarium for 15 days at 18 C (35& salinity,1.5 L/animal). Mussels were then treated for 4 days, under natural light and without any food source. Experiments have been realized at five different temperatures (18 C, 20 C, 22 C, 24 C and 26 C). Groups of mussels were kept in 20 L polypropylene plastic vessels (1 L per animal) (4 replicates per treatment) and exposed to 13 mM Ni (daily theoretical nominal dose of 770 mg/L per animal). The exposure level represents the EC50 for the effect on digestive gland lysosomal membrane stability (LMS) (mussels were exposed to a range of 0.01e15 mg/L for 96 h) (Dondero et al., 2011). A set of animals were maintained in seawater with no addition of Ni at the five different experimental temperatures and considered as relative controls. Sea water at the desired temperature was renewed every day and Ni was added. After treatments, a first set of mussel’s digestive gland tissue (10 individual fractions per exposure period considered separately) were rapidly dissected out, washed into artificial seawater buffered with 20 mM Hepes, pH 7.4, flash frozen into liquid nitrogen, and stored at 80 C until biochemical analysis. A second set of tissues (10 individual digestive glands) were flash frozen into liquid nitrogen and stored at 80 C until Ni analysis. 2.2. Nickel analysis The content of Ni in the digestive gland fractions was determined by atomic absorption spectrophotometry (AAS) after acid digestion of 0.5 g dry tissues with 4 ml of 65% HNO3 and 1 ml of 70% HCIO4 for 24 h at 80 C (Amiard et al., 1987). The acid was removed from the samples by evaporation and the residues were diluted in 10 ml of 1 N HNO3. Ni was determined by flame and flameless AAS using a varian spectrophotometer Vectra 250 Plus with ZEEMAN correction. Blanks and reference materials (dogfish liver DOLT-2, NRCC) were assessed through the procedure in the same way as the sample. Our results (21.0 1.1 mg/g dry weight, n ¼ 3) were in good agreement with certified values (20.8 0.5 mg/g dry weight). Levels of Ni are given relative to the dry weight of tissue. Analytical confirmation of nickel concentrations in reconstituted water was performed by flame atomic absorption spectroscopy. 2.3. Biochemical analysis Before biochemical analysis, digestive glands were homogenized in phosphate buffer (0.1 M, pH 7.5). The homogenate
obtained was centrifuged at 9000 g for cytosolic fractions (S9). The quantities of proteins present in S9 fraction were determined according to the Bradford (1976) method using Coomassie Blue reagent. Metallothioneins (MTs) content was evaluated in digestive gland according to the spectrophotometric method described in Viarengo et al. (1997) based on cysteine residues titration of a partially purified metallothionein extract. MTs protein levels were determined using a spectrophotometric assay for MTs using Ellman’s reagent (0.4 mM DTNB in 100 mM KH2PO4) at pH 8.5 in a solution containing 2 M NaCl and 1 mM EDTA. Reduced GSH standard solutions were used for calibration (2e100 mM) and data were expressed as mg MT per mg of protein taking into consideration mussel’s MT molecular weight and number of cysteine residues. CAT was determined according to Clairbone’s method (1985). Reaction mixture (final volume of 1 ml) contained 0.78 ml 0.1 M phosphate buffer (pH 7.5) and 0.2 ml 0.5 mM H2O2. After 30 s preincubation, the reaction was started by the addition of 0.02 ml of the (S9) solution containing CAT fractions. CAT activity was evaluated by kinetic measurement at 20 C using a Jenway 6105 spectrophotometer (l ¼ 240 nm). Results were expressed as mmoles hydrogen peroxide transformed per min and per mg protein. GST activity was measured in digestive gland cytosol by the method of Habig et al. (1974) using 10 mg of cytosolic protein, 1chloro-2,4-dinitrobenzene (CDNB) (Sigma-Aldrich, Saint Louis, MO, USA) as substrate, and glutathione reduced form GSH (1 and 4 mM final concentration, respectively), in 100 mM sodium phosphate buffer, pH 7.5. GST activity was determined by kinetic measurement at 20 C using a Jenway 6105 spectrophotometer (l ¼ 340 nm). Results were expressed as mmoles GSHeCDNB produced per min and per mg protein. Lipid peroxidation was estimated in terms of thiobarbituric acid reactive species (TBARS) with use of 1,1,3,3-treaethyloxypropane as a standard. The reaction was assessed at 532 nm, using TBA reagent as described by Buege and Aust (1978). MDA content was expressed as nmoles equivalent MDA/mg proteins. AChE was determined accordingly to Ellman et al. (1961). Reaction mixture (final volume of 1 ml) contained 0.85 ml 0.1 M phosphate buffer (pH 7.5), 0.05 ml 8 mM DNTB (Sigma-Aldrich, Saint Louis, MO, USA) and 0.05 ml of the (S9) solution containing acetylcholinesterase fractions. After pre-incubation, the reaction was started by the addition of 0.05 ml 8.25 mM acetylthiocholine (Sigma-Aldrich, Saint Louis, MO, USA). Acetylcholinesterase activity was determined by kinetic measurement at 20 C using a Jenway 6105 spectrophotometer (l ¼ 420 nm). Results were expressed as mmoles thiocholine produced per min and per mg protein. 2.4. Statistical analysis Statistical analyses were performed with SP SS/PC (SP SS, Microsoft, and Redmond, WA). Significant differences between means were determined using one-way ANOVAs and the Duncan’s test for multiple range comparison with significance level established at P < 0.05. Spearman Correlation matrix was also calculated to study the relationships between the different biomarkers measured (Statistica Soft Inc.). Factor analysis of the variables analyzed was carried out by means of the principal component analysis (PCA) method with orthogonal rotation (Varimax) using the Systat 11 software (SYSTAT Software Inc.) (Varimax minimizes the number of variables that have high loadings on each factor). The PCA analysis was used as an effective technique simplifying the correlation structure through linear transformation of the original variables (Jolliffe, 1986).
Please cite this article in press as: Attig, H., et al., Effects of thermal stress and nickel exposure on biomarkers responses in Mytilus galloprovincialis (Lam), Marine Environmental Research (2013), http://dx.doi.org/10.1016/j.marenvres.2013.12.006
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3. Results The amounts of Ni in M. galloprovincialis digestive gland after 4 days exposure to the metals along with a temperature gradient (18 C, 20 C, 22 C, 24 C and 26 C) are provided in Fig. 1. Data show an increasing Ni accumulation in animals exposed to temperature ranging from 18 C to 24 C with a maximum reached at 24 C (4.57 0.56 mg/g dry weight). The minimal Ni accumulation was observed in mussels exposed to 18 C (2.99 0.23 mg/g dry weight). Interestingly, mussels exposed to 26 C exhibited the lowest Ni uptake (1.93 0.48 mg/g dry weight). The results relative to the MT concentrations are reported in Fig. 2. Our data show that MT concentrations significantly increased along with the temperature gradient in digestive gland tissues of mussels. The same trend was observed in musses exposed to increasing temperature and Ni. The maximum was observed after in animals exposed to Ni at 24 C with up to 115.45 9.21 mg/mg proteins Ni (83% increase respect to the absolute control). Moreover, at 20 C, 22 C and 24 C a significant difference was observed between mussels exposed to only heat stress and those exposed to heat stress and Ni in term of MT concentrations. The results relative to the CAT activity in mussels exposed to Ni and temperatures are reported in Fig. 3. Our data indicated that CAT activity maximum response was reached in animals exposed to Ni and 20 C with respectively 122.31 18.58 mmol/min/mg proteins. Animals co-exposed to 26 C and Ni showed the lowest CAT activity being inhibited respect to that registered in the absolute control animals (18 C) (61.49 8.23 mmol/min/mg proteins and 83.39 10.33 mmol/min/mg proteins respectively). The effect of Ni exposure along with the temperature gradient on the mussel digestive gland phase II enzyme activity is shown in Fig. 4. A significant response respect to relative control animals was recorded for all temperatures except for animals exposed to 26 C. A significant decrease in GST activity was recorded in animals coexposed to 26 C and Ni in comparison with animals exposed to 18 C (101.10 9.48 mmol/min/mg proteins and 123.41 10.53 mmol/ min/mg proteins respectively). In animals exposed only to the temperature gradient the GST activity was significantly increased at 22 C and 24 C when compared to animals exposed to 18 C.
Fig. 1. Ni accumulation in Mytilus galloprovincialis digestive gland in animals exposed for 4 days to Ni (770 mg/L) along with a temperature gradient (18 C, 20 C, 22 C, 24 C and 26 C). Data, expressed in mg/g dry weight (n ¼ 10), were analyzed by ANOVA þ Tukey’s post test. a: Statistically significant differences (P < 0.01) in comparison with control condition (18 C without Ni supply). b: Statistically significant differences (P < 0.01) in comparison with animals exposed to18 C/Ni for 4 days.
Fig. 2. Metallothioneins protein content in digestive gland of Mytilus galloprovincialis exposed to Ni and temperature. Mussels were exposed for 4 days to Ni (770 mg/L) along with a temperature gradient (18 C, 20 C, 22 C, 24 C and 26 C). Data, expressed as nmole/mn/mg proteins (n ¼ 10), were analyzed by ANOVA þ Tukey’s post test. a: Statistically significant differences (P < 0.01) in comparison with control condition (18 C without Ni supply). b: Statistically significant differences (P < 0.01) in comparison with animals exposed to18 C/Ni for 4 days.
The MDA accumulation evaluated as thiobarbituric acid reactive species (TBARS) in mussel digestive gland after 4 days exposure to Ni along with the temperature gradient is reported in Fig. 5. A temperature-dependent accumulation of MDA was observed being more pronounced in animals co-exposed to Ni with a maximum of 27.79 3.13 nmol/mg proteins reached after 4 days in mussels exposed to 26 C and Ni and a minimum in animals maintained at 18 C without Ni supply (14.67 1.13 nmol/mg proteins). The response of AChE activity in mussel’s digestive gland after exposure to Ni along with the temperature gradient is reported in Fig. 6. AChE activity was unchanged in mussels exposed to Ni for all the tested temperatures respect to mussels exposed to heat stress only. 24 C and 26 C exposure rendered a significant inhibition of the AChE activity when compared with absolute control animals (18 C). The correlation coefficients obtained between the investigated biomarkers in digestive gland of mussels exposed to the temperature gradient and to Ni along with the temperature gradient are reported respectively in Table 1A and B. MT, MDA and AChE activity were significantly correlated to the exposure temperature in animals exposed to heat stress (Table 1A). However, significant correlation was recorded between exposure temperature and MT, MDA, AChE and CAT activities in organisms exposed to the metals along with a temperature gradient (Table 1B). Results from PCA using biomarkers data in animals exposed to the heat stress revealed that the first axis (47.29% of the overall variance) was mainly influenced by CAT and GST activities (Fig. 7A). MDA accumulation and AChE activity and MT content mainly composed the second axis (23.76% of the variance). This axis appeared associated with a heat specific response since the five exposure temperatures were clearly separated. PCA was also obtained from biomarkers data in animals exposed to Ni along with the temperature gradient depicting a first axis (54.20% of the variance) mainly associated with AChE and GST activities (Fig. 7B). 4. Discussion The presence of contaminants in aquatic environments may results in an additional stress when coinciding with a-biotic
Please cite this article in press as: Attig, H., et al., Effects of thermal stress and nickel exposure on biomarkers responses in Mytilus galloprovincialis (Lam), Marine Environmental Research (2013), http://dx.doi.org/10.1016/j.marenvres.2013.12.006
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Fig. 3. Catalase activity in digestive gland of Mytilus galloprovincialis exposed to Ni and temperature. Mussels were exposed for 4 days to Ni (770 mg/L) along with a temperature gradient (18 C, 20 C, 22 C, 24 C and 26 C). Data, expressed as nmole/mn/mg proteins (n ¼ 10), were analyzed by ANOVA þ Tukey’s post test. a: Statistically significant differences (P < 0.01) in comparison with control condition (18 C without Ni supply). b: Statistically significant differences (P < 0.01) in comparison with animals exposed to18 C/Ni for 4 days.
stressors such as heat stress. Recent reports indicated that heat stress increased the toxicity of polycyclic hydrocarbons and metals (Kamel et al., 2012; Negri et al., 2013; Sokolova and Lannig, 2008). Although bivalves are reported to have a higher tolerance to environmental changes, as compared with other marine organisms (Wang et al., 2011; Tomanek and Zuzow, 2010; Negri et al., 2013), reduction in clearance rates and respiration were observed upon in bivalves exposed to hypoxia and temperature fluctuations. The metal accumulation in mollusks was reported to be dependent of dose, exposure time and ambient water conditions
Fig. 4. Glutathione es e Transferase activity in digestive gland of Mytilus galloprovincialis exposed to Ni and temperature. Mussels were exposed for 4 days to Ni (770 mg/L) along with a temperature gradient (18 C, 20 C, 22 C, 24 C and 26 C). Data, expressed as nmole/mn/mg proteins (n ¼ 10), were analyzed by ANOVA þ Tukey’s post test. a: Statistically significant differences (P < 0.01) in comparison with control condition (18 C without Ni supply). b: Statistically significant differences (P < 0.01) in comparison with animals exposed to18 C/Ni for 4 days.
(Negri et al., 2013; Attig et al., 2010; Das and Jana, 1999). In the present study, Ni accumulation in the digestive gland tissue of mussels e metabolically the most active organ e was found to be modulated by the water temperature. Our data showed that the bioaccumulation was function of temperature until reaching 24 C. The reduction of Ni uptake at 26 C could be due to the reduction of the mussel ventilation rate at higher temperature. Indeed, Lannig et al. (2008) observed a synergistic effect of elevated temperatures and cadmium exposure that led to oxygen limitation by impaired performance in oxygen supply through ventilation and circulation in the eastern oysters Crassostrea verginica. Moreover, Anestis et al. (2007) reported that mussels M. galloprovincialis increased the duration of valve closure by about six fold when acclimated to 24 C rather than to 17 C. In the present work, MT concentration was found to be positively regulated by the ambient temperature in absence and in presence of Ni (r ¼ 0.84 in animals exposed only to heat stress and r ¼ 0.79 in animals exposed Ni along with heat stress (Table 1). However, no significant correlation was found between Ni loads in the DG and the MT concentrations (Table 1). Moreover, for a same exposure temperature, our data showed significant differences of MT concentration in the DG of mussels treated with Ni when compared to those expose only to heat stress (except at 26 C). From data obtained in the present work it appears clear that MT concentration do not rely directly on intracellular concentration of Ni but probably on the oxidative stress status of the cell as observed for heat stressed mussels. Indeed, recent reports indicated that MTs concentrations were significantly up-regulated by temperature increase and by a combination of heat stress and metal exposure (Ivanina et al., 2008, 2009). Therefore, it is of interest to establish the relation between metallothionein concentration and the oxidative stress status of the DG cells. Indeed the metal transcription factor 1 (MTF-1), functioning as a sensor of intracellular Zn, is responsible for both basal and Zn/Cu mediated expression of MT-1 in mammalian (Sadhu and Gedamu, 1988). This transcription factor may function in mussels as a sensor of the redox state of the cell, triggering a rapid and specific response to the presence of
Fig. 5. Malondialdehyde accumulation in digestive gland of Mytilus galloprovincialis exposed to Ni and temperature. Mussels were exposed for 4 days to Ni (770 mg/L) along with a temperature gradient (18 C, 20 C, 22 C, 24 C and 26 C). Data, expressed as nmole/mn/mg proteins (n ¼ 10), were analyzed by ANOVA þ Tukey’s post test. a: Statistically significant differences (P < 0.01) in comparison with control condition (18 C without Ni supply). b: Statistically significant differences (P < 0.01) in comparison with animals exposed to18 C/Ni for 4 days.
Please cite this article in press as: Attig, H., et al., Effects of thermal stress and nickel exposure on biomarkers responses in Mytilus galloprovincialis (Lam), Marine Environmental Research (2013), http://dx.doi.org/10.1016/j.marenvres.2013.12.006
H. Attig et al. / Marine Environmental Research xxx (2013) 1e7
Fig. 6. Acetylcholinesterase activity in digestive gland of Mytilus galloprovincialis exposed to Ni and temperature. Mussels were exposed for 4 days to Ni (770 mg/L) along with a temperature gradient (18 C, 20 C, 22 C, 24 C and 26 C). Data, expressed as nmole/mn/mg proteins (n ¼ 10), were analyzed by ANOVA þ Tukey’s post test. a: Statistically significant differences (P < 0.01) in comparison with control condition (18 C without Ni supply). b: Statistically significant differences (P < 0.01) in comparison with animals exposed to18 C/Ni for 4 days.
unbuffered, highly electrophilic metalsor oxidants (Dondero et al., 2005; Viarengo et al., 1999). Correlation between Ni exposure and MT induction is controversial. Ni ions are known to have a high affinity for sulfydrylic groups of proteins (Costa et al., 1994) and MT induction by Ni was reported not only in vertebrates such as the cod Eleginus navaga (Eriksen et al., 1991) but also in the copepod Tigriopus brevicornis (Barka et al., 2001), although with less extent than other metals (Cu, Zn, Hg, Cd, Ag) tested in the same organism (Barka et al., 2001). Recently our research group demonstrated that mussels exposed to Ni displayed a significant increase of mt10 mRNA abundance and no effects on the cognate mt20 (Dondero et al., 2011), indicating that Ni ions behave in the same way as copper to which mt20 is almost insensitive (Dondero et al., 2005). In mussels, the antioxidant defense system includes enzymes such as CAT, GST, superoxide dismutase (SOD) and other low molecular weight scavengers such as reduced glutathione (GSH) (Livingstone, 2001). Our data provided some evidences about the occurrence of oxidative stress in the digestive gland tissue of mussels exposed to Ni along with a heat stress gradient. Indeed a temperature dependent rise of MDA was observed along with a positive and significant correlation between MDA accumulation and temperature without or in presence of Ni supply (r ¼ 0.82 and 0.78, respectively). Catalase activity is considered as a key antioxidant enzymatic activity of the cell. The production of oxyradicals in mussels has been reported to be mediated by a higher range of contaminants including Ni (Attig et al., 2010; Dondero et al., 2011). Recent reports indicate the implication of higher temperatures in the enhancement of cellular (ROS) release in mussels, thereby increasing the risk of oxidative alterations (Kefaloyianni et al., 2005; Verlecar et al., 2007; Lockwood et al., 2010). Our data show a bell shape response of the catalase activity in animals exposed to the temperature gradient and a decreasing trend to the Ni along with the temperature gradient. This may suggest a potent cytotoxicity effect of Ni when associated to higher temperature (24 C and 26 C) (r ¼ 0.71) (Table 1B). GST activity has already been characterized in mussels (Fitzpatrick et al., 1995, 1997). From data discussed in this study, it appears clear that GST activity was not implicated in the response to heat stress as the correlation
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coefficient was not significant with Temperature increase (Table 1A). However, GST activity was positively and significantly correlated with Ni loads in Ni exposed mussels along with the temperature gradient (r ¼ 0.82) (Table 1B), suggesting the implication of GST in Ni homeostasis as previously reported with other metals such as cadmium (Banni et al., 2009). AChE is considered highly responsive to biotic and abiotic changes in different marine species (Dimitriadis et al., 2012; Greco et al., 2011; Banni et al., 2010). Moreover, AChE was reported to be inhibited in mussels exposed to acute and subacute environmental contaminants including metals (Jebali et al., 2006; Viarengo et al., 2007; Banni et al., 2010). In the present study, we observed a significant increase of the AChE activity in mussels exposed to Ni along with the heat stress gradient (18 Ce24 C). However a significant inhibition of the AChE enzyme was depicted in mussels exposed to Ni at the highest temperature (26 C). AChE activity was found to be negatively correlated with temperature and Ni accumulation with a correlation coefficient of (r ¼ 0.62 and r ¼ 0.72 respectively (Table 1)). Even if little is known on Ni effects on AChE activity in mussels, our data provided clues about the occurrence of a negative effect of Ni; a non-conventional inhibitor of the AChE activity at higher temperatures. Biomarker data and Ni accumulation in mussels exposed to Ni along with the heat stress gradient were summarized by means of PCA (Fig. 7). In mussels exposed only to heat stress (Fig. 7A), PC1 discriminated data mainly following exposure temperature: results from heat stresses organisms showed positive scores in PC1 for temperatures 18 C, 20 C and 22 C; differently, data showed negative scores for temperatures 24 C and 26 C (Table 1A). PC1 was able to summarize biomarker responses in mussels: in particular eigenvectors loadings for PC1 indicated negative values to biomarkers such as GST, MT, MDA and a positive loading to AChE and CAT (Table 1B). For mussels exposed to Ni along with the heat stress gradient, the PCA analysis (Fig. 7B) showed that PC1 discriminated data also following Temperature. PC1 was able to discriminate the effects of higher temperatures (24 C and 26 C) mainly characterized by an increase in MDA and MT accumulation with respect to the other temperatures. PC2 described only a
Table 1 Correlation matrix of studied biomarkers and exposure time in heat stressed animals (A) and in Ni exposed M. galloprovincialis along with temperature gradient (B). Spearman’s correlation coefficient is provided as the top number and the significance of the correlation is provided as the bottom number for each comparison. CAT GST MDA AChE MT
[Ni] CAT GST MDA AChE MT
0.36 0.08 0.41 0.04 0.82* 0.001 L0.62* 0.001 0.84* 0.001 T 0.11 0.60 L0.75* 0.001 0.20 0.33 0.78* 0.001 L0.72* 0.001 0.79* 0.001 T
A 0.27 0.19 0.14 0.51 0.12 0.57 0.17 0.41 CAT
0.17 0.42 0.12 0.55 0.47 0.02 GST
0.49 0.06 0.69* 0.001 MDA
0.44 0.03 AChE B
0.03 0.90 0.64* 0.001 0.11 0.60 0.03 0.89 0.13 0.54 [Ni]
0.19 0.36 L0.58 0.001 0.46 0.04 L0.72 0.001 CAT
0.06 0.77 0.07 0.75 0.40 0.05 GST
0.48 0.02 0.68* 0.001 MDA
L0.62* 0.001 AChE
Please cite this article in press as: Attig, H., et al., Effects of thermal stress and nickel exposure on biomarkers responses in Mytilus galloprovincialis (Lam), Marine Environmental Research (2013), http://dx.doi.org/10.1016/j.marenvres.2013.12.006
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Fig. 7. Biplot from additional principal component analysis of all variables studied heat stressed animals (A) and in Ni exposed M. galloprovincialis along with temperature gradient (B).
minimal amount of data variance in both experiments (with/ without Ni supply).
and particularly in organisms challenging extreme temperature fluctuations.
5. Conclusion
Acknowledgments
The study of interactive effects of temperature and other environmental stressors such as metals on marine organisms especially those inhabiting intertidal zones may represent an important issue in future ecotoxicological research. The joint effect of stressors similar to that described in this study is not specific to metals and can be extended to other contaminants or factors that affect metals uptake and cellular response of exposed organisms. As a result, stress tolerance to environmental parameters such as temperatures increase will be reduced in animals inhabiting polluted areas of intertidal and coastal regions. The data reported in this work should be carefully considered in view of the biological effects of metals
This work was supported by funds from “Ministère de l’Enseignement Supérieur et de la Recherche Scientifique; UR04A6R05. Biochimie et Toxicologie Environnementale” and by Theme 6 of the EC seventh framework program through the Marine Ecosystem Evolution in a Changing Environment (MEECE No 212085) Collaborative Project.
References Amiard, J.C., Pineau, A., Boiteau, H.-L., Metayer, C., Amiard-Triquet, C., 1987. Application de la spectrométrie d’absorption atomique Zeeman aux dosages de huit
Please cite this article in press as: Attig, H., et al., Effects of thermal stress and nickel exposure on biomarkers responses in Mytilus galloprovincialis (Lam), Marine Environmental Research (2013), http://dx.doi.org/10.1016/j.marenvres.2013.12.006
H. Attig et al. / Marine Environmental Research xxx (2013) 1e7 éléments traces (Ag, Cd, Cr, Cu, Mn, Ni, Pb et Se) dans les matrices biologiques solides. Water Res. 21, 693e697. Anestis, A., Lazou, A., Pörtner, H.O., Michaelidis, B., 2007. Behavioral, metabolic, and molecular stress responses of marine bivalve Mytilus galloprovincialis during long-term acclimation at increasing ambient temperature. Am. J. Physiol. Regul. Integr. Comp. Physiol 293 (2), R911eR921. Attig, H., Dagnino, A., Negri, A., Jebali, J., Boussetta, H., Viarengo, A., Dondero, F., Banni, M., 2010. Uptake and biochemical responses of mussels Mytilus galloprovincialis exposed to sublethal nickel concentrations. Ecotoxicol. Environ. Saf. 73 (7), 1712e1719. Banni, M., Jebali, J., Daubez, M., Clerandeaux, C., Guerbej, H., Narbonne, J.F., Boussetta, H., 2005. Monitoring pollution in Tunisian coasts: application of a classification scale based on biochemical markers. Biomarkers 10 (2e3), 105e 116. Banni, M., Dondero, F., Jebali, J., Guerbej, H., Boussetta, H., Viarengo, A., 2007. Assessment of heavy metal contamination using real-time PCR analysis of mussel metallothionein mt10 and mt20 expression: a validation along the Tunisian coast. Biomarkers 12 (4), 369e383. Banni, M., Bouraoui, Z., Clerandeau, C., Narbonne, J.F., Boussetta, H., 2009. Mixture toxicity assessment of cadmium and benzo[a]pyrene in the sea worm Hediste diversicolor. Chemosphere 77, 902e906. Banni, M., Negri, A., Dagnino, A., Jebali, J., Ameur, S., Boussetta, 2010. Acute effects of benzo[a]pyrene on digestive gland enzymatic biomarkers and DNA damage on mussel Mytilus galloprovincialis. Ecotoxicol. Environ. Saf. 73 (5), 842e848. Banni, M., Negri, A., Mignone, F., Boussetta, H., Viarengo, A., Dondero, F., 2011. Gene expression rhythms in the mussel Mytilus galloprovincialis (Lam.) across an annual cycle. PLoS One 6 (5), e18904. Barka, S., Pavillon, J.F., Amiard, J.C., 2001. Influence of different essential and nonessential metals on MTLP levels in the Copepod Tigriopus brevicornis. Comp. Biochem. Physiol. C 128, 479e493. Bradford, M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248e254. Buege, J.A., Aust, S.D., 1978. Microsomal lipid peroxidation. In: Packer, L. (Ed.), Meth. Enzymol, vol. 52, pp. 302e310. Canesi, L., Negri, A., Barmo, C., Banni, M., Gallo, G., Viarengo, A., Dondero, F., 2011. The organophosphate chlorpyrifos interferes with the responses to 17b-estradiol in the digestive gland of the marine mussel Mytilus galloprovincialis. PLoS One 6 (5), e19803. Claiborne, A., 1985. Catalase activity. In: Greenwald, R.A. (Ed.), CRC Handbook of Methods in Oxygen Radical Research. CRC Press, Florida, pp. 283e294. Costa, M., Zhuang, Z., Huang, X., Cosentino, S., Klein, C.B., Salnikov, K., 1994. Molecular mechanisms of nickel carcinogenesis. Sci. Total Environ. 148, 191e199. Das, S., Jana, B.B., 1999. Dose-dependent uptake and Eichhornia-induced elimination of cadmium in various organs of the freshwater mussel, Lamellidens marginalis (Linn.). Ecol. Eng. 12, 207e229. Denkhaus, E., Salnikov, K., 2002. Nickel essentiality, toxicity, and carcinogenicity. Crit. Rev. Oncol. Hematol. 42, 35e56. Dimitriadis, V.K., Gougoula, C., Anestis, A., Pörtner, H.O., Michaelidis, B., 2012. Monitoring the biochemical and cellular responses of marine bivalves during thermal stress by using biomarkers. Mar. Environ. Res. 73, 70e77. Dondero, F., Piacentini, L., Banni, M., Rebelo, M., Burlando, B., Viarengo, A., 2005. Quantitative PCR analysis of two molluscan metallothionein genes unveils differential expression and regulation. Gene 345, 259e270. Dondero, F., Banni, M., Negri, A., Boatti, L., Dagnino, A., Viarengo, A., 2011. Interactions of a pesticide/heavy metal mixture in marine bivalves: a transcriptomic assessment. BMC Genomics 12, 195. Ellman, G.L., Courtney, K.O., Andres, V., Featherstone, R.M., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88e95. Eriksen, K.D.H., Andersen, T., Stenersen, J., 1991. Iso-metallothionein in cod induced by cadmium, zinc and nickel. In: Proceedings of the 12th Annual Conference on Physiological and Biochemical Approaches to the Toxicological Assessment of Environmental Pollution. Fitzpatrick, P.J., O’Halloran, J., Sheehan, D., Walsh, A.R., 1997. Assessment of a glutathione S-transferase and related proteins in the gill and digestive gland of Mytilus edulis (L.), as potential organic pollution biomarkers. Biomarkers 2, 51e 56. Fitzpatrick, P.J., Sheehan, D., Livingstone, D.R., 1995. Studies on isoenzymes of glutathione S transferase in the digestive gland of Mytilus galloprovincialis with exposure to pollution. Mar. Environ. Res. 39, 241e244. Greco, L., Pellerin, J., Capri, E., Garnerot, F., Louis, S., Fournier, M., Sacchi, A., Fusi, M., Lapointe, D., 2011. Physiological effects of temperature and a herbicide mixture on the soft-shell clam Mya arenaria (Mollusca, Bivalvia). Environ. Toxicol. Chem. 30 (1), 132e141. Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione S-transferases, the first step in mercapturic acid formation. J. Biol. Chem. 249, 7130e7139.
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Ivanina, A.V., Cherkasov, A.S., Sokolova, I.M., 2008. Effect of cadmium on cellular protein and glutathione synthesis and expression of stress proteins in eastern oysters, Crassostrea virginica Gmelin. J. Exp. Biol. 211, 577e586. Ivanina, A.V., Taylor, C., Sokolova, I.M., 2009. Effects of elevated temperature and cadmium exposure on stress protein response in eastern oysters Crassostrea virginica (Gmelin). Aquat. Toxicol. 91, 245e254. Jebali, J., Banni, M., DE Alemeida, E.-A., Bannaoui, A., Boussetta, H., 2006. Effects of malathion and cadmium on acetylcholinesterase activity and metallothionein levels in the fish Seriola dumerilli. Fish Physiol. Biochem. 32, 93e98. Jolliffe, I.T., 1986. Principal Component Analysis. Springer Verlag, New York, NY, USA. Kamel, N., Attig, H., Dagnino, A., Boussetta, H., Banni, M., 2012. Increased temperatures affect oxidative stress markers and detoxification response to benzo[a] pyrene exposure in mussel Mytilus galloprovincialis. Arch. Environ. Contam. Toxicol. 63 (4), 534e543. Kefaloyianni, E., Gourgou, E., Ferle, V., Kotsakis, E., Gaitanaki, C., Beis, I., 2005. Acute thermal stress and various heavy metals induce tissue-specific pro- or antiapoptotic events via the p38-MAPK signal transduction pathway in Mytilus galloprovincialis (Lam.). J. Exp. Biol. 208 (23), 4427e4436. Kienle, C., Kohler, H.R., Gerhardt, A., 2009. Behavioural and developmental toxicity of chlorpyrifos and nickel chloride to zebra fish (Danio rerio) embryos and larvae. Ecotoxicol. Environ. Saf. 72, 1740e1747. Lannig, G., Cherkasov, A.S., Pörtner, H.O., Bock, C., Sokolova, I.M., 2008. Cadmiumdependent oxygen limitation affects temperature tolerance in eastern oysters (Crassostrea virginica Gmelin). Am. J. Physiol. Regul. Integr. Comp. Physiol. 294, R1338eR1346. Livingstone, D.R., 2001. Contaminant-stimulated reactive oxygen species production and oxidative damage in aquatic organisms. Mar. Pollut. Bull. 42, 656e666. Lockwood, B.L., Sanders, J.G., Somero, G.N., 2010. Transcriptomic responses to heat stress in invasive and native blue mussels (genus Mytilus): molecular correlates of invasive success. J. Exp. Biol. 213, 3548e3558. Narbonne, J.F., Daubeze, M., Clérandeau, C., Garrigues, P., 1999. Scale of classification based on biochemical markers in mussels: application to pollution monitoring in European coasts. Biomarkers 4, 415e424. Negri, A., Oliveri, C., Sforzini, S., Mignione, F., Viarengo, A., Banni, M., 25 2013. Transcriptional response of the mussel Mytilus galloprovincialis (Lam.) following exposure to heat stress and copper. PLoS One 8 (6), e66802. Pane, E.F., Smith, C., McGeer, J.C., Wood, C.M., 2003. Mechanism of acute and chronic waterborne nickel toxicity in the freshwater cladoceran Daphnia magna. Environ. Sci. Technol. 37 (19), 4382e4389. Papachristou, P., Haralambous, K.J., Loizidou, N., Spyrellis, N., 1993. Studies on the nickel removal from aqueous solutions. J. Environ. Sci. Health A 28 (1), 135e142. Sadhu, C., Gedamu, L., 1988. Regulation of human metallothionein (MT) genes. Differential expression of MTI-F, MTI-G, and MTII-A genes in the hepatoblastoma cell line (HepG2). J. Biol. Chem. 263, 2679e2684. Scholze, M., Knorr, W., Arnell, N.W., Prentice, I.C., 2006. A climate-change risk analysis for world ecosystems. Proc. Natl. Acad. Sci. U. S. A. 103 (35), 13116e13120. Sokolova, I.M., Lannig, G., 2008. Interactive effects of metal pollution and temperature on metabolism in aquatic ectotherms: implications of global climate change. Clim. Res. 37, 181e201. Tlili, S., Métais, I., Boussetta, H., Mouneyrac, C., 2010. Linking changes at subindividual and population levels in Donax trunculus: assessment of marine stress. Chemosphere 81 (6), 692e700. Tomanek, L., Zuzow, M.J., 2010. The proteomic response of the mussel congeners Mytilus galloprovincialis and M. trossulus to acute heat stress: implications for thermal tolerance limits and metabolic costs of thermal stress. J. Exp. Biol. 213 (Pt 20), 3559e3574. Vandenbrouck, T., Soetaert, A., Van der Ven, K., Blust, R., De Coen, W., 2009. Nickel and binary metal mixture responses in Daphnia magna: molecular fingerprints and (sub) organismal effects. Aquat. Toxicol. 92 (1), 18e29. Verlecar, X.N., Jena, K.B., Chainy, G.B., 2007. Biochemical markers of oxidative stress in Perna viridis exposed to mercury and temperature. Chem. Biol. Interact. 167 (3), 219e226. Viarengo, A., Burlando, B., Cavaletto, M., Marchi, B., Ponzano, E., Blasco, J., 1999. Role of metallothionein against oxidative stress in the mussel Mytilus galloprovincialis. Am. J. Physiol. 277, 1612e1619. Viarengo, A., Ponzano, E., Dondero, F., Fabbri, R., 1997. A simple spectrophotometric method for metallothionein evaluation in marine organisms: an application to Mediterranean and Antarctic molluscs. Mar. Environ. Res. 44, 69e84. Viarengo, A., Lowe, D., Bolognesi, C., Fabbri, E., Koehler, A., 2007. The use of biomarkers in biomonitoring: a 2-tier approach assessing the level of pollutantinduced stress syndrome in sentinel organisms. Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 146 (3), 281e300. Wang, Y., Hu, M., Wong, W.H., Shin, P.K., Cheung, S.G., 2011. The combined effects of oxygen availability and salinity on physiological responses and scope for growth in the green-lipped mussel Perna viridis. Mar. Pollut. Bull. 63 (5e12), 255e261.
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Effects of thermal stress and nickel exposure on biomarkers responses in Mytilus galloprovincialis (Lam) Hajer Attig a, Naouel Kamel a, Susanna Sforzini b, Alessandro Dagnino b, Jebali Jamel a, Hamadi Boussetta a, Aldo Viarengo b, Mohamed Banni a, b, * a b
Laboratory of Biochemistry and Environmental Toxicology, ISA, Chott-Mariem, 4042 Sousse, Tunisia Department of Environmental and Life Sciences, University of Piemonte Orientale Amedeo Avogadro, Via Bellini 25 G, 15100 Alessandria, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 October 2013 Received in revised form 7 December 2013 Accepted 9 December 2013
The present work aimed to assess the Mytilus galloprovincialis digestive gland biomarkers responses to nickel (Ni) exposure along with a heat stress gradient. Mussels were exposed to a sublethal dose of nickel (13 mM) along with a temperature gradient (18 C, 20 C, 22 C, 24 C and 26 C) for 4 days. Metallothionein (MTs) content was assessed as specific response to metals. Catalase (CAT), glutathione Stransferase (GST) activities and malondialdehyde (MDA) were measured as biomarkers of oxidative stress and lipid peroxidation. The cholinergic system was monitored using the acetylcholinesterase activity (AChE). Moreover, Ni uptakes along with the exposure temperatures were assessed. A correlation matrix (CM) between the investigated biomarkers and the exposure temperatures and a Principal Component Analysis (PCA) were achieved. Our data showed a negative effect of temperature increase on mussel’s antioxidant and detoxification response to Ni exposure being more pronounced in animals exposed to the 24 C and 26 C. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Heat stress Nickel Global warming Biomarker response Oxidative stress Mussels
1. Introduction Human activity results in the deposition of a large variety of xenobiotics into the environment on a continual basis, which effectively ensures wildlife exposure to complex mixtures of environmental stressors (Narbonne et al., 1999). Marine environment and especially intertidal zones receive chemical input from many diverse sources of contamination, such as wastewater and industrial discharge, agricultural and urban runoff (Banni et al., 2005; Tlili et al., 2010). Moreover, one consequence of the increase of CO2 levels, recognized as a major environmental problem at the global level, is climate change; continuous temperature increases may represent an important risk to marine ecosystems, especially coastal areas (Scholze et al., 2006). In recent years, a growing attention on the ecological and ecotoxicological effects of nickel (Ni) contamination has been considered (Kienle et al., 2009; Banni et al., 2011; Dondero et al., 2011). Indeed, as the result of accelerated consumption of nickelcontaining products nickel compounds are released to the
* Corresponding author. Laboratory of Biochemistry and Environmental Toxicology, Higher Institute of Agronomy, Chott Mariem, Sousse 4042, Tunisia. Tel.: þ216 73 327 544; fax: þ216 73 327591. E-mail address: [email protected] (M. Banni).
environment at all stages of production and utilization. Their accumulation in the environment may represent a serious hazard to wild life and human health (Papachristou et al., 1993; Kienle et al., 2009; Vandenbrouck., 2009; Attig et al., 2010). Ni is a metal of high environmental relevance that has been shown to exert longterm toxic effects to aquatic organisms including bivalves and fish (Pane et al., 2003; Attig et al., 2010; Banni et al., 2011; Dondero et al., 2011). In addition to the direct production of free radicals, Ni was suspected to cause depletion of the antioxidant enzymes system (Denkhaus and Salnikov, 2002) and therefore it should be considered a pro-oxidant agent. Although scientists generally have a good understanding of the toxicity of individual environmental stressors, there is a great need to bridge the gap between our understanding of the toxic effects of exposure to individual stressor and those effects from exposure to mixtures of these stressors. The study of the biological responses of organisms to different environmental conditions and the quantitative evaluation of their physiological status are being considered as a successful approach for the assessment of environmental quality (Banni et al., 2009, 2010; Viarengo et al., 2007). Mussels, Mytilus galloprovincialis are filter feeding organisms, often used in monitoring programs to evaluate the accumulation of contaminants in their tissues and the consequent effects on biological processes (Banni et al., 2011; Canesi et al., 2011; Viarengo et al., 2007). The digestive gland of mussels has been used as a model system for studying the
0141-1136/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.marenvres.2013.12.006
Please cite this article in press as: Attig, H., et al., Effects of thermal stress and nickel exposure on biomarkers responses in Mytilus galloprovincialis (Lam), Marine Environmental Research (2013), http://dx.doi.org/10.1016/j.marenvres.2013.12.006
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H. Attig et al. / Marine Environmental Research xxx (2013) 1e7
biomarkers response to several environmental stressors and chemicals loads in this organism (Banni et al., 2007; Viarengo et al., 2007; Dondero et al., 2011; Banni et al., 2011). Indeed, Biomarkers, considered as the changes that may occur, from the molecular to the organism level, are often employed to evaluate the physiological status of organisms exposed to environmental stressors (Viarengo et al., 2007). The present work sought to identify the effects of simultaneous changes in temperature and Ni supply on conventional biomarker of exposure in the digestive gland tissues of mussels. Le selected biomarkers were; metallothionein content, catalase, glutathione Stransferase (GST) and acetylcholinesterase activities. Moreover, the levels of malondialdehyde (MDA) were determined. We also investigated the Ni loads in digestive gland tissues along with the temperature gradient. 2. Material and methods 2.1. Animals and treatments Specimens of M. galloprovincialis (Lam) 4e5 cm shell length were purchased from an aquaculture farm in Bizerta Lagoon (Tunisia) and further acclimatised to aerated and daily renewed clean sea water in an aquarium for 15 days at 18 C (35& salinity,1.5 L/animal). Mussels were then treated for 4 days, under natural light and without any food source. Experiments have been realized at five different temperatures (18 C, 20 C, 22 C, 24 C and 26 C). Groups of mussels were kept in 20 L polypropylene plastic vessels (1 L per animal) (4 replicates per treatment) and exposed to 13 mM Ni (daily theoretical nominal dose of 770 mg/L per animal). The exposure level represents the EC50 for the effect on digestive gland lysosomal membrane stability (LMS) (mussels were exposed to a range of 0.01e15 mg/L for 96 h) (Dondero et al., 2011). A set of animals were maintained in seawater with no addition of Ni at the five different experimental temperatures and considered as relative controls. Sea water at the desired temperature was renewed every day and Ni was added. After treatments, a first set of mussel’s digestive gland tissue (10 individual fractions per exposure period considered separately) were rapidly dissected out, washed into artificial seawater buffered with 20 mM Hepes, pH 7.4, flash frozen into liquid nitrogen, and stored at 80 C until biochemical analysis. A second set of tissues (10 individual digestive glands) were flash frozen into liquid nitrogen and stored at 80 C until Ni analysis. 2.2. Nickel analysis The content of Ni in the digestive gland fractions was determined by atomic absorption spectrophotometry (AAS) after acid digestion of 0.5 g dry tissues with 4 ml of 65% HNO3 and 1 ml of 70% HCIO4 for 24 h at 80 C (Amiard et al., 1987). The acid was removed from the samples by evaporation and the residues were diluted in 10 ml of 1 N HNO3. Ni was determined by flame and flameless AAS using a varian spectrophotometer Vectra 250 Plus with ZEEMAN correction. Blanks and reference materials (dogfish liver DOLT-2, NRCC) were assessed through the procedure in the same way as the sample. Our results (21.0 1.1 mg/g dry weight, n ¼ 3) were in good agreement with certified values (20.8 0.5 mg/g dry weight). Levels of Ni are given relative to the dry weight of tissue. Analytical confirmation of nickel concentrations in reconstituted water was performed by flame atomic absorption spectroscopy. 2.3. Biochemical analysis Before biochemical analysis, digestive glands were homogenized in phosphate buffer (0.1 M, pH 7.5). The homogenate
obtained was centrifuged at 9000 g for cytosolic fractions (S9). The quantities of proteins present in S9 fraction were determined according to the Bradford (1976) method using Coomassie Blue reagent. Metallothioneins (MTs) content was evaluated in digestive gland according to the spectrophotometric method described in Viarengo et al. (1997) based on cysteine residues titration of a partially purified metallothionein extract. MTs protein levels were determined using a spectrophotometric assay for MTs using Ellman’s reagent (0.4 mM DTNB in 100 mM KH2PO4) at pH 8.5 in a solution containing 2 M NaCl and 1 mM EDTA. Reduced GSH standard solutions were used for calibration (2e100 mM) and data were expressed as mg MT per mg of protein taking into consideration mussel’s MT molecular weight and number of cysteine residues. CAT was determined according to Clairbone’s method (1985). Reaction mixture (final volume of 1 ml) contained 0.78 ml 0.1 M phosphate buffer (pH 7.5) and 0.2 ml 0.5 mM H2O2. After 30 s preincubation, the reaction was started by the addition of 0.02 ml of the (S9) solution containing CAT fractions. CAT activity was evaluated by kinetic measurement at 20 C using a Jenway 6105 spectrophotometer (l ¼ 240 nm). Results were expressed as mmoles hydrogen peroxide transformed per min and per mg protein. GST activity was measured in digestive gland cytosol by the method of Habig et al. (1974) using 10 mg of cytosolic protein, 1chloro-2,4-dinitrobenzene (CDNB) (Sigma-Aldrich, Saint Louis, MO, USA) as substrate, and glutathione reduced form GSH (1 and 4 mM final concentration, respectively), in 100 mM sodium phosphate buffer, pH 7.5. GST activity was determined by kinetic measurement at 20 C using a Jenway 6105 spectrophotometer (l ¼ 340 nm). Results were expressed as mmoles GSHeCDNB produced per min and per mg protein. Lipid peroxidation was estimated in terms of thiobarbituric acid reactive species (TBARS) with use of 1,1,3,3-treaethyloxypropane as a standard. The reaction was assessed at 532 nm, using TBA reagent as described by Buege and Aust (1978). MDA content was expressed as nmoles equivalent MDA/mg proteins. AChE was determined accordingly to Ellman et al. (1961). Reaction mixture (final volume of 1 ml) contained 0.85 ml 0.1 M phosphate buffer (pH 7.5), 0.05 ml 8 mM DNTB (Sigma-Aldrich, Saint Louis, MO, USA) and 0.05 ml of the (S9) solution containing acetylcholinesterase fractions. After pre-incubation, the reaction was started by the addition of 0.05 ml 8.25 mM acetylthiocholine (Sigma-Aldrich, Saint Louis, MO, USA). Acetylcholinesterase activity was determined by kinetic measurement at 20 C using a Jenway 6105 spectrophotometer (l ¼ 420 nm). Results were expressed as mmoles thiocholine produced per min and per mg protein. 2.4. Statistical analysis Statistical analyses were performed with SP SS/PC (SP SS, Microsoft, and Redmond, WA). Significant differences between means were determined using one-way ANOVAs and the Duncan’s test for multiple range comparison with significance level established at P < 0.05. Spearman Correlation matrix was also calculated to study the relationships between the different biomarkers measured (Statistica Soft Inc.). Factor analysis of the variables analyzed was carried out by means of the principal component analysis (PCA) method with orthogonal rotation (Varimax) using the Systat 11 software (SYSTAT Software Inc.) (Varimax minimizes the number of variables that have high loadings on each factor). The PCA analysis was used as an effective technique simplifying the correlation structure through linear transformation of the original variables (Jolliffe, 1986).
Please cite this article in press as: Attig, H., et al., Effects of thermal stress and nickel exposure on biomarkers responses in Mytilus galloprovincialis (Lam), Marine Environmental Research (2013), http://dx.doi.org/10.1016/j.marenvres.2013.12.006
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3. Results The amounts of Ni in M. galloprovincialis digestive gland after 4 days exposure to the metals along with a temperature gradient (18 C, 20 C, 22 C, 24 C and 26 C) are provided in Fig. 1. Data show an increasing Ni accumulation in animals exposed to temperature ranging from 18 C to 24 C with a maximum reached at 24 C (4.57 0.56 mg/g dry weight). The minimal Ni accumulation was observed in mussels exposed to 18 C (2.99 0.23 mg/g dry weight). Interestingly, mussels exposed to 26 C exhibited the lowest Ni uptake (1.93 0.48 mg/g dry weight). The results relative to the MT concentrations are reported in Fig. 2. Our data show that MT concentrations significantly increased along with the temperature gradient in digestive gland tissues of mussels. The same trend was observed in musses exposed to increasing temperature and Ni. The maximum was observed after in animals exposed to Ni at 24 C with up to 115.45 9.21 mg/mg proteins Ni (83% increase respect to the absolute control). Moreover, at 20 C, 22 C and 24 C a significant difference was observed between mussels exposed to only heat stress and those exposed to heat stress and Ni in term of MT concentrations. The results relative to the CAT activity in mussels exposed to Ni and temperatures are reported in Fig. 3. Our data indicated that CAT activity maximum response was reached in animals exposed to Ni and 20 C with respectively 122.31 18.58 mmol/min/mg proteins. Animals co-exposed to 26 C and Ni showed the lowest CAT activity being inhibited respect to that registered in the absolute control animals (18 C) (61.49 8.23 mmol/min/mg proteins and 83.39 10.33 mmol/min/mg proteins respectively). The effect of Ni exposure along with the temperature gradient on the mussel digestive gland phase II enzyme activity is shown in Fig. 4. A significant response respect to relative control animals was recorded for all temperatures except for animals exposed to 26 C. A significant decrease in GST activity was recorded in animals coexposed to 26 C and Ni in comparison with animals exposed to 18 C (101.10 9.48 mmol/min/mg proteins and 123.41 10.53 mmol/ min/mg proteins respectively). In animals exposed only to the temperature gradient the GST activity was significantly increased at 22 C and 24 C when compared to animals exposed to 18 C.
Fig. 1. Ni accumulation in Mytilus galloprovincialis digestive gland in animals exposed for 4 days to Ni (770 mg/L) along with a temperature gradient (18 C, 20 C, 22 C, 24 C and 26 C). Data, expressed in mg/g dry weight (n ¼ 10), were analyzed by ANOVA þ Tukey’s post test. a: Statistically significant differences (P < 0.01) in comparison with control condition (18 C without Ni supply). b: Statistically significant differences (P < 0.01) in comparison with animals exposed to18 C/Ni for 4 days.
Fig. 2. Metallothioneins protein content in digestive gland of Mytilus galloprovincialis exposed to Ni and temperature. Mussels were exposed for 4 days to Ni (770 mg/L) along with a temperature gradient (18 C, 20 C, 22 C, 24 C and 26 C). Data, expressed as nmole/mn/mg proteins (n ¼ 10), were analyzed by ANOVA þ Tukey’s post test. a: Statistically significant differences (P < 0.01) in comparison with control condition (18 C without Ni supply). b: Statistically significant differences (P < 0.01) in comparison with animals exposed to18 C/Ni for 4 days.
The MDA accumulation evaluated as thiobarbituric acid reactive species (TBARS) in mussel digestive gland after 4 days exposure to Ni along with the temperature gradient is reported in Fig. 5. A temperature-dependent accumulation of MDA was observed being more pronounced in animals co-exposed to Ni with a maximum of 27.79 3.13 nmol/mg proteins reached after 4 days in mussels exposed to 26 C and Ni and a minimum in animals maintained at 18 C without Ni supply (14.67 1.13 nmol/mg proteins). The response of AChE activity in mussel’s digestive gland after exposure to Ni along with the temperature gradient is reported in Fig. 6. AChE activity was unchanged in mussels exposed to Ni for all the tested temperatures respect to mussels exposed to heat stress only. 24 C and 26 C exposure rendered a significant inhibition of the AChE activity when compared with absolute control animals (18 C). The correlation coefficients obtained between the investigated biomarkers in digestive gland of mussels exposed to the temperature gradient and to Ni along with the temperature gradient are reported respectively in Table 1A and B. MT, MDA and AChE activity were significantly correlated to the exposure temperature in animals exposed to heat stress (Table 1A). However, significant correlation was recorded between exposure temperature and MT, MDA, AChE and CAT activities in organisms exposed to the metals along with a temperature gradient (Table 1B). Results from PCA using biomarkers data in animals exposed to the heat stress revealed that the first axis (47.29% of the overall variance) was mainly influenced by CAT and GST activities (Fig. 7A). MDA accumulation and AChE activity and MT content mainly composed the second axis (23.76% of the variance). This axis appeared associated with a heat specific response since the five exposure temperatures were clearly separated. PCA was also obtained from biomarkers data in animals exposed to Ni along with the temperature gradient depicting a first axis (54.20% of the variance) mainly associated with AChE and GST activities (Fig. 7B). 4. Discussion The presence of contaminants in aquatic environments may results in an additional stress when coinciding with a-biotic
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Fig. 3. Catalase activity in digestive gland of Mytilus galloprovincialis exposed to Ni and temperature. Mussels were exposed for 4 days to Ni (770 mg/L) along with a temperature gradient (18 C, 20 C, 22 C, 24 C and 26 C). Data, expressed as nmole/mn/mg proteins (n ¼ 10), were analyzed by ANOVA þ Tukey’s post test. a: Statistically significant differences (P < 0.01) in comparison with control condition (18 C without Ni supply). b: Statistically significant differences (P < 0.01) in comparison with animals exposed to18 C/Ni for 4 days.
stressors such as heat stress. Recent reports indicated that heat stress increased the toxicity of polycyclic hydrocarbons and metals (Kamel et al., 2012; Negri et al., 2013; Sokolova and Lannig, 2008). Although bivalves are reported to have a higher tolerance to environmental changes, as compared with other marine organisms (Wang et al., 2011; Tomanek and Zuzow, 2010; Negri et al., 2013), reduction in clearance rates and respiration were observed upon in bivalves exposed to hypoxia and temperature fluctuations. The metal accumulation in mollusks was reported to be dependent of dose, exposure time and ambient water conditions
Fig. 4. Glutathione es e Transferase activity in digestive gland of Mytilus galloprovincialis exposed to Ni and temperature. Mussels were exposed for 4 days to Ni (770 mg/L) along with a temperature gradient (18 C, 20 C, 22 C, 24 C and 26 C). Data, expressed as nmole/mn/mg proteins (n ¼ 10), were analyzed by ANOVA þ Tukey’s post test. a: Statistically significant differences (P < 0.01) in comparison with control condition (18 C without Ni supply). b: Statistically significant differences (P < 0.01) in comparison with animals exposed to18 C/Ni for 4 days.
(Negri et al., 2013; Attig et al., 2010; Das and Jana, 1999). In the present study, Ni accumulation in the digestive gland tissue of mussels e metabolically the most active organ e was found to be modulated by the water temperature. Our data showed that the bioaccumulation was function of temperature until reaching 24 C. The reduction of Ni uptake at 26 C could be due to the reduction of the mussel ventilation rate at higher temperature. Indeed, Lannig et al. (2008) observed a synergistic effect of elevated temperatures and cadmium exposure that led to oxygen limitation by impaired performance in oxygen supply through ventilation and circulation in the eastern oysters Crassostrea verginica. Moreover, Anestis et al. (2007) reported that mussels M. galloprovincialis increased the duration of valve closure by about six fold when acclimated to 24 C rather than to 17 C. In the present work, MT concentration was found to be positively regulated by the ambient temperature in absence and in presence of Ni (r ¼ 0.84 in animals exposed only to heat stress and r ¼ 0.79 in animals exposed Ni along with heat stress (Table 1). However, no significant correlation was found between Ni loads in the DG and the MT concentrations (Table 1). Moreover, for a same exposure temperature, our data showed significant differences of MT concentration in the DG of mussels treated with Ni when compared to those expose only to heat stress (except at 26 C). From data obtained in the present work it appears clear that MT concentration do not rely directly on intracellular concentration of Ni but probably on the oxidative stress status of the cell as observed for heat stressed mussels. Indeed, recent reports indicated that MTs concentrations were significantly up-regulated by temperature increase and by a combination of heat stress and metal exposure (Ivanina et al., 2008, 2009). Therefore, it is of interest to establish the relation between metallothionein concentration and the oxidative stress status of the DG cells. Indeed the metal transcription factor 1 (MTF-1), functioning as a sensor of intracellular Zn, is responsible for both basal and Zn/Cu mediated expression of MT-1 in mammalian (Sadhu and Gedamu, 1988). This transcription factor may function in mussels as a sensor of the redox state of the cell, triggering a rapid and specific response to the presence of
Fig. 5. Malondialdehyde accumulation in digestive gland of Mytilus galloprovincialis exposed to Ni and temperature. Mussels were exposed for 4 days to Ni (770 mg/L) along with a temperature gradient (18 C, 20 C, 22 C, 24 C and 26 C). Data, expressed as nmole/mn/mg proteins (n ¼ 10), were analyzed by ANOVA þ Tukey’s post test. a: Statistically significant differences (P < 0.01) in comparison with control condition (18 C without Ni supply). b: Statistically significant differences (P < 0.01) in comparison with animals exposed to18 C/Ni for 4 days.
Please cite this article in press as: Attig, H., et al., Effects of thermal stress and nickel exposure on biomarkers responses in Mytilus galloprovincialis (Lam), Marine Environmental Research (2013), http://dx.doi.org/10.1016/j.marenvres.2013.12.006
H. Attig et al. / Marine Environmental Research xxx (2013) 1e7
Fig. 6. Acetylcholinesterase activity in digestive gland of Mytilus galloprovincialis exposed to Ni and temperature. Mussels were exposed for 4 days to Ni (770 mg/L) along with a temperature gradient (18 C, 20 C, 22 C, 24 C and 26 C). Data, expressed as nmole/mn/mg proteins (n ¼ 10), were analyzed by ANOVA þ Tukey’s post test. a: Statistically significant differences (P < 0.01) in comparison with control condition (18 C without Ni supply). b: Statistically significant differences (P < 0.01) in comparison with animals exposed to18 C/Ni for 4 days.
unbuffered, highly electrophilic metalsor oxidants (Dondero et al., 2005; Viarengo et al., 1999). Correlation between Ni exposure and MT induction is controversial. Ni ions are known to have a high affinity for sulfydrylic groups of proteins (Costa et al., 1994) and MT induction by Ni was reported not only in vertebrates such as the cod Eleginus navaga (Eriksen et al., 1991) but also in the copepod Tigriopus brevicornis (Barka et al., 2001), although with less extent than other metals (Cu, Zn, Hg, Cd, Ag) tested in the same organism (Barka et al., 2001). Recently our research group demonstrated that mussels exposed to Ni displayed a significant increase of mt10 mRNA abundance and no effects on the cognate mt20 (Dondero et al., 2011), indicating that Ni ions behave in the same way as copper to which mt20 is almost insensitive (Dondero et al., 2005). In mussels, the antioxidant defense system includes enzymes such as CAT, GST, superoxide dismutase (SOD) and other low molecular weight scavengers such as reduced glutathione (GSH) (Livingstone, 2001). Our data provided some evidences about the occurrence of oxidative stress in the digestive gland tissue of mussels exposed to Ni along with a heat stress gradient. Indeed a temperature dependent rise of MDA was observed along with a positive and significant correlation between MDA accumulation and temperature without or in presence of Ni supply (r ¼ 0.82 and 0.78, respectively). Catalase activity is considered as a key antioxidant enzymatic activity of the cell. The production of oxyradicals in mussels has been reported to be mediated by a higher range of contaminants including Ni (Attig et al., 2010; Dondero et al., 2011). Recent reports indicate the implication of higher temperatures in the enhancement of cellular (ROS) release in mussels, thereby increasing the risk of oxidative alterations (Kefaloyianni et al., 2005; Verlecar et al., 2007; Lockwood et al., 2010). Our data show a bell shape response of the catalase activity in animals exposed to the temperature gradient and a decreasing trend to the Ni along with the temperature gradient. This may suggest a potent cytotoxicity effect of Ni when associated to higher temperature (24 C and 26 C) (r ¼ 0.71) (Table 1B). GST activity has already been characterized in mussels (Fitzpatrick et al., 1995, 1997). From data discussed in this study, it appears clear that GST activity was not implicated in the response to heat stress as the correlation
5
coefficient was not significant with Temperature increase (Table 1A). However, GST activity was positively and significantly correlated with Ni loads in Ni exposed mussels along with the temperature gradient (r ¼ 0.82) (Table 1B), suggesting the implication of GST in Ni homeostasis as previously reported with other metals such as cadmium (Banni et al., 2009). AChE is considered highly responsive to biotic and abiotic changes in different marine species (Dimitriadis et al., 2012; Greco et al., 2011; Banni et al., 2010). Moreover, AChE was reported to be inhibited in mussels exposed to acute and subacute environmental contaminants including metals (Jebali et al., 2006; Viarengo et al., 2007; Banni et al., 2010). In the present study, we observed a significant increase of the AChE activity in mussels exposed to Ni along with the heat stress gradient (18 Ce24 C). However a significant inhibition of the AChE enzyme was depicted in mussels exposed to Ni at the highest temperature (26 C). AChE activity was found to be negatively correlated with temperature and Ni accumulation with a correlation coefficient of (r ¼ 0.62 and r ¼ 0.72 respectively (Table 1)). Even if little is known on Ni effects on AChE activity in mussels, our data provided clues about the occurrence of a negative effect of Ni; a non-conventional inhibitor of the AChE activity at higher temperatures. Biomarker data and Ni accumulation in mussels exposed to Ni along with the heat stress gradient were summarized by means of PCA (Fig. 7). In mussels exposed only to heat stress (Fig. 7A), PC1 discriminated data mainly following exposure temperature: results from heat stresses organisms showed positive scores in PC1 for temperatures 18 C, 20 C and 22 C; differently, data showed negative scores for temperatures 24 C and 26 C (Table 1A). PC1 was able to summarize biomarker responses in mussels: in particular eigenvectors loadings for PC1 indicated negative values to biomarkers such as GST, MT, MDA and a positive loading to AChE and CAT (Table 1B). For mussels exposed to Ni along with the heat stress gradient, the PCA analysis (Fig. 7B) showed that PC1 discriminated data also following Temperature. PC1 was able to discriminate the effects of higher temperatures (24 C and 26 C) mainly characterized by an increase in MDA and MT accumulation with respect to the other temperatures. PC2 described only a
Table 1 Correlation matrix of studied biomarkers and exposure time in heat stressed animals (A) and in Ni exposed M. galloprovincialis along with temperature gradient (B). Spearman’s correlation coefficient is provided as the top number and the significance of the correlation is provided as the bottom number for each comparison. CAT GST MDA AChE MT
[Ni] CAT GST MDA AChE MT
0.36 0.08 0.41 0.04 0.82* 0.001 L0.62* 0.001 0.84* 0.001 T 0.11 0.60 L0.75* 0.001 0.20 0.33 0.78* 0.001 L0.72* 0.001 0.79* 0.001 T
A 0.27 0.19 0.14 0.51 0.12 0.57 0.17 0.41 CAT
0.17 0.42 0.12 0.55 0.47 0.02 GST
0.49 0.06 0.69* 0.001 MDA
0.44 0.03 AChE B
0.03 0.90 0.64* 0.001 0.11 0.60 0.03 0.89 0.13 0.54 [Ni]
0.19 0.36 L0.58 0.001 0.46 0.04 L0.72 0.001 CAT
0.06 0.77 0.07 0.75 0.40 0.05 GST
0.48 0.02 0.68* 0.001 MDA
L0.62* 0.001 AChE
Please cite this article in press as: Attig, H., et al., Effects of thermal stress and nickel exposure on biomarkers responses in Mytilus galloprovincialis (Lam), Marine Environmental Research (2013), http://dx.doi.org/10.1016/j.marenvres.2013.12.006
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H. Attig et al. / Marine Environmental Research xxx (2013) 1e7
Fig. 7. Biplot from additional principal component analysis of all variables studied heat stressed animals (A) and in Ni exposed M. galloprovincialis along with temperature gradient (B).
minimal amount of data variance in both experiments (with/ without Ni supply).
and particularly in organisms challenging extreme temperature fluctuations.
5. Conclusion
Acknowledgments
The study of interactive effects of temperature and other environmental stressors such as metals on marine organisms especially those inhabiting intertidal zones may represent an important issue in future ecotoxicological research. The joint effect of stressors similar to that described in this study is not specific to metals and can be extended to other contaminants or factors that affect metals uptake and cellular response of exposed organisms. As a result, stress tolerance to environmental parameters such as temperatures increase will be reduced in animals inhabiting polluted areas of intertidal and coastal regions. The data reported in this work should be carefully considered in view of the biological effects of metals
This work was supported by funds from “Ministère de l’Enseignement Supérieur et de la Recherche Scientifique; UR04A6R05. Biochimie et Toxicologie Environnementale” and by Theme 6 of the EC seventh framework program through the Marine Ecosystem Evolution in a Changing Environment (MEECE No 212085) Collaborative Project.
References Amiard, J.C., Pineau, A., Boiteau, H.-L., Metayer, C., Amiard-Triquet, C., 1987. Application de la spectrométrie d’absorption atomique Zeeman aux dosages de huit
Please cite this article in press as: Attig, H., et al., Effects of thermal stress and nickel exposure on biomarkers responses in Mytilus galloprovincialis (Lam), Marine Environmental Research (2013), http://dx.doi.org/10.1016/j.marenvres.2013.12.006
H. Attig et al. / Marine Environmental Research xxx (2013) 1e7 éléments traces (Ag, Cd, Cr, Cu, Mn, Ni, Pb et Se) dans les matrices biologiques solides. Water Res. 21, 693e697. Anestis, A., Lazou, A., Pörtner, H.O., Michaelidis, B., 2007. Behavioral, metabolic, and molecular stress responses of marine bivalve Mytilus galloprovincialis during long-term acclimation at increasing ambient temperature. Am. J. Physiol. Regul. Integr. Comp. Physiol 293 (2), R911eR921. Attig, H., Dagnino, A., Negri, A., Jebali, J., Boussetta, H., Viarengo, A., Dondero, F., Banni, M., 2010. Uptake and biochemical responses of mussels Mytilus galloprovincialis exposed to sublethal nickel concentrations. Ecotoxicol. Environ. Saf. 73 (7), 1712e1719. Banni, M., Jebali, J., Daubez, M., Clerandeaux, C., Guerbej, H., Narbonne, J.F., Boussetta, H., 2005. Monitoring pollution in Tunisian coasts: application of a classification scale based on biochemical markers. Biomarkers 10 (2e3), 105e 116. Banni, M., Dondero, F., Jebali, J., Guerbej, H., Boussetta, H., Viarengo, A., 2007. Assessment of heavy metal contamination using real-time PCR analysis of mussel metallothionein mt10 and mt20 expression: a validation along the Tunisian coast. Biomarkers 12 (4), 369e383. Banni, M., Bouraoui, Z., Clerandeau, C., Narbonne, J.F., Boussetta, H., 2009. Mixture toxicity assessment of cadmium and benzo[a]pyrene in the sea worm Hediste diversicolor. Chemosphere 77, 902e906. Banni, M., Negri, A., Dagnino, A., Jebali, J., Ameur, S., Boussetta, 2010. Acute effects of benzo[a]pyrene on digestive gland enzymatic biomarkers and DNA damage on mussel Mytilus galloprovincialis. Ecotoxicol. Environ. Saf. 73 (5), 842e848. Banni, M., Negri, A., Mignone, F., Boussetta, H., Viarengo, A., Dondero, F., 2011. Gene expression rhythms in the mussel Mytilus galloprovincialis (Lam.) across an annual cycle. PLoS One 6 (5), e18904. Barka, S., Pavillon, J.F., Amiard, J.C., 2001. Influence of different essential and nonessential metals on MTLP levels in the Copepod Tigriopus brevicornis. Comp. Biochem. Physiol. C 128, 479e493. Bradford, M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248e254. Buege, J.A., Aust, S.D., 1978. Microsomal lipid peroxidation. In: Packer, L. (Ed.), Meth. Enzymol, vol. 52, pp. 302e310. Canesi, L., Negri, A., Barmo, C., Banni, M., Gallo, G., Viarengo, A., Dondero, F., 2011. The organophosphate chlorpyrifos interferes with the responses to 17b-estradiol in the digestive gland of the marine mussel Mytilus galloprovincialis. PLoS One 6 (5), e19803. Claiborne, A., 1985. Catalase activity. In: Greenwald, R.A. (Ed.), CRC Handbook of Methods in Oxygen Radical Research. CRC Press, Florida, pp. 283e294. Costa, M., Zhuang, Z., Huang, X., Cosentino, S., Klein, C.B., Salnikov, K., 1994. Molecular mechanisms of nickel carcinogenesis. Sci. Total Environ. 148, 191e199. Das, S., Jana, B.B., 1999. Dose-dependent uptake and Eichhornia-induced elimination of cadmium in various organs of the freshwater mussel, Lamellidens marginalis (Linn.). Ecol. Eng. 12, 207e229. Denkhaus, E., Salnikov, K., 2002. Nickel essentiality, toxicity, and carcinogenicity. Crit. Rev. Oncol. Hematol. 42, 35e56. Dimitriadis, V.K., Gougoula, C., Anestis, A., Pörtner, H.O., Michaelidis, B., 2012. Monitoring the biochemical and cellular responses of marine bivalves during thermal stress by using biomarkers. Mar. Environ. Res. 73, 70e77. Dondero, F., Piacentini, L., Banni, M., Rebelo, M., Burlando, B., Viarengo, A., 2005. Quantitative PCR analysis of two molluscan metallothionein genes unveils differential expression and regulation. Gene 345, 259e270. Dondero, F., Banni, M., Negri, A., Boatti, L., Dagnino, A., Viarengo, A., 2011. Interactions of a pesticide/heavy metal mixture in marine bivalves: a transcriptomic assessment. BMC Genomics 12, 195. Ellman, G.L., Courtney, K.O., Andres, V., Featherstone, R.M., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88e95. Eriksen, K.D.H., Andersen, T., Stenersen, J., 1991. Iso-metallothionein in cod induced by cadmium, zinc and nickel. In: Proceedings of the 12th Annual Conference on Physiological and Biochemical Approaches to the Toxicological Assessment of Environmental Pollution. Fitzpatrick, P.J., O’Halloran, J., Sheehan, D., Walsh, A.R., 1997. Assessment of a glutathione S-transferase and related proteins in the gill and digestive gland of Mytilus edulis (L.), as potential organic pollution biomarkers. Biomarkers 2, 51e 56. Fitzpatrick, P.J., Sheehan, D., Livingstone, D.R., 1995. Studies on isoenzymes of glutathione S transferase in the digestive gland of Mytilus galloprovincialis with exposure to pollution. Mar. Environ. Res. 39, 241e244. Greco, L., Pellerin, J., Capri, E., Garnerot, F., Louis, S., Fournier, M., Sacchi, A., Fusi, M., Lapointe, D., 2011. Physiological effects of temperature and a herbicide mixture on the soft-shell clam Mya arenaria (Mollusca, Bivalvia). Environ. Toxicol. Chem. 30 (1), 132e141. Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione S-transferases, the first step in mercapturic acid formation. J. Biol. Chem. 249, 7130e7139.
7
Ivanina, A.V., Cherkasov, A.S., Sokolova, I.M., 2008. Effect of cadmium on cellular protein and glutathione synthesis and expression of stress proteins in eastern oysters, Crassostrea virginica Gmelin. J. Exp. Biol. 211, 577e586. Ivanina, A.V., Taylor, C., Sokolova, I.M., 2009. Effects of elevated temperature and cadmium exposure on stress protein response in eastern oysters Crassostrea virginica (Gmelin). Aquat. Toxicol. 91, 245e254. Jebali, J., Banni, M., DE Alemeida, E.-A., Bannaoui, A., Boussetta, H., 2006. Effects of malathion and cadmium on acetylcholinesterase activity and metallothionein levels in the fish Seriola dumerilli. Fish Physiol. Biochem. 32, 93e98. Jolliffe, I.T., 1986. Principal Component Analysis. Springer Verlag, New York, NY, USA. Kamel, N., Attig, H., Dagnino, A., Boussetta, H., Banni, M., 2012. Increased temperatures affect oxidative stress markers and detoxification response to benzo[a] pyrene exposure in mussel Mytilus galloprovincialis. Arch. Environ. Contam. Toxicol. 63 (4), 534e543. Kefaloyianni, E., Gourgou, E., Ferle, V., Kotsakis, E., Gaitanaki, C., Beis, I., 2005. Acute thermal stress and various heavy metals induce tissue-specific pro- or antiapoptotic events via the p38-MAPK signal transduction pathway in Mytilus galloprovincialis (Lam.). J. Exp. Biol. 208 (23), 4427e4436. Kienle, C., Kohler, H.R., Gerhardt, A., 2009. Behavioural and developmental toxicity of chlorpyrifos and nickel chloride to zebra fish (Danio rerio) embryos and larvae. Ecotoxicol. Environ. Saf. 72, 1740e1747. Lannig, G., Cherkasov, A.S., Pörtner, H.O., Bock, C., Sokolova, I.M., 2008. Cadmiumdependent oxygen limitation affects temperature tolerance in eastern oysters (Crassostrea virginica Gmelin). Am. J. Physiol. Regul. Integr. Comp. Physiol. 294, R1338eR1346. Livingstone, D.R., 2001. Contaminant-stimulated reactive oxygen species production and oxidative damage in aquatic organisms. Mar. Pollut. Bull. 42, 656e666. Lockwood, B.L., Sanders, J.G., Somero, G.N., 2010. Transcriptomic responses to heat stress in invasive and native blue mussels (genus Mytilus): molecular correlates of invasive success. J. Exp. Biol. 213, 3548e3558. Narbonne, J.F., Daubeze, M., Clérandeau, C., Garrigues, P., 1999. Scale of classification based on biochemical markers in mussels: application to pollution monitoring in European coasts. Biomarkers 4, 415e424. Negri, A., Oliveri, C., Sforzini, S., Mignione, F., Viarengo, A., Banni, M., 25 2013. Transcriptional response of the mussel Mytilus galloprovincialis (Lam.) following exposure to heat stress and copper. PLoS One 8 (6), e66802. Pane, E.F., Smith, C., McGeer, J.C., Wood, C.M., 2003. Mechanism of acute and chronic waterborne nickel toxicity in the freshwater cladoceran Daphnia magna. Environ. Sci. Technol. 37 (19), 4382e4389. Papachristou, P., Haralambous, K.J., Loizidou, N., Spyrellis, N., 1993. Studies on the nickel removal from aqueous solutions. J. Environ. Sci. Health A 28 (1), 135e142. Sadhu, C., Gedamu, L., 1988. Regulation of human metallothionein (MT) genes. Differential expression of MTI-F, MTI-G, and MTII-A genes in the hepatoblastoma cell line (HepG2). J. Biol. Chem. 263, 2679e2684. Scholze, M., Knorr, W., Arnell, N.W., Prentice, I.C., 2006. A climate-change risk analysis for world ecosystems. Proc. Natl. Acad. Sci. U. S. A. 103 (35), 13116e13120. Sokolova, I.M., Lannig, G., 2008. Interactive effects of metal pollution and temperature on metabolism in aquatic ectotherms: implications of global climate change. Clim. Res. 37, 181e201. Tlili, S., Métais, I., Boussetta, H., Mouneyrac, C., 2010. Linking changes at subindividual and population levels in Donax trunculus: assessment of marine stress. Chemosphere 81 (6), 692e700. Tomanek, L., Zuzow, M.J., 2010. The proteomic response of the mussel congeners Mytilus galloprovincialis and M. trossulus to acute heat stress: implications for thermal tolerance limits and metabolic costs of thermal stress. J. Exp. Biol. 213 (Pt 20), 3559e3574. Vandenbrouck, T., Soetaert, A., Van der Ven, K., Blust, R., De Coen, W., 2009. Nickel and binary metal mixture responses in Daphnia magna: molecular fingerprints and (sub) organismal effects. Aquat. Toxicol. 92 (1), 18e29. Verlecar, X.N., Jena, K.B., Chainy, G.B., 2007. Biochemical markers of oxidative stress in Perna viridis exposed to mercury and temperature. Chem. Biol. Interact. 167 (3), 219e226. Viarengo, A., Burlando, B., Cavaletto, M., Marchi, B., Ponzano, E., Blasco, J., 1999. Role of metallothionein against oxidative stress in the mussel Mytilus galloprovincialis. Am. J. Physiol. 277, 1612e1619. Viarengo, A., Ponzano, E., Dondero, F., Fabbri, R., 1997. A simple spectrophotometric method for metallothionein evaluation in marine organisms: an application to Mediterranean and Antarctic molluscs. Mar. Environ. Res. 44, 69e84. Viarengo, A., Lowe, D., Bolognesi, C., Fabbri, E., Koehler, A., 2007. The use of biomarkers in biomonitoring: a 2-tier approach assessing the level of pollutantinduced stress syndrome in sentinel organisms. Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 146 (3), 281e300. Wang, Y., Hu, M., Wong, W.H., Shin, P.K., Cheung, S.G., 2011. The combined effects of oxygen availability and salinity on physiological responses and scope for growth in the green-lipped mussel Perna viridis. Mar. Pollut. Bull. 63 (5e12), 255e261.
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