Abstract
This study investigates cadmium effects on key messenger RNA (mRNA) expression (MT, MnSOD, CuZnSOD, CAT, ABCB1, HSP70, and CO1) by qPCR in the cockle Cerastoderma glaucum after chronic exposure to two high but environmentally relevant concentrations of CdCl2 (50 μg/L and 5 mg/L) for 12 h to 18 days. Cd accumulation measured in cockles’ tissues is significantly higher in both treatment conditions compared to controls and in a dose-dependent manner. Stress on stress tests performed at different times of the experiment clearly demonstrated that exposure to both concentrations of Cd significantly affects cockle survival time in air. Important changes in gene transcription were also highlighted. In particular, MT, HSP70, CAT, and CuZnSOD seem to be relevant biomarkers of Cd exposure because (1) their mRNA levels increase upon exposure and (2) they are highly correlated to Cd accumulation in tissues. Results may be useful for control strategies and for the use of cockles as sentinel organisms.
Similar content being viewed by others
Explore related subjects
Discover the latest articles, news and stories from top researchers in related subjects.Avoid common mistakes on your manuscript.
Introduction
Cadmium is released in the aquatic environment from both anthropogenic and natural sources (Choi et al. 2007). It is one of the most toxic metals principally obtained as a by-product in zinc refining (Pinot et al. 2000) and found in phosphate fertilizers (Cupit et al. 2002). Cadmium can enter the aquatic food chain through direct consumption of water or biota and has the potential to accumulate at each level of the food chain so that human may amass large quantity of Cd through diet (Zhu et al. 2012). Therefore, Cd is considered as a serious environmental health threat. Major programs of freshwater safeguard have studied the deleterious impacts of Cd at the ecosystem, community, or population levels of organization (Brooks et al. 2004; Al Kaddissi et al. 2012).
Many aquatic organisms are of potential interest as ecologically sensitive targets of metallic pollution. Among them, bivalves are regularly used as bioindicators of water metal pollution because of their high bioaccumulation capacities for heavy metals (Inza et al. 1997) and because they are well represented in aquatic ecosystems. Responses to Cd exposure have been largely investigated in different bivalve species such as cockles (Ladhar-Chaabouni et al. 2008; Paul-Pont et al. 2012), zebra mussels (Navarro et al. 2011), oysters (Ivanina et al. 2008a, b; Sanni et al. 2008), bay scallops (Wang et al. 2009), clams (Wang et al. 2010), and freshwater bivalves (Bigot et al. 2009). To highlight the response to Cd exposure, authors used different biomarkers at various biological organization levels, i.e., biochemical, transcriptional, proteomic, and histochemical. These studies showed that Cd exposure could inhibit the activity of some mitochondrial enzymes and usually induces expression of detoxification and other defense genes, but their regulation remains complex. In Tunisia, the Gulf of Gabès was shown to be contaminated by trace metals, especially Cd, which originated from the crude phosphate treatment from plants (Hamza-Chaffai et al. 2003; Smaoui-Damak et al. 2003). Cadmium concentrations in bivalves collected from some localities along this area exceeded the consumption safety level currently authorized by the World Health Organization (Smaoui-Damak et al. 2003; Machreki-Ajmi and Hamza-Chaffai 2006).
The cockle Cerastoderma glaucum is widely distributed from the Baltic to the Mediterranean Sea in a wide range of ecological conditions (Derbali et al. 2012) and has been validated as a bioindicator organism reflecting the pollution state of the coast. This sedentary and filter-feeding species lives in the superficial sediment and is known to be suitable for toxicology and risk assessment studies since it tends to accumulate large amounts of pollutants and especially metals such as Cd (Szefer et al. 1999; Cheggour et al. 2001; Machreki-Ajmi et al. 2008). Cockles are major preys for diverse animal groups such as crustaceans, fishes, and wading birds (Paul-Pont et al. 2010a), and they may also contribute to reduce the particulate organic load (Derbali et al. 2012). Beyond these ecological roles, they are also commercially important resources (Paul-Pont et al. 2010a). Some investigators have recently focused on the biology of Cerastoderma glaucum and the responses to different biochemical, immunotoxicological, and endocrine disruptors were studied using biomarkers that have been employed for the analysis of pollution impacts in estuaries and toxicity risk assessment of specific contaminants (Matozzo et al. 2007; Machreki-Ajmi and Hamza-Chaffai 2008; Marin et al. 2008). The qRT-PCR analysis of gene transcription has become one of the most robust tools of molecular biology. Changes in gene expression are likely to play a critical role in both acclimation and adaptation to environmental changes (Schulte 2004). Key changes in gene expression are also thought to precede the manifestation of strong functional alterations, giving expression profiling a great potential for early detection of alterations (Steiner and Anderson 2000; Schulte 2001; Mirbahai and Chipman 2014).
The present work aims to unravel the modifications of key messenger RNA (mRNA) expression underlying physiological responses of the cockle Cerastoderma glaucum to Cd exposure. The mRNA transcripts used for this analysis encode proteins involved in diverse pathways such as metal and xenobiotic detoxification (metallothionein, MT; ATP-binding cassette xenobiotic transporter, ABCB1), protection against oxidative stress (superoxide dismutases, MnSOD and CuZnSOD; catalase, CAT), general stress (heat shock protein, HSP70), and mitochondrial alterations (cytochrome c oxidase 1, CO1). In this study, gills were chosen for gene transcription monitoring. This organ constitutes a key interface for the uptake of dissolved metal ions from water and therefore was considered as the main target organ in metal exposure studies (Marigomez et al. 2002; Paul-Pont et al. 2010b).
Two concentrations of cadmium were used during an experimentation of 18 days: the lowest CdCl2 dose employed in this study (50 μg/L) is a high environmentally relevant concentration since it has been found in several contaminated sites (Blackmore 1998; Serafim and Bebianno 2007; Zhang et al. 2011; Zhao et al. 2014). The highest CdCl2 dose employed (5 mg/L) is less encountered, but it might be found in very highly polluted sites and especially during accidental pollution (Henry et al. 1984; Belabed et al. 1994). Survival rates of Cerastoderma glaucum and stress on stress tests (survival to air exposure) were assessed. Cd bioaccumulation levels were examined in the same individuals, and relationships between gene transcription and Cd levels were further examined in an attempt to highlight an mRNA expression pattern in cockles exposed in vivo to Cd.
Materials and methods
Animals and treatments
Cockles (Cerastoderma glaucum) (28–32 mm) were collected from the “Luza” site (located 45 km in the north of Sfax, Tunisia; Fig. 1), which is considered as a reference site with a low pollution level (Barhoumi et al. 2009; Kessabi et al. 2010). After an acclimation period of 24 h, cockles were assigned to three polyethylene tanks (30 × 30 × 20 cm), each one containing a layer of autoclaved sediment collected from the Luza site and 6 L of aerated seawater. The first tank contained seawater without CdCl2 (control), the second tank contained seawater with 50 μg/L of CdCl2 (low concentration), and the third tank contained seawater with 5 mg/L of CdCl2 (high concentration). Eighty to 90 cockles were held in each tank (experimental design schematized in Fig. 1), and exposure time varied between 12 h and 18 days (Fig. 1). During the experiment, seawater and CdCl2 were renewed twice a week.
Verification of metal content in the sediment used for the experimentation
A lyophilized sediment (0.5 g) from the Luza site was placed in glass tubes previously washed with hydrochloric acid (10 %) and then mineralized using 5 mL of nitric acid (HNO3) and 3 mL of hydrochloric acid (HCl). Ten milliliters of hydrochloric acid is then added to the dry residue. Determination of trace concentrations of Cd, Cu, Zn, Ni, Cr, Mn, and Pb was performed using inductively coupled plasma atomic emission spectrophotometry (ICP AES, Thermo Scientific iCAP 6000 series) (Geffard et al. 2001).
Stress on stress test
Ten animals of each condition (controls and CdCl2 contaminations) were sampled after 0, 1, 5, and 15 days of exposure and submitted to anoxia by air exposure at 15 °C (Fig. 1). Survival was assessed daily according to the method of Viarengo et al. (1995). Death symptoms were considered to be open valves and absence of muscular activity. Lethal time corresponding to 50 % of dead animals (LT50) was measured.
Condition index of cockles
Condition index (CI) was calculated individually on five cockles at each sampling time (Fig. 1). Before dissection, all cockles were weighted (total and soft weight), and the CI was expressed as a percentage of the ratio of fresh weight of soft tissues to total weight (Lobel et al. 1991).
Determination of cadmium content in cockles
Measurement of Cd concentration in cockles was carried out as previously described (Geffard et al. 2010) by electrothermic atomic absorption spectrophotometry with Zeeman correction, using a graphite furnace (SpectrAA Zeeman 220). The measure was done individually on five cockles at each sampling time (Fig. 1) on the remaining animal tissues including digestive gland, muscle, mantle, and foot.
RNA extraction and cDNA synthesis
Gene transcription analyses were carried out individually on the same five individuals for which Cd concentration and CI were measured and at each sampling time (Fig. 1). Gills of each sample were dissected, conserved in RNA later (Sigma-Aldrich), and stored at −20 °C. Total RNA was isolated from 100 mg of gills using Tri-Reagent (Invitrogen). RNA concentration and quality were determined using the Experion system (Bio-Rad). Three micrograms of total RNA was added to dTRace primer (5′-GACCACGCGTATCGATGTCGACTTTTTTTTTTTTTTTTT-3′) and nuclease free water in a final volume of 12.25 μL. A denaturation at 70 °C was carried out for 5 min and the mixture kept on ice. First-strand cDNAs were then synthetized using dTRace, RNAsin (Promega), and MMLV reverse transcriptase (Promega) in a final volume of 25 μL for 90 min at 42 °C.
Real-time quantitative PCR
Real-time PCR reactions were performed using a StepOnePlus apparatus (Applied Biosystems) with Fast SYBR Green Master Mix (Applied Biosystems) and specific primers (Table S1 and S2 in Supplementary data) designed on the basis of previously characterized sequences as described in details in Supplementary Data. The best housekeeping gene was selected among four genes tested using the Bestkeeper Software (Pfaffl et al. 2004), and the relative expression was determined by the comparative Ct method (Livak and Schmittgen 2001) using control cockles at time 0 (T 0) as calibrator (cf Supplementary Data).
Statistical analyses
All statistical analyses were performed using the R statistical Software version 2.14.1. In the stress on stress experiment, influence of Cd concentration and exposure time on ability to survive under anoxia was analyzed using the SURVREG function (survival package, R; Lumley and Therneau 2003). To test for significant differences, t tests were used for bioaccumulation values and Kolmogorov-Smirnov tests for gene transcription values. After normalization of the data by log(x + 1) transformation and stabilization of their variances by square-root transformation, influences of Cd treatment and exposure time on gene transcription, Cd bioaccumulation, and CI were assessed by independent ANOVA significance tests. Principal component analyses (PCAs) were performed and correlations were tested statistically using Pearson’s method to highlight correlations between gene transcription, Cd bioaccumulation, and CI.
Results
Heavy metal content analyzed from the sediment used for the experimentation showed that all the concentrations (Cd 0.109, Cu 0.941, Zn 7.082, Ni 0.727, Cr 2.717, Mn 6.301, Pb 1.654 μg/g of dry weight) were considerably below the maximum levels authorized by the Tunisian and French norms (T.N 106.002 and French Ministerial Decree of 22 February 1998).
Survival, stress on stress response, and CI
Survival rates of Cerastoderma glaucum were assessed for each exposure time, and the Cd-induced mortality remain below 10 % for each concentration and time point. Survival under anoxia (i.e., stress on stress assay) is significantly affected by Cd exposure (Fig. 2). Indeed, a significant decrease of lethal time (LT50) values is observed with the increase of Cd concentration (p < 0.001; SURVREG function, R) and exposure time (p < 0.001; Fig. 2). LT50 of controls reaches 8 days while decreasing from 8 days (after 1 day of exposure) to 2 days (after 15 days of exposure) for the concentration of 50 μg/L and from 6 days (at day 1) to 1 day (at day 15) for the concentration of 5 mg/L.
Neither low nor high exposure to CdCl2 affected the condition index (CI) of Cerastoderma glaucum after 18 days. No significant difference was observed comparing the mean CI for control (9.14 ± 2.42) and exposed groups (50 μg/L, 7.39 ± 1.75; 5 mg/L, 8.39 ± 1.98).
Cd bioaccumulation in cockles’ tissues
Cadmium concentration remains low and relatively constant in tissues of control cockles (Fig. 3). Following exposure to 50 μg/L of CdCl2, the tissue cadmium concentration was significantly increased to about 0.5 μg/g and displayed no significant variation in time (p > 0.05). On the contrary, the treatment with 5 mg/L of CdCl2 resulted in a dramatic accumulation of Cd that increased with exposure time (p < 0.001) up to 35.9 ± 10.850 μg/g at day 18 (Fig. 3).
Effect of Cd on relative gene transcription
Relative expression of the seven stress-regulated genes is represented as box plots with respect to the calibrator (controls at time 0) in Figs. 4 and 5. The global comparison of the gene transcriptions indicates a lower variability within sampling points in controls compared to exposed cockles (either 50 μg/L and 5 mg/L) for all the target genes studied (Figs. 4 and 5). However, exposition to CdCl2 globally led to an increase in gene transcription for CuZnSOD, CAT, MT, and HSP70 (up to +243, +49, +44, and +379 fold the mean expression level of control samples respectively; Fig. 4a–d, Table 1). Gene transcription was dependent of exposure time in some cases. In particular, gene transcription globally increased with exposure time for CuZnSOD, HSP70, and MT in cockles exposed to 50 μg/L of CdCl2 and for CAT and MT in cockles exposed to 5 mg/L of CdCl2 (Fig. 4a–d, Table 1). However, variation was rarely linear: Gene transcriptions of HSP70 and MT at Cd concentration of 50 μg/L and MT at Cd concentration of 5 mg/L first decreased to a minimum (−3, −19, and −47 fold the mean expression level of control samples respectively, after 1 to 3 days of exposure) and then increased (up to +379, +16, and +44 fold the mean expression level of control samples respectively; Fig. 4c, d). Concerning CO1, MnSOD, and ABCB1, expression was not influenced by the CdCl2 treatment in itself, but some of them were influenced by exposure time. Gene transcription globally decreased with time for MnSOD in cockles exposed to 50 μg/L of CdCl2 and for CO1 in cockles exposed to 5 mg/L of CdCl2 (Table 1). As previously, this decrease was nonlinear; expression first increased to a maximum, decreased abruptly, and slightly increased again (Fig. 5a, b).
PCAs
To highlight relationships between mRNA levels (ΔCt = Ctsample − CHKG), CI, and Cd bioaccumulation, two different PCAs were carried out, one considering controls and cockles exposed to 50 μg/L and the other one considering controls and cockles exposed to 5 mg/L (Fig. 6). All pair-wise correlations were tested statistically (Pearson’s correlation test), and the results are presented in Supplementary data (Tables S2 and S3).
In the 50 μg/L analyses (Fig. 6a, c), 58.3 % of the total variance is explained by the two main axes. Positive correlations are observed between Cd bioaccumulation and MT, HSP70, CuZnSOD, CO1, CAT, and ABCB1 mRNA levels (Fig. 6a, Table S3). No correlation is found between this cluster and MnSOD and CI (Fig. 6a and Table S3). The projection of the samples on the two main axes (Fig. 6c) indicates that all the control samples are found quite grouped on the right of the figure with the lowest Cd bioaccumulation and the lowest mRNA levels of HSP70, MT, CuZnSOD, CO1, CAT, and ABCB1. On the contrary, the cockles exposed to 50 μg/L of CdCl2 are found mostly on the left of the figure with the highest Cd bioaccumulation and the highest mRNA expression of the biomarkers cited above (Fig. 6c).
In the 5 mg/L analysis (Fig. 6b, d), 61.4 % of the total variance is explained by the two main axes represented in the figure. Strong positive correlations (p < 0.05, Table S4) are observed between Cd bioaccumulation, CAT, HSP70, MT, and CuZnSOD mRNA levels (Fig. 6b). This cluster is negatively correlated to MnSOD and CI while not correlated to CO1 and ABCB1 (Fig. 6b, Table S4). CO1 and ABCB1 are however clearly correlated with each other (Fig. 6b, Table S4, p < 0.05). The projection of the samples on the two main axes (Fig. 6d) indicates that all the control samples are found quite grouped on the right of the figure with the lowest Cd bioaccumulation and the lowest mRNA levels of HSP70, MT, CuZnSOD, and CAT and the highest CI and mRNA levels of MnSOD. On the contrary, the cockles exposed to 5 mg/L of CdCl2 are found mostly on the left of the figure with the highest Cd bioaccumulation and the highest mRNA expression of the biomarkers cited above (Fig. 6d). Two of the exposed samples are closely related to the CO1/ABCB1 cluster.
Discussion
The global physiological state of cockles was assessed along experiments by performing stress on stress tests and measuring condition index. According to Bayne (1986), stress results in a reduced capacity of individuals to adapt to changes in their environment. This concept was applied in this study through the surimposition of exposure to air (a natural stressor) over the effect of Cd stress (stress on stress tests) (Viarengo et al. 1995). The LT50 data demonstrates that exposure to both concentrations of CdCl2 significantly affects cockle survival time in air. Such trend probably reflects metabolic perturbations following CdCl2 exposure and a decrease of anoxia tolerance in accordance with previous data obtained on bivalves (Viarengo et al. 1995; Hamza-Chaffai et al. 1998; Ladhar-Chaabouni et al. 2008). This may suggest a decreased general health status among cockles exposed to CdCl2 and imply potential “trade-offs”, e.g., physiological allocations between detoxification and other processes linked to the survival of the organism, as previously described (Handy et al. 1999; Forbes 2000; Van Straalen and Hoffman 2000).
Measures of bivalve’s condition based on weight are thought to be reliable indicators of the energetic health status and energy reserves in mollusks. Lannig et al. (2006) found significant variations in condition index in oysters (Crassotrea virginica) after 20 days of exposure to both CdCl2 (50 μg/L) and temperature (28 °C). The results obtained in our study do not show any difference in the condition index between controls and exposed cockles. This finding probably highlights that this parameter is too much integrative and that only 18 days of exposition might not be enough to perceive or to produce variations between exposed cockles and control ones.
Because bivalves are filter-feeding organisms able to accumulate high concentrations of metals (Paul-Pont et al. 2012), a dose-dependent Cd accumulation in treated cockles was an expected result. Moreover, our study indicates that at a high level of CdCl2 exposure (5 mg/l), Cd accumulation is time-dependent. Ladhar-Chaabouni et al. (2008) also demonstrated in Cerastoderma glaucum that Cd accumulation was time- and dose-dependent for concentrations ranging from 50 to 150 μg/L, giving this species a great potential for long-term ecotoxicological studies. This bioaccumulation capacity of bivalves exposed to metallic contaminations was already highlighted in several species such as Cerastoderma edule (Paul-Pont et al. 2012), Ruditapes decussatus (Bebianno and Langston 1993; Smaoui-Damak et al. 2003, 2004), Corbicula fluminea (Legeay et al. 2005), and Dreissena polymorpha (Marie et al. 2006a, b). The present study adds the fact that Cerastoderma glaucum are particularly tolerant to higher cadmium concentration (5 mg/L) without exhibiting dramatic effects, indicating a strong resistance and the establishment of effective acclimation strategies.
The protection and/or detoxification processes probably settled by cockles may be studied at different levels and especially at the transcriptional level. Indeed, changes in gene expression are likely to play critical roles in acclimation of organisms (Schulte 2004). The aim of our study was to highlight modifications in mRNA expression of several genes involved in the tolerance to Cd exposure. Defense proteins involved in xenobiotic detoxification (MT, ABCB1), protection against oxidative stress (MnSOD, CuZnSOD, CAT), general stress (HSP70), and mitochondrial alterations (CO1) were chosen because their functions make them primary candidates for cellular protection against stress and because modifications of their gene expression had already been shown in invertebrates (Achard-Joris et al. 2006; Ivanina et al. 2008a, b; Navarro et al. 2011; Al Kaddissi et al. 2012).
A relatively low variation is observed in gene transcription along the experiment in control cockles indicating that mRNA expression is not affected by the experimental procedure. A high variability of gene transcription is observed for all target genes in exposed cockle biological variability that reflects the natural genotypic and phenotypic variation among individuals (Bustin 2010), but the much higher variability observed in exposed individuals suggests the existence of different types of response among individuals exposed to the same contaminant. Elements confirming this hypothesis may be found with the projections of individuals into the PCAs (Fig. 6c, d). For both concentrations of CdCl2, control individuals are found quite grouped, while exposed individuals are more scattered, some even found close to the controls (Fig. 6c, d).
Exposure to 50 μg/L and 5 mg/L resulted in important variations in the mRNA expression of four genes (MT, HSP70, CAT, and CuZnSOD). A time-dependent increase of MT mRNA abundance is observed after exposure to the two levels of Cd toxicity. MT gene transcription is highly correlated to Cd accumulation in the cockles’ tissues, meaning that individuals that accumulate the most Cd are also the ones with the highest MT mRNA levels. The MT isoform studied here is the MT1 that has been reported to be specific to Cd contamination (Gonzalez et al. 2006). The upregulation of this gene is in accordance with previous experiments performed with different invertebrates following metal exposure (Lecoeur et al. 2004; Dondero et al. 2005; Zorita et al. 2007; Navarro et al. 2011; Al Kaddissi et al. 2012). It may reflect the role of MTs in uptake and handling of metals, as well as antioxidants reacting with free radicals and reactive oxygen species (ROS), preventing interactions with critical cellular components such as enzymes, structural proteins, DNA, and membrane lipids (Andrews 2000; Amiard et al. 2006). The response of HSP70 is quite similar to that of MT after CdCl2 exposure and also highly correlated to Cd accumulation in tissues. Some studies have shown that HSP70s are synthesized intensively in the cell in response to a variety of harmful stimuli, including heat, heavy metals (Pb2+, Cd2+), organic contaminants, injuries, diseases, and other stressors (Singer et al. 2005). Navarro et al. (2011) and Ivanina et al. (2008a) also showed a strong increase in the response of this gene after Cd exposure in D. polymorpha and in Crassostrea virginica, respectively. HSP70 are involved in folding/refolding of newly synthesized and damaged proteins as well as in sequestering and degradation of proteins that are damaged beyond repair (Mayer and Bukau 2005). A strong upregulation of its gene (reaching +379 and +366 fold the mean expression level of controls for 50 μg/L and 5 mg/L of CdCl2, respectively) reflects a mechanism highly settled to protect cells against stress-induced damage. However, variations in expression of genes coding for MT and HSP70 were not completely linear (with a decrease in gene transcription following the first increase), reflecting a potential time-lag between gene and protein regulation. Analyses of protein concentrations would be particularly useful to give complementary data and to disentangle more precisely the regulation.
Several investigations have shown that Cd can indirectly promote oxidative stress in cells. This metal can bind to antioxidants or to their substrates. Consequently, impairment of the defense mechanism against ROS leads to an increase in ROS concentration (Wang et al. 2004; Cao et al. 2010). This fact could explain the increase in gene transcription of the antioxidant enzymes CAT and CuZnSOD following high and low CdCl2 exposure and their correlation to Cd accumulation in tissues. CuZnSOD (the cytosolic form of SODs) is known to catalyze the dismutation of superoxide anion into hydrogen peroxide, which is in turn reduced by CAT into water and molecular oxygen. The increase of these antioxidant enzymes in response to Cd exposure constitutes an adaptation in gills to prevent and repair metal-induced damage in cellular component (Fernandez et al. 2010). Similar results were reported for the clam larvae Meretrix meretrix; the SOD and CAT activities increased after exposure to 25 μg/L of Cd (Wang et al. 2010). Jo et al. (2008) also found that SOD and CAT mRNA expression levels increased significantly after 24 h of exposure to 0.1 ppm of Cd in the oyster Crassostrea gigas. In our study, the expression of MnSOD (the mitochondrial form of SODs) is not influenced by the CdCl2 treatment whatever its concentration and reflects natural physiological differences between cockles rather than a response to Cd. It can also be explained by the presence of sufficient basal concentration of antioxidant enzymes to protect cockles against oxidative stress.
Earlier studies have shown that energy misbalance is implicated in Cd-induced stress and toxicity in aquatic organisms, especially mitochondrial dysfunctions that lead to impaired ATP production, reduced aerobic capacity, and elevated oxidative stress (Sokolova et al. 2005; Cherkasov et al. 2007). As part of the mitochondrial respiratory chain, CO1 expression may be an interesting biomarker of the metabolic activity. Results showed that the CO1 mRNA expression is correlated to Cd accumulation at the concentration of 50 μg/L of CdCl2 with however no clear pattern along the 18 days of the experiment (an increase in expression might be observed but not significant). At the concentration of 5 mg/L of CdCl2, no correlation between CO1 expression and Cd accumulation was observed, and a downregulation of this gene along the experiment is even observed. The expression of CO1 thus appears to be modulated by the level of Cd concentration. Lerebours et al. (2010) previously stated that the intensity of gene response may not correlate positively with toxicant concentrations and that different gene expression patterns are expected for different concentrations of exposure. This is in concordance with our findings and tends to indicate that a modification in the metabolism might occur. Low concentration of Cd exposure tends to increase the level of CO1 mRNA and thus the mitochondrial metabolism. This could be a cellular strategy (1) to compensate for the decrease in the number of functional mitochondria by increasing ATP production, so as to provide enough energy for cellular needs, especially to tolerate Cd (Al Kaddissi et al. 2012), and (2) to efficiently consume O2 to limit Cd-induced oxidative damage in cells (Wang et al. 2004). It has to be noted that the transcription level of CO1 correlated well with those of CuZnSOD and CAT. Achard-Joris et al. (2006) found the same upregulation of the CO1 gene in three mollusk species following high Cd exposure. In contrast, in our study, exposure to the high concentration of CdCl2 (5 mg/L) led to a progressive decrease of the CO1 mRNA content, reflecting a potential decrease in the metabolic activity. This result tends to indicate a strong disturbance of cellular energy balance, with individuals no more able to compensate for the probable inhibition of the mitochondria function. Negative impacts on physiological performances and survival of organisms are expected (Sokolova and Lanning 2008). This is partially observed with the mortality rate higher in the 5 mg/L tank compared to the others and the LT50 (stress on stress) lower in this condition. Cases of repression/decrease of CO1 mRNA/activity were also recorded in the crayfish Procambarus clarkii and in Crassostrea gigas after acute and medium-term Cd exposure (Ivanina et al. 2008b; Al Kaddissi et al. 2012).
The ATP-binding cassette xenobiotic transporter ABCB1 gene transcription is significantly correlated to Cd accumulation at the concentration of 50 μg/L of CdCl2 but not at the concentration of 5 mg/L. However, it does not exhibit a clear pattern along the experiments. This protein, which acts by transporting toxic compounds out of the cell (Minier et al. 2006), seems to be an actor of the response to the medium exposure to CdCl2 (50 μg/L), but a strong variability in expression between individuals reduces the power of this biomarker.
In conclusion, the physiological trend detected in Cerastoderma glaucum exposed to low and high concentration of CdCl2 suggests a general decreased health status along the experiment. Gene transcription monitoring performed on seven genes potentially involved in the tolerance to Cd exposure showed that MT, HSP70, CuZnSOD, and CAT are relevant biomarkers of Cd exposure because of (1) their relative gene transcription higher in exposed individuals compared to controls and (2) their significant correlation to Cd accumulation. The upregulation of these genes indicates that Cd detoxification is promoted as well as protection against free radicals and intracellular oxidative damage. Following the mRNA expression of CO1 and ABCB1 gives complementary information on the status of exposed individuals, in particular their metabolic activity, but these biomarkers do not seem to be relevant with a chronic exposure. The molecular mechanisms underlined in this study imply energetic costs and therefore might explain the reduced tolerance of cockles to anoxia (stress on stress). The influence of contaminants other than metals of the environment from which the cockles were harvested as well as the influence of the experiment itself should however not be discarded. The tools developed may be useful both for future control strategies and for the use of the cockle Cerastoderma glaucum as a sentinel species. However, future investigations should be performed to validate these biomarkers in field conditions. Moreover, parallel biochemical, physiological, immunological, and morphological/pathological data should complement this study to better assess Cd toxicity and its mechanism of action.
References
Achard-Joris M, Gonzalez P, Marie V, Baudrimont M, Bourdineaud JP (2006) Cytochrome c oxydase subunit I gene is up-regulated by cadmium in freshwater and marine bivalves. Biometals 19:237–244
Al Kaddissi S, Legeay A, Elia AC, Gonzalez P, Floriani M, Cavalie I, Massabuau JC, Gilbin R, Simon O (2012) Mitochondrial gene expression, antioxidant responses, and histopathology after cadmium exposure. Environ Toxicol. doi:10.1002/tox.21817
Amiard JC, Amiard-Triquet C, Barka S, Pellerin J, Rainbow PS (2006) Metallothioneins in aquatic invertebrates: their role in metal detoxification and their use as biomarkers. Aquat Toxicol 76:160–202
Andrews GK (2000) Regulation of metallothionein gene expression by oxidative stress and metal ions. Biochem Pharmacol 59:95–104
Barhoumi S, Messaoudi I, Deli T, Said K, Kerkeni A (2009) Cadmium bioaccumulation in three benthic fish species, Salaria basilisca, Zosterisessor ophiocephalus and Solea vulgaris collected from the Gulf of Gabes in Tunisia. J Environ Sci 21:980–984
Bayne BL (1986) Measuring the effects of pollution at the cellular and organism level. In: Kullemberg G (ed) The role of the oceans as a waste disposal option. Springer, Netherlands, pp 617–634
Bebianno MJ, Langston WJ (1993) Turnover rate of metallothionein and cadmium in Mytilus edulis. Biometals 6:239–244
Belabed W, Kestali N, Semsari S, Gaid A (1994) Toxicity study of some heavy metals with daphnia test. Tech Sci Methods 6:331–336
Bigot A, Doyen P, Vasseur P, Rodius F (2009) Metallothionein coding sequence identification and seasonal mRNA expression of detoxification genes in the bivalve Corbicula fluminea. Ecotoxicol Environ Saf 72:382–387
Blackmore G (1998) An overview of trace metal pollution in the coastal waters of Hong Kong. Sci Total Environ 214:21–48
Brooks BW, Stanley JK, White JC, Turner PK, Wu KB, La Point TW (2004) Laboratory and field responses to cadmium: an experimental study in effluent-dominated stream mesocosms. Environ Toxicol Chem 23:1057–1064
Bustin SA (2010) Why the need for qPCR publication guidelines?—The case for MIQE. Methods 50:217–226
Cao L, Huang W, Liu J, Yin X, Dou S (2010) Accumulation and oxidative stress biomarkers in Japanese flounder larvae and juveniles under chronic cadmium exposure. Comp Biochem Physiol C Toxicol Pharmacol 151:386–392
Cheggour M, Chafik A, Langston WJ, Burt GR, Benbrahim S, Texier H (2001) Metals in sediments and the edible cockle Cerastoderma edule from two Moroccan Atlantic lagoons: Moulay Bou Selham and Sidi Moussa. Environ Pollut 115:149–160
Cherkasov AA, Overton RA, Sokolov EP, Sokolova IM (2007) Temperature-dependent effects of cadmium and purine nucleotides on mitochondrial aconitase from a marine ectotherm, Crassostrea virginica: a role of temperature in oxidative stress and allosteric enzyme regulation. J Exp Biol 210:46–55
Choi CY, An KW, Nelson ER, Habibi HR (2007) Cadmium affects the expression of metallothionein (MT) and glutathione peroxidase (GPX) mRNA in goldfish, Carassius auratus. Comp Biochem Physiol C Toxicol Pharmacol 145:595–600
Cupit M, Larsson O, de Meeus C, Eduljee GH, Hutton M (2002) Assessment and management of risks arising from exposure to cadmium in fertilisers—II. Sci Total Environ 291:189–206
Derbali A, El Hasni K, Jarboui O, Ghorbel M (2012) Distribution, abundance and biological parameters of Cerastoderma glaucum (Mollusca: Bivalvia) along the Gabes coasts (Tunisia, Central Mediterranean). Acta Adriat 53(3):363–374
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 345:259–270
Fernandez B, Campillo JA, Martinez-Gomez C, Benedicto J (2010) Antioxidant responses in gills of mussel (Mytilus galloprovincialis) as biomarkers of environmental stress along the Spanish Mediterranean coast. Aquat Toxicol 99:186–197
Forbes VE (2000) Is hormesis an evolutionary expectation? Funct Ecol 14:12–24
Geffard O, Budzinski H, Augagneur S, Seaman M, His E (2001) Assessment of sediment contamination by spermiotoxicity and embryotoxicity bioassays with sea urchins (Paracentrotus lividus) and oysters (Crassostrea gigas). Environ Toxicol Chem 20(7):1605–1611
Geffard A, Sartelet H, Garric J, Biagianti-Risbourg S, Delahaut L, Geffard O (2010) Subcellular compartimentalization of cadmium, nickel, and lead in Gammarus fossarum: comparison of methods. Chemosphere 78:822–829
Gonzalez P, Baudrimont M, Boudou A, Bourdineaud JP (2006) Comparative effects of direct cadmium contamination on gene expression in gills, liver, skeletal muscles and brain of the zebrafish (Danio rerio). Biometals 19:225–235
Hamza-Chaffai A, Romeo M, Gnassia-Barelli M, El Abed A (1998) Effect of copper and lindane on some biomarkers measured in the clam Ruditapes decussatus. Bull Environ Contam Toxicol 61:397–404
Hamza-Chaffai A, Pellerin J, Amiard JC (2003) Health assessment of a marine bivalve Ruditapes decussatus from the Gulf of Gabes (Tunisia). Environ Int 28:609–617
Handy RD, Sims DW, Giles A, Campbell HA, Musonda MM (1999) Metabolic trade-off between locomotion and detoxification for maintenance of blood chemistry and growth parameters by rainbow trout (Oncorhynchus mykiss) during chronic dietary exposure to copper. Aquat Toxicol 47:23–41
Henry M, Huang W, Cornet C, Belluau M, Durbec JP (1984) Accidental contamination by cadmium of the mollusc Ruditapes deeussatus: bioaccumulation and toxicity (LD 50, 96 H). Oceanol Acta 7(3):329–336
Inza B, Ribeyre F, Maury-Brachet R, Boudou A (1997) Tissue distribution of inorganic mercury, methylmercury and cadmium in the Asiatic clam (Corbicula fluminea) in relation to the contamination levels of the water column and sediment. Chemosphere 35:2817–2836
Ivanina AV, Cherkasov AS, Sokolova IM (2008a) Effects of cadmium on cellular protein and glutathione synthesis and expression of stress proteins in eastern oysters, Crassostrea virginica Gmelin. J Exp Biol 211:577–586
Ivanina AV, Habinck E, Sokolova IM (2008b) Differential sensitivity to cadmium of key mitochondrial enzymes in the eastern oyster, Crassostrea virginica Gmelin (Bivalvia: Ostreidae). Comp Biochem Physiol C Toxicol Pharmacol 148:72–79
Jo PG, Choi YK, Choi CY (2008) Cloning and mRNA expression of antioxidant enzymes in the Pacific oyster, Crassostrea gigas in response to cadmium exposure. Comp Biochem Physiol C Toxicol Pharmacol 147:460–469
Kessabi K, Navarro A, Casado M, Said K, Messaoudi I, Pina B (2010) Evaluation of environmental impact on natural populations of the Mediterranean killifish Aphanius fasciatus by quantitative RNA biomarkers. Mar Environ Res 70:327–333
Ladhar-Chaabouni R, Mokdad-Gargouri R, Denis F, Hamza-Chaffai A (2008) Cloning and characterization of cDNA probes for the analysis of metallothionein gene expression in the Mediterranean bivalves: Ruditapes decussatus and Cerastoderma glaucum. Mol Biol Rep 36:1007–1014
Lannig G, Flores JF, Sokolova IM (2006) Temperature-dependent stress response in oysters, Crassostrea virginica: pollution reduces temperature tolerance in oysters. Aquat Toxicol 79:278–287
Lecoeur S, Videmann B, Berny P (2004) Evaluation of metallothionein as a biomarker of single and combined Cd/Cu exposure in Dreissena polymorpha. Environ Res 94:184–191
Legeay A, Achard-Joris M, Baudrimont M, Massabuau JC, Bourdineaud JP (2005) Impact of cadmium contamination and oxygenation levels on biochemical responses in the Asiatic clam Corbicula fluminea. Aquat Toxicol 74:242–253
Lerebours A, Adam-Guillermin C, Brèthes D, Frelon S, Floriani M, Camilleri V, Garnier-Laplace J, Bourdineaud J-P (2010) Mitochondrial energetic metabolism perturbations in skeletal muscles and brain of zebrafish (Danio rerio) exposed to low concentrations of waterborne uranium. Aquat Toxicol 100:66–74
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods 25:402–408
Lobel PB, Bajdik CD, Belkhode SP, Jackson SE, Longerich HP (1991) Improved protocol for collecting mussel watch specimens taking into account sex, size, condition, shell shape, and chronological age. Arch Environ Contam Toxicol 21:409–414
Lumley T, Therneau T (2003) The survival package. The Comprehensive R Archive Network
Machreki-Ajmi M, Hamza-Chaffai A (2006) Accumulation of cadmium and lead in Cerastoderma glaucum originating from the Gulf of Gabes, Tunisia. Bull Environ Contam Toxicol 76:529–537
Machreki-Ajmi M, Hamza-Chaffai A (2008) Assessment of sediment/water contamination by in vivo transplantation of the cockles Cerastoderma glaucum from a non contaminated to a contaminated area by cadmium. Ecotoxicology 17:802–810
Machreki-Ajmi M, Ketata I, Ladhar-Chaabouni R, Hamza-Chaffai A (2008) The effect of in situ cadmium contamination on some biomarkers in Cerastoderma glaucum. Ecotoxicology 17:1–11
Marie V, Baudrimont M, Boudou A (2006a) Cadmium and zinc bioaccumulation and metallothionein response in two freshwater bivalves (Corbicula fluminea and Dreissena polymorpha) transplanted along a polymetallic gradient. Chemosphere 65:609–617
Marie V, Gonzalez P, Baudrimont M, Bourdineaud JP, Boudou A (2006b) Metallothionein response to cadmium and zinc exposures compared in two freshwater bivalves, Dreissena polymorpha and Corbicula fluminea. Biometals 19:399–407
Marigomez I, Soto M, Cajaraville MP, Angulo E, Giamberini L (2002) Cellular and subcellular distribution of metals in molluscs. Microsc Res Tech 56:358–392
Marin MG, Rigato S, Ricciardi F, Matozzo V (2008) Lethal and estrogenic effects of 4-nonylphenol in the cockle Cerastoderma glaucum. Mar Pollut Bull 57:552–558
Matozzo V, Rova G, Marin MG (2007) Haemocytes of the cockle Cerastoderma glaucum: morphological characterisation and involvement in immune responses. Fish Shellfish Immunol 23:732–746
Mayer MP, Bukau B (2005) Hsp70 chaperones: cellular functions and molecular mechanism. Cell Mol Life Sci 62:670–684
Minier C, Abarnou A, Jaouen-Madoulet A, Le Guellec AM, Tutundjian R, Bocquene G, Leboulenger F (2006) A pollution-monitoring pilot study involving contaminant and biomarker measurements in the Seine Estuary, France, using zebra mussels (Dreissena polymorpha). Environ Toxicol Chem 25:112–119
Mirbahai L, Chipman JK (2014) Epigenetic memory of environmental organisms: a reflection of lifetime stressor exposures. Mutat Res 13:157–171
Navarro A, Faria M, Barata C, Pina B (2011) Transcriptional response of stress genes to metal exposure in zebra mussel larvae and adults. Environ Pollut 159:100–107
Paul-Pont I, Gonzalez P, Baudrimont M, Jude F, Raymond N, Bourrasseau L, Le Goïc N, Haynes F, Legeay A, Paillard C, de Montaudouin X (2010a) Interactive effects of metal contamination and pathogenic organisms on the marine bivalve Cerastoderma edule. Mar Pollut Bull 60(4):515–525
Paul-Pont I, Gonzalez P, Baudrimont M, Nili H, de Montaudouin X (2010b) Short-term metallothionein inductions in the edible cockle Cerastoderma edule after cadmium or mercury exposure: discrepancy between mRNA and protein responses. Aquat Toxicol 97:260–267
Paul-Pont I, Gonzalez P, Montero N, de Montaudouin X, Baudrimont M (2012) Cloning, characterization and gene expression of a metallothionein isoform in the edible cockle Cerastoderma edule after cadmium or mercury exposure. Ecotoxicol Environ Saf 75:119–126
Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper–Excel-based tool using pair-wise correlations. Biotechnol Lett 26:509–515
Pinot F, Kreps SE, Bachelet M, Hainaut P, Bakonyi M, Polla BS (2000) Cadmium in the environment: sources, mechanisms of biotoxicity, and biomarkers. Rev Environ Health 15:299–323
Sanni B, Williams K, Sokolov EP, Sokolova IM (2008) Effects of acclimation temperature and cadmium exposure on mitochondrial aconitase and LON protease from a model marine ectotherm, Crassostrea virginica. Comp Biochem Physiol C Toxicol Pharmacol 147:101–112
Schulte PM (2001) Environmental adaptations as windows on molecular evolution. Comp Biochem Physiol B Biochem Mol Biol 128:597–611
Schulte PM (2004) Changes in gene expression as biochemical adaptations to environmental change: a tribute to Peter Hochachka. Comp Biochem Physiol B Biochem Mol Biol 139:519–529
Serafim A, Bebianno MJ (2007) Kinetic model of cadmium accumu- lation and elimination and metallothionein response in Ruditapes decussatus. Environ Toxicol Chem 26:960–969
Singer C, Zimmermann S, Sures B (2005) Induction of heat shock proteins (hsp70) in the zebra mussel (Dreissena polymorpha) following exposure to platinum group metals (platinum, palladium and rhodium): comparison with lead and cadmium exposures. Aquat Toxicol 75:65–75
Smaoui-Damak W, Hamza-Chaffai A, Berthet B, Amiard JC (2003) Preliminary study of the clam Ruditapes decussatus exposed in situ to metal contamination and originating from the Gulf of Gabes, Tunisia. Bull Environ Contam Toxicol 71:961–970
Smaoui-Damak W, Hamza-Chaffai A, Bebianno MJ, Amiard JC (2004) Variation of metallothioneins in gills of the clam Ruditapes decussatus from the Gulf of Gabes (Tunisia). Comp Biochem Physiol C Toxicol Pharmacol 139:181–188
Sokolova IM, Lanning G (2008) Interactive effects of metal pollution and temperature on metabolism in aquatic ectotherms: implications of global climate change. Climate Res 37:181–201
Sokolova IM, Sokolov EP, Ponnappa KM (2005) Cadmium exposure affects mitochondrial bioenergetics and gene expression of key mitochondrial proteins in the eastern oyster Crassostrea virginica Gmelin (Bivalvia: Ostreidae). Aquat Toxicol 73:242–255
Steiner S, Anderson NL (2000) Expression profiling in toxicology: potentials and limitations. Toxicol Lett 112–113:467–471
Szefer P, Wolowicz M, Kusak A, Deslous-Paoli J, Czarnowski W, Frelek K, Belzunce M (1999) Distribution of mercury and other trace metals in the cockle Cerastoderma glaucum from the Mediterranean Lagoon Etang de Thau. Arch Environ Contam Toxicol 36:56–63
Van Straalen NM, Hoffman AA (2000) Review of experimental evidence for physiological costs of tolerance to toxicants. In: Kammenga J, Laskowski R (eds) Demography in Ecotoxicology. John Wiley & Sons, pp 147–161
Viarengo A, Canesi L, Pertica M, Mancinelli G, Accomando A, Smaalb AC, Orunesu M (1995) Stress on stress response: a simple monitoring tool in the assessment of a general stress syndrome in mussels. Mar Environ Res 39:245–248
Wang Y, Fang J, Leonard SS, Rao KM (2004) Cadmium inhibits the electron transfer chain and induces reactive oxygen species. Free Radic Biol Med 36:1434–1443
Wang L, Song L, Ni D, Zhang H, Liu W (2009) Alteration of metallothionein mRNA in bay scallop Argopecten irradians under cadmium exposure and bacteria challenge. Comp Biochem Physiol C Toxicol Pharmacol 149:50–57
Wang Q, Wang X, Yang H, Liu B (2010) Analysis of metallotionein expression and antioxidant enzyme activities in Meretrix meretrix larvae under sublethal cadmium exposure. Aquat Toxicol 100:321–328
Zhang L, Liu X, Zhou D, Yu J, Zhao J, Wu H (2011) Toxicological effects induced by cadmium in gills of Manila clam Ruditapes 189 philippinarum using NMR-based metabolomics. Clean Soil Air Water 39:989–995
Zhao L, Zhang Y, Liang J, Xu X, Wang H, Yang F, Yan X (2014) Environmental cadmium exposure impacts physiological responses in Manila clams. Biol Trace Elem Res 159:241–253
Zhu B, Gao KS, Wang KJ, Ke CH, Huang HQ (2012) Gonad differential proteins revealed with proteomics in oyster (Saccostrea cucullata) using alga as food contaminated with cadmium. Chemosphere 87:397–403
Zorita I, Bilbao E, Schad A, Cancio I, Soto M, Cajaraville MP (2007) Tissue- and cell-specific expression of metallothionein genes in cadmium- and copper-exposed mussels analyzed by in situ hybridization and RT-PCR. Toxicol Appl Pharmacol 220:186–196
Acknowledgments
This research was supported by the European Program FP7 PEOPLE-IRSES GENERA (use of genomic and proteomic tools for the development of contaminant-specific biomarkers for the environmental risk assessment of aquatic ecosystems).
Author information
Authors and Affiliations
Corresponding author
Additional information
Responsible editor: Philippe Garrigues
Electronic supplementary material
Below is the link to the electronic supplementary material.
ESM 1
(DOCX 32 kb)
Rights and permissions
About this article
Cite this article
Karray, S., Marchand, J., Moreau, B. et al. Transcriptional response of stress-regulated genes to cadmium exposure in the cockle Cerastoderma glaucum from the gulf of Gabès area (Tunisia). Environ Sci Pollut Res 22, 17290–17302 (2015). https://doi.org/10.1007/s11356-014-3971-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11356-014-3971-8