Importance of metallothioneins in the cadmium detoxification process in Daphnia magna

Comparative Biochemistry and Physiology, Part C 144 (2006) 286 – 293 www.elsevier.com/locate/cbpc

Importance of metallothioneins in the cadmium detoxification process in Daphnia magna B. Fraysse a , O. Geffard b , B. Berthet c,d , H. Quéau b , S. Biagianti-Risbourg a , A. Geffard a,⁎ a

Laboratoire d'Eco-toxicologie, EA 2069 URVVC, faculté des Sciences, Université de Reims Champagne Ardenne Moulin de la Housse BP 1039, 51687 Reims Cedex2, France b Laboratoire d'écotoxicologie, Cemagref, 3 bis quai Chauveau, CP 220, 69336 Lyon, Cedex 9, France c Service d'Ecotoxicologie, SMAB, Pôle Mer et Littoral, 2 rue de la Houssinière, BP 92208, 44322 Nantes Cedex 3, France d Institut Catholique d'Etudes Supérieures, 85000 La Roche sur Yon, France Received 4 July 2006; received in revised form 11 October 2006; accepted 12 October 2006 Available online 19 October 2006

Abstract Good knowledge of the relationship between toxic metals and biological systems, particularly the sub-cellular fraction, could be a suitable early indicator of toxic effects. These effects and the sub-cellular behaviour of cadmium were studied with a widely used species in freshwater toxicity bioassays, Daphnia magna. In spite of this very commonplace usage in ecotoxicological studies, very few data are available on its toxicant metabolism and in particular metal homeostasis. Combining multi-tools analysis, a soluble protein was found: it is heat-stable, rich in sulfhydryl groups (differential pulse polarography), characterised by a molecular mass of approximately 6.5 kDa, with a G-75 chromatographic profile corresponding to the rabbit metallothioneins monomer, with few if any aromatic-containing amino acids, it binds metals (e.g. Cd, Cu), and its concentration increases with Cd exposure. This evidence led us to hypothesise that metallothioneins (MTs) are present in D. magna. Up to 75% of the Cd body burden with Cd exposure is bound to the MTs fraction. The increase in the Cd concentration in the surrounding medium and concomitantly in daphnids induces sub-cellular reorganisation of essential metals such as Cu and Zn. The rate of metals in the soluble cellular fraction and associated with MTs increases with the Cd body burden. Monitoring sub-cellular distribution of metals after exposure in the natural environment could be very useful for ecotoxicological assessment. © 2006 Elsevier Inc. All rights reserved. Keywords: Cadmium; Daphnia magna; Metallothioneins; Sub-cellular distribution

1. Introduction The knowledge of the sub-cellular behaviour of toxicants, mainly metals, in living organisms is fundamental to understanding their toxicological consequences at individual and trans-generation levels and along the food web (Barata et al., 2002; Wallace et al., 2003; Bragigand et al., 2004; Campbell et al., 2005). Tolerance or resistance to metal toxicity is based at least partly on controlling intracellular metal speciation. Sequestration by cellular ligands such as metallothioneins, lysosomes, and mineralised organically based concretions appears to be one of the most commonly adopted strategies by invertebrates (Langston et al., 1998; Marigomez et al., 2002; Wallace ⁎ Corresponding author. Tel.: +33 326 913 328; fax: +33 326 913 342. E-mail address: [email protected] (A. Geffard). 1532-0456/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2006.10.003

et al., 2003). The results of these different detoxification processes could be separated, after compartmentalisation, between different tissular fractions defined as insoluble fraction (metal concretions, organelles, nucleus, etc.) and soluble (i.e. cytosolic) fraction, including proteins of detoxification (metallothioneins) and target molecules such as enzymes (Mouneyrac et al., 1998; Ettajani et al., 2001). The cytosolic fraction was known to have an important role in the toxicokinetics (sequestration, elimination) and toxicodynamics of metals (Wang et al., 1999). One of these detoxification processes implies a metalloprotein, the metallothioneins (MTs), the presence of which has been recognised in numerous phyla, including vertebrates, invertebrates and micro-organisms (Amiard et al., 2006). MTs are metal-binding proteins characterised by a low molecular mass (6–7 kDa), a high content of cysteine (20–30%), which is a sulfhydryl group rich protein, with no aromatic amino acids,

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and heat-stable (Roesijadi, 1992). It is generally thought that these molecules play a role in the homeostasis of the essential metals zinc (Zn) and copper (Cu). The detoxification processes for non-essential trace elements such as cadmium (Cd) is mainly due to the binding potency of MTs, leading to chemical inactivation before sequestration in lysosomes or in mineral concretions, or excretion via body fluids (Klaassen et al., 1999). The literature shows that in invertebrates the synthesis of metallothioneins is often induced when organisms are exposed to metal-contaminated environments (Amiard et al., 2006). Aquatic ecosystems are the final sink for potentially all toxic metals in the environment, via transfer from natural and/or anthropogenic sources. Moreover, one of the freshwater species most recommended and used as standard bioindicator organisms for both water and sediment toxicity bioassays is the cladoceran Daphnia magna (Maltby and Calow, 1989; Persoone and Janssen, 1993). The common end-points are survival, growth and reproduction (Leppàen and Kukkonen, 1998). During the last decade there has been an increasing interest in investigating other end-points, especially in relation to those biochemical responses that may be considered early biomarkers of contamination (Huggett et al., 1992), as well as in predicting the long-term effects at higher biological levels of organisation (De Coen and Janssen, 2003a,b). In the case of metal-specific biomarkers such as MTs, very few studies have been published on daphnids. Only the presence of metallothionein-like proteins (MT-LPs) was indicated based on the nonheritability (Bodar et al., 1990) of Cd tolerance, associated with the observation of a low-molecular-mass protein that is heatresistant and binds cadmium (Stuhlbacher et al., 1992; Barata et al., 2002; Tsui and Wang, 2005). The present study sought to specify the toxicological responses of D. magna exposed to an experimental cadmium concentration gradient. The effect on the growth, reproduction and biochemical markers, and the MT-LPs level were recorded. In the second part, this paper describes the importance of MTLPs in Cd sequestration compared to various sub-cellular metal burdens during a cadmium exposure concentration representative of real environmental conditions. Third, the consequences of exposure to Cd, a non-essential metal, are investigated through the modifications of sub-cellular speciation for two essential metals linked to MT-LPs metabolism: copper and zinc. 2. Materials and methods 2.1. Daphnia exposure 2.1.1. Culture conditions A single clone of D. magna (CL 5 from Sheffield University) was used throughout the study. The daphnids were cultured at a constant temperature of 21° ± 1 °C and a 16:8 light:dark cycle. Natural uncontaminated drilling water was used in both daphnid cultures and the experiments. This water was previously mixed with water treated by osmosis system in order to reach a total hardness of 200 mg CaCO3 L− 1 and a conductivity of 600 μs cm− 1 in a pH between 7.8 and 8.2. Daphnid cultures

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consisted of 1-L glass bottles containing approximately 900 mL of culture medium and 20 organisms. Culture medium was renewed twice a week and the offspring produced were discarded every day. The animals were fed daily using unicellular green algae Selenastrum capricornicum (2 × 107 cell daphnid− 1). The algal culture medium was the LC medium, which does not contain EDTA. 2.1.2. Cd solutions All Cd solutions were obtained from a certified solution (Certipur, 1 g L− 1, Merck, Germany). Cd solutions were prepared in high-density polyethylene bottles and acidified (0.2% of HNO3) for storage. The volume of acidified Cd solutions used for the experimental exposure was up to 200 μL L− 1 of culture medium, with no pH variations. In all controls, 200 μL of acidified ultrapure water (0.2%) were added. 2.1.3. Experimental design Experiment 1: D. magna exposure to a Cd concentration gradient (Exp. 1). The aim of this first experiment was to study MT-LPs induction and the toxic effect (size, reproduction rate) of chronic exposure to a Cd contamination gradient in D. magna. Based on the standard chronic bioassay (ISO 10706), the real source of metal contamination was not controlled (water + food), since the use of algae as food may change the speciation and the bioavailability of Cd. Consequently, for this first experiment, done in triplicate, 20 juvenile animals (b24 h old) per concentration were exposed daily to dissolved Cd (8 h) and then to uncontaminated algae (16 h) for 21 days. Media were spiked with the following nominal Cd concentrations of 0 (control), 3.12, 6.25, 12.5 and 25 μg L− 1. Every day, daphnids were fed with increasing amount of algae to ensure a good growth within the exposure period (De Schamphelaere and Janssen, 2004), with respectively 8 × 106 cells per daphnid from day 0 to day 4, 1.2 × 107 cells per daphnid from 5 to 7, and 2 × 107 cells per daphnid from day 8 to 21. The medium was renewed every day and parent mortality and the number of juveniles were recorded. At the end of the 21-day exposure period, mortality and the number of juveniles were noted for the last time, and parent daphnids were collected to determine size and wet weight measurements, Cd body burden and MT-LPs concentration. To assess the body length, the organisms were photographed (PowerShot S50, Canon) and the size was estimated from the compound eye to the apical spine using image analysis software (SigmaScan Pro 8.0 sofware, SPSS Inc.). Experiment 2: characterisation of cadmium-binding compounds (Exp. 2). For this second experiment, 20 adult animals (15 days old) were transferred to 1-L polypropylene beakers containing a Cd contamination of 0 (control) or 0.5 μg L− 1. This experiment was conducted in 10 replicates with Cd concentration representative of environmental concentrations of impacted sites. The medium was renewed every day and daphnids were fed with 2 × 107 cells per organism. At the end of the 7-day exposure period, daphnids from the 10 replicates were mixed to obtain four samples with approximately 50 organisms.

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All samples of D. magna (Exp. 1 and 2) were frozen, freezedried and stored at ambient temperature in a dry atmosphere prior to being used for metal (Cd, Cu, Zn) and MT-LPs determination. The freeze-dried storage does not induce changes in MT-LPs and metal determination and in metal sub-cellular distribution (unpublished data). 2.2. Characterisation of metal-binding compounds and –SH groups and MT-LPs determination In order to characterise metal binding compounds, sulfhydryl groups, MT-LPs and metals concentrations, we applied the experimental protocol described by Berthet et al. (2005). Daphnid samples were homogenised with a hand-held glass-grinder in a TRIS buffer solution of 20 mM, NaCl 150 mM, pH 8.6, β-mercaptoethanol (10 mM L− 1 ) at 4 °C (4 mL g− 1 fresh weight). For samples used in gel permeation, PMSF (0.1 mM L− 1 ) was added to ensure molecular integrity of metallothioneins. Soluble (S1) and insoluble (P1) fractions were separated by initial centrifugation (25,000 g; 55 min at 4 °C). An aliquot fraction of the S1 fraction (1.2 mL for samples provided for gel permeation and 150 μL for other samples) was then heat-denatured (75 °C, 15 min) to precipitate the heat-denaturable compounds (C2) that were then separated from the heat-stable compounds (S2, containing MT-LPs) by centrifugation (15,000 g, 10 min at 4 °C). The S1 and S2 fractions of samples provided for gel permeation were stored at 4 °C until analysis, the same day that compartmentation for S2 and within 24 h for S1. Concentrations of metals were measured in P1 and S1 fractions, whereas MT-LPs were determined on the S2 fraction. Metals were not determined in S2 fractions because the fate of metals during heating is uncertain (Bragigand and Berthet, 2003). 2.2.1. Sephadex G-75 SF gel permeation Aliquots (1 mL) of the S1 and S2 fractions were fractionated by gel permeation using a Sephadex G-75 SF (Pharmacia) column with gel, 20 × 700 mm, and eluted with the same buffer used for sample preparation (TRIS 20 mM, NaCl 150 mM, pH 8.6). A flow rate of 0.3 mL min− 1 was used to elute the samples, which were collected as 2.3-mL fractions. Four replicates were used for each type of sample (S1 or S2). S1 molecular mass fractionation allowed us to evaluate the metal distribution and S2 fractionation was used for sulfhydryl group analysis. S2 fractionation was not used for metal determination for the reasons mentioned above (Bragigand and Berthet, 2003). The column was calibrated using known molecular mass standards ranging from 66 to 6.5 kDa (MW-GF-70; Sigma), and metallothioneins from rabbit liver as standard (Sigma). Column eluents were monitored continuously by UV absorbance at 280 nm (Hg lamp, Bio-Rad).

insoluble (P1) fractions were determined by flame (Zn) or electrothermal (Cd and Cu) atomic absorption spectrophotometry using Zeeman effect. The analytical method has been described previously by Amiard et al. (1987). The chromatographic fractions were acidified with 200 μL of HNO3 (65%, pro analysis, Merck). For the other samples (S1 and P1), an acid digestion step at 90 °C was required. This step lasted 12 h and involved the addition of nitric acid at a ratio of 1:1 S1 supernatant and 1 mL nitric acid per 0.5 g of P1 fraction. The solutions obtained were brought to a known volume (3 mL) with deionised water. The total metal body burden in daphnids was calculated by summing up the metal amounts in the soluble (S1) and insoluble (P1) fractions. Analytical methods were validated using certified reference material (lobster hepatopancreas: TORT 2, National Research Council of Canada). The percent recovery of TORT 2 reference samples (mean ± SD) was within the certified range for the three metals (Cd: 98 ± 5%; Cu: 100 ± 1%; Zn: 98 ± 1%; n = 3). Concerning the validation of metal analysis in S1 fractionation, the percent recovery compared to the global S1 value was determined. Based on a mean of the four samples, the mean recovery was 129 ± 48%, 110 ± 4%, and 100 ± 50% for Cd, Cu and Zn, respectively, in control individuals. For the exposed sampled, the mean recovery was 104 ± 10%, 114 ± 25% and 112 ± 14% for Cd, Cu and Zn, respectively. 2.4. Sulfhydryl groups and MTs concentration measurements Differential pulse polarography (DPP) was used to measure the sulfhydryl group concentration in S2 fractions, applied or not on the G75 SF column, corresponding to MTs or metallothionein-like proteins (MT-LPs). Sulfhydryl groups (–SH) were determined using Brdickà reagent (Bridckà, 1933) according to the method described by Thompson and Cosson (1984). This method has been validated within the BEQUALM (Biological Effects Quality Assurance in Monitoring Programmes; Allen et al., 2002). Measurements were performed at a constant temperature (4 °C) on a Metrohm 797 VA Computrace controlled by a personal computer, using the Metrodata 797 PC software (Metrohm). Quantification was based on rabbit liver MTs, using the method of standard addition. Sulfhydryl groups were determined in fractions collected after gel permeation (S2) on the same day as the chromatography. The high similarity between the polarogram generated by rabbit MTs and daphnid MTLPs confirmed the suitability of using rabbit MTs to calibrate the assay (Fig. 4). Concerning rabbit MTs, the elution recovery is approximately 106 × 6% (n = 2). The data obtained by DPP were expressed as micrograms of rabbit MTs equivalent (μg) per grams of fresh daphnid weight. 2.5. Statistical analysis

2.3. Metal determinations Metal (Cd, Cu, Zn) concentrations in the fractions obtained by gel permeation of cytosol (S1), in gross cytosolic (S1) and

Values were compared using one-way ANOVA (Statistica software) after checking the homogeneity of variances (Cochran's test). Significant differences (at the 95% level)

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were then determined using Tukey's HSD test. The results are presented as mean ± SD. 3. Results 3.1. Cadmium bioaccumulation, toxic effects and MT-LPs concentrations For the selected experimental conditions in Exp. 1, there were no significant effects on mortality whatever the Cd concentration. A slight reduction in growth is observed for the highest tested concentration, 25 μg L− 1 (Fig. 1). For the reproduction rate, a lower neonate production was observed for a Cd concentration higher than 3.13 μg L− 1 but the difference is only significant ( p b 0.05) in the higher concentration (25 μg L− 1). As shown in Fig. 2, Cd was bioaccumulated by D. magna in each fraction considered (whole body, soluble and insoluble fractions) according to the degree of exposure. The Cd burden in these fractions was linearly correlated with the exposure concentration ( p b 0.01). The weakest exposure concentration inducing a significant increase in Cd concentration for the three daphnid fractions was 6.25 μg L− 1. The distribution of Cd between the soluble and insoluble fractions was constant. Metal was stored mainly in soluble form, with 76 ± 2% of the total body burden in this sub-cellular fraction. Daphnids exposed for a shorter time (7 days) and to a lower Cd concentration (0.5 μg L− 1, Exp. 2) also showed a significant Cd accumulation in the different fractions, with a majority in the soluble fraction (75%, Table 1). The concentration of MT-LPs in daphnids exposed to the Cd gradient (Exp. 1) also increased with the Cd exposure concentration (data not shown): the lowest Cd concentration inducing such a significant effect was 12.5 μg L− 1. This MT-LPs concentration showed a good linear correlation with the soluble Cd burden (Fig. 3, p b 0.0001). Whereas the soluble Cd concentration was multiplied by approximately 20, comparing the values of the control and 25 μg L− 1 exposed groups, MT-LPs concentration increased only by a factor of 1.2. In addition, the

Fig. 1. Biological effects (size and reproduction rate) on D. magna after 21 days of Cd exposure (8 h/day) via the water route. The same letter indicates no significant difference for a given end-point (ANOVA, Tukey's HSD, p b 0.05).

Fig. 2. Concentration of Cd in whole body (WB), soluble (SF) and insoluble (IF) sub-cellular fractions in D. magna after exposure to Cd for 21 days (8 h/day) via the water route (wr) (mean ± SD, n = 3). Dotted lines: linear correlation for each fraction ( p b 0.005). Symbols with the same letter are not significantly different for a given fraction (ANOVA, Tukey's HSD, p b 0.01).

shape of this relation was weak (0.05) but significant. Contrary to daphnids exposed to the Cd gradient experiment, no induction was observed in daphnids exposed for 7 days to a low Cd concentration (Exp. 2). Concentrations of 592 μg (±122) and 540 μg (± 90) of rabbit MTs equivalent per g of D. magna were measured in controls and exposed individuals, respectively. 3.2. Thermoresistant and sulfhydryl groups contain proteins in the soluble fraction The S2 supernatant from daphnids of the second experiment (controls and individuals exposed to 0.5 μg Cd L− 1, Exp. 2), representing the cellular soluble fraction containing the thermoresistant proteins, was eluted with the Sephadex G-75 SF column. The sulfhydryl group burden contained by the eluted fractions was evaluated with DPP. Fig. 4A shows the sulfhydryl group elution profile of the rabbit MTs liver used as standard. Two peaks were observed, at approximately 6.5 and 13 kDa, corresponding to the MTs monomer (peak 2) and dimer (peak 1), respectively. This profile defines three different molecular areas of interest: (i) the area containing the highmolecular-weight proteins (HMW N 30 kDa), (ii) the area containing the MTs proteins (30 kDa N MT-MW N 2 kDa) and (iii) the area containing the low-molecular-weight compounds (LMW b 2 kDa). The elution profile of thermoresistant sulfhydryl-rich proteins obtained with D. magna heat-treated samples was not as well defined as the rabbit MTs profile (Fig. 4B). As noted before for the MT-LPs concentration, no difference occurred in shape and intensity between daphnids exposed or not exposed to Cd. Thiolic compounds were mainly (90%) present in fractions corresponding to a molecular mass between 2 and 20 kDa, with a maximum peak at 6.5 kDa and a second at 13 kDa, principally in control daphnids. The remaining 10% were distributed between the background and a weak peak in the LMW area. The latter is doubtless mainly attributable to β-mercaptoethanol, used during the homogenisation step (Bragigand and Berthet, 2003).

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Table 1 Distribution of Cd, Cu and Zn in sub-cellular fractions of D. magna exposed or not exposed to Cd (0.5 μg L− 1) for 7 days (mean ± SD, n = 4) Cd

Cu

Zn

Control

Cd exposure

Control

Cd exposure

Control

Cd exposure

Whole body Concentration (ng/g, fw)

61.7 ± 25.7

1294 ± 142.6⁎⁎

1212 ± 57.9

1239 ± 77.5

5832 ± 1415

6361 ± 574

Insoluble fraction Concentration (ng/g, fw) Contribution (%)

27.2 ± 15.3 42.3+ 6.9

313.7 ± 88.9⁎ 25.4 ± 9.6⁎

397.7 ± 16.5 32.9 ± 1.9

309.4 ± 64.0⁎ 25.0 ± 5.2⁎

2244 ± 588 38.8 ± 7.3

2351 ± 586 37.1 ± 9.3

Soluble fraction Concentration (ng/g, fw) Contribution (%)

34.5 ± 10.4 57.7+ 6.9

975.5 ± 224.1⁎⁎ 74.6 ± 9.6⁎

814.0 ± 58.1 67.1 ± 1.9

929.6 ± 96.2 75.0 ± ± 5.2⁎

3587 ± 1035 61.2 ± 7.3

4010 ± 711 62.9 ± 9.3

14.7 ± 3.4⁎ 76.2 ± 2.8⁎⁎ 9.1 ± 5.2

46.1 ± 9.0 41.5 ± 5.4 12.4 ± 5.5

34.8 ± 3.3 50.5 ± 2.0⁎ 14.7 ± 2.9

54.6 ± 18.6 10.3 ± 4.9 35.0 ± 21.9

48.3 ± 16.4 18.4 ± 6.3 33.2 ± 15.5

Soluble molecular mass distribution (%) HMW 33.1 ± 8.1 MT-MW 54.7 ± 4.3 LMW 12.2 ± 6.4

Footnote: asterisks indicate a statistical difference between the two treatments for a given metal (⁎: p b 0.05; ⁎⁎: p b 0.001).

During gel permeation of rabbit MTs liver or S2 heatdenatured supernatant of control or exposed daphnids (0.5 μg Cd L− 1), no absorbance was observed at 280 nm wave length in the molecular mass range of 2–30 kDa (data not shown).

(LMW), corresponding to the macromolecules (e.g. metalloenzyme), with 33 ± 8% of the eluted metal, and the small biomolecules (e.g. peptides, amino acids) and salts, containing 12 ± 6% of Cd. For Cd-treated organisms, the three main peaks were found again, with the same order of importance: MT-MW N

3.3. Distribution of Cd in the soluble fraction As shown in Table 1, the Cd concentration in each subcellular fraction increased notably after the Cd exposure. As in the first experiment, approximately 75% of the Cd was detected in the soluble fraction in exposed daphnids (Table 1). Fig. 5A presents the distribution of Cd in the soluble fraction according to the compound's molecular mass. For control organisms, the Cd was eluted in three main peaks. The highest, representing 55± 4% of the Cd soluble burden, was eluted at approximately 6.5 kDa (MT-MW) (Table 1). The second and third peaks in magnitude were the exclusion (HMW) and total volumes

Fig. 3. Relationship between soluble Cd fraction (SF) and metallothionein-like protein (MT-LPs) concentrations in D. magna after exposure to Cd for 21 days (8 h/day) via the water route (mean ± SD, n = 3). Dotted lines: linear correlation ( p b 0.001).

Fig. 4. Distribution of sulf hydryl groups in parts of molecular mass obtained from a standard rabbit MTs (A; peak 1: dimer, peak 2: monomer) and heattreated soluble faction from D. magna (B) exposed or not exposed to Cd (0.5 μg.L− 1, 7 days), using a Sephadex G75-SF column. HMW: high molecular mass (N30 kDa); MT-MW: metallothioneins molecular mass (30 kDa to 2 kDa); LMW: low-molecular-mass pool (b2 kDa). The MT-LPs concentrations are expressed as equivalent standard MTs rabbit signal obtained by differential pulse polarography.

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differed drastically from the two other metals. Zinc was divided into two main peaks, in the HMW and LMW areas, with a mean of 51% and 34% of the total soluble Zn burden, respectively (Fig. 5B, Table 1). The metal eluted in the MT-MW fraction corresponded to a mean of only 14% and can be considered as the background level. Whatever sub-cellular fraction was considered, no difference was observed in the Zn distribution between Cd-treated and untreated daphnids, perhaps in relation with the wide distribution of the data. The coefficient of variation (CV% = SD / mean × 100) calculated for Zn data ranged from 33% to 63%, whereas this value varied between 4% and 24% for Cd and Cu. 4. Discussion

Fig. 5. Distribution of Cd, Cu and Zn in parts of molecular mass obtained from soluble fractions from D. magna exposed (A, B) or not exposed (A) to Cd (0.5 μg L− 1, 7 days), using a Sephadex G75-SF column. HMW: high molecular mass (N30 kDa); MT-MW: metallothionein molecular mass (30 kDa to 2 kDa); LMW: low-molecular-mass pool (b2 kDa).

HMW N LMW. The major effect of the Cd treatment, compared to the control group was a shift in the metal associated with HMW proteins to the MTs fraction, with a contribution of 15 ± 3% and 76 ± 3%, respectively, compared with 33% and 55%, respectively, in these same fractions in controls (Table 1). The MT-MW fraction contained around 40 times more Cd after metal treatment, increasing from 19 to 743 ng g− 1 fw. 3.4. Distribution of Cu and Zn in the soluble fraction The whole body burden of Cu and Zn was not affected by the Cd treatment (Table 1). However, the distribution of Cu between the soluble and insoluble fractions was modified. Cu burden in the soluble fraction increased slightly but significantly. In the non-Cd-treated daphnids, this fraction contained 67 ± 2% of the Cu, versus 75 ± 5% in the Cd-treated organisms. For Zn, the distribution between soluble and insoluble fractions was constant, with roughly 60% and 40%, respectively. Differences occurred in the metal distribution in the soluble fraction between Cu and Zn in terms of molecular mass distribution and effects of Cd exposure. For Cu, the elution profiles were similar to those obtained with Cd, i.e. three main peaks distributed in each molecular-mass area (HMW, MT-MW, LMW; Fig. 5B). With Cd exposure, as observed for the Cd distribution, the part of Cu associated with the HMW fraction decreased from 46% to 35% of the soluble Cu burden, in parallel with the increase in the percentage of the MT-MW fraction, from 41% to 50% (Table 1). The elution profile for Zn

Previous studies on the toxicity of Cd to D. magna assumed the presence of MT-LPs compound family, based on heat-stable, low molecular mass and Cd-binding capacity criteria, particularly to explain a non-genetic tolerance to Cd exposure (Stuhlbacher et al., 1992; Barata et al., 2002; Guan and Wang, 2006). In addition, works demonstrated the inducibility of MTLPs synthesis in daphnids in relation with the exposure to Cd (Guan and Wang, 2004) and Hg (Tsui and Wang, 2005), using the Ag+ saturation method for protein quantification (the measurement is based on the very high affinity of soluble and heat-stable proteins for Ag+). The different criteria defined in these previous studies correspond to several properties of the metallothioneins according to Klaassen et al. (1999) and Chan et al. (2002). The present study on D. magna has shown the presence of proteins with similar criteria (heat-stable, low molecular mass, cytosolic location, metal-binding compounds) but specify several other properties in concordance with metallothionein characteristics. In this study, metal-binding proteins present molecular mass principally around 6.5 kDa and 13 kDa corresponding to the G-75 SF profile of rabbit MTs monomer and dimer. In addition, this molecular mass protein pool i) was rich in sulfhydryl groups appropriate to the substantial cysteine amino acid in these proteins, ii) presented no aromatic containing amino acids (absence of absorbance at 280 nm) and iii) presented an essential (Cu and Zn) and nonessential (Cd) metal-binding character. Even if most studies must be conducted to confirm the molecular mass using dimensional electrophoresis (SDS-page) and to specify the amino acid composition, protein characteristics observed in this study seem to show that metallothioneins are present in D. magna. We will discuss the use of metallothioneins (MTs) expression rather than metallothionein-like proteins (MT-LPs). The metal-binding capacities of metallothioneins resulted in several authors following two main rules, first in the control of homeostasis of essential metals such as zinc and copper and second in the detoxification process of non-essential metals and an excess of essential metals (Amiard et al., 2006 and the references cited therein). Similarly, the metal-binding properties of these proteins explain the possible MTs synthesis induction by these elements. Therefore, metal induction of MTs synthesis in several invertebrate species has led some authors to propose it as a metal-exposure biomarker in the biomonitoring context

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(Amiard et al., 2006). The exposure of daphnids to a Cd concentration gradient induced an increase in the Cd body burden and metallothioneins synthesis. The concentration of metallothioneins is linearly correlated with the concentration of Cd in the soluble fraction of the cladocera (Fig. 2). These results suggest the possible use of metallothioneins in D. magna as a Cd-exposure biomarker. However, the induction of MTs synthesis was observed in the first experiment for a relatively high Cd-exposure concentration (higher than 6.5 μg Cd L− 1) and not in the case of daphnids exposed to a low Cd concentration (0.5 μg L− 1) or during a short time of exposure (Exp. 2). Moreover, when the soluble Cd concentration is multiplied by 20, comparing the values of the control and 25 μg Cd L− 1 exposed group (Exp. 1), MTs concentrations increase only by a factor 1.2. Each metallothionein molecule can bind a large number of metal atoms (Cosson and Amiard, 2000) so that MTs concentrations may never increase in the same proportion as Cd concentrations. This may be a limit of the use of MTs concentrations as a biomarker of metal contaminants in the case of a moderate degree of metal exposure, as was observed in our second experiment. Consequently, the potential use of MTs in D. magna must be studied in more detailed laboratory experiments as well as in environmental conditions. After having specified the presence of MTs in D. magna, the second objective of this study was to evaluate its role in the subcellular distribution of Cd. Whatever the Cd exposure conditions, for 21 days, 8 h per day and a water-borne exposure (Exp. 1) or continuously over 7 days with a dietary and water-borne exposure (water and trophic; Exp. 2), 75% of the bioaccumulated Cd was in the soluble fraction (i.e. a mean of 976 ng g− 1 ; Exp. 2); in addition, 76% of this soluble Cd was found in the MT-MW area (i.e. 743 ng g− 1 ; Exp. 2), so the Cd bound to MTs represents more than half (i.e. 57%, corresponding to the 743 ng g− 1 compared to the 1294 ng g− 1 see Table 1) of the total Cd body burden in exposed daphnids. However, Cd bound to MTs represents only one-third (31%, i.e. 18.9 ng g− 1 ) of the total Cd body burden for non-exposed daphnids. We recall that the increase in Cd storage capability of the MTs fraction after a short exposure to a low Cd concentration (Exp. 2) was not related to an induction of the synthesis of these proteins. The total Cd body burden was multiplied by 20, comparing control daphnids and those exposed to 0.5 μg Cd L− 1 , and concerning the MT-MW proteins the Cd quantity was multiplied by 39. This was also observed by Guan and Wang (2004). These results suggest that the increasing capacity of Cd storage by the background level of MTs could be due to the displacement of essential metals, such as Zn or Cu, from MTs to other biological ligands. The same hypothesis is commonly suggested to explain this observation (Klaassen et al., 1999; Yang et al., 2000; Chan et al., 2002; Perceval et al., 2004). Another explanation could be the binding of Cd to free sulfhydryl groups available on the apo-proteins. Indeed, a low amount of apo-MTs traps the Zn ions newly transported to the cytosol cell (Suhy et al., 1999). This phenomenon is most probably concomitant to the first one and less important in Cdbinding capability.

When the Cd burden increases in cytosol (observed for both experiments), stored metal in each MW fraction also increases but within different ranges. Indeed, following Cd exposure, the metal concentration in the soluble fraction is multiplied by nearly 25, and Cd concentration in HMW compounds is multiplied by 10, by 25 for LMW compounds and by 40 for MT-MW compounds. Thus, the behaviour of the three compound fractions towards Cd affinity is quite distinct. Cd does not bind preferentially to HMW proteins, which gather functional biomolecules, and mainly enzymes. Cd's link to these molecules can lead to functional loss, via three-dimensional structure modifications and/or active site disruption (Perceval et al., 2004). It seems that when the metal concentration, linked to the protein fraction, exceeds a threshold value metal cellular toxicity occurs (Giguère et al., 2003). The quantity of Cd found in the LMW seems to be in equilibrium with the metal load present in cytosol. This fraction is composed of small biomolecules (e.g. polypeptides, amino acids) and salts. The association of Cd with these compounds limits its impact on the cellular biochemical activity by reducing its availability for macro-biomolecules (Giguère et al., 2003; Perceval et al., 2004). These findings confirm that monitoring the metal burden and distribution in the various subcellular fractions can be very useful in the context of ecotoxicological assessment of the natural environment. Indeed, D. magna, given its short life cycle, can be valuable in estimating the level of metal contamination (MTs concentration and induction rate, Campbell et al., 2005), the potential metal toxic impact on organisms (metal concentration bound to HMW proteins, Wallace et al., 2003; Perceval et al., 2004), and the bioavailability of the metal and its transfer to higher trophic levels (soluble and insoluble fractions of metals; Bragigand et al., 2004). Acknowledgements The authors would like to thank Laurence Delahaut for her technical support. Thanks are due to Linda Northrup for her careful English revision of manuscript. This study is part of the PNETOX Project EMMCC with financial support granted by the French Ministry of Ecology (Convention n°04 000 165). References Allen, Y., Balk, L., Colijn, F., Davies, I., Feist, S., Hylland, K., Lowe, D., Reckerman, M., Rumohr, H., Thain, J.E., 2002. Biological effects quality assurance in monitoring programmes (BEQUALM). EU Contract N°:SMT4-CT98-2241. Final Report V2, CEFAS Contract Report C0629, pp. 19–23. Amiard, J.-C., Pineau, A., Boiteau, H.L., Métayer, C., Amiard-Triquet, C., 1987. Application de la spectrométrie d'absorption atomique Zeeman aux dosages de huit éléments traces (Ag, Cd, Cr, Cu, Mn, Ni, Pb et Se) dans des matières biologiques solides. Water Res. 21, 693–697. Amiard, J.-C., Amiard-Triquet, C., Barka, S., Pellerin, J., Rainbow, P.S., 2006. Metallothioneins in aquatic invertebrates: their role in metal detoxification and their use as biomarkers. Aquat. Toxicol. 76, 160–202. Barata, C., Markich, S.J., Baird, D.J., Taylor, G., Soares, A.M.V.M., 2002. Genetic variability in sublethal tolerance to mixtures of cadmium and zinc in clones of Daphnia magna Straus. Aquat. Toxicol. 60, 85–99.

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