Environ. Sci. Technol. 2004, 38, 1073-1078
Cadmium Induction of Metallothionein Isoforms in Juvenile and Adult Mussel (Mytilus edulis) C O R I N A M . C I O C A N ‡,§ A N D J E A N E T T E M . R O T C H E L L * ,‡ Department of Biology and Environmental Science, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QJ, U.K., and National Institute for Marine Research and Development “Grigore Antipa”, 300 Mamaia Boulevard, 8700 Constanta, Romania
Isoforms of metallothionein in the digestive gland of control and experimentally Cd-exposed mussels (Mytilus edulis) (200 µg L-1 Cd2+ and 400 µg L-1 Cd2+; 20 days) were studied using the reverse transcriptase-polymerase chain reaction. In addition, glutathione S-transferase (GSTpi) primers were designed to evaluate the reduction in the antioxidant defense systems (glutathione) accompanying the aging process in the same organisms. Following experimental exposure, an accumulation of Cd was observed in the digestive gland of exposed mussels, both adults and juveniles, up to 500 times higher than in the control. An induction of the dimeric form MT20 II was detected in 400 µg L-1 exposed mussels, as well as a visible inhibition of the monomeric form MT10 IV. After 20 days of exposure juveniles expressed increased GSTpi compared with adults. Results reveal individual variation of both metallothioneins and GSTpi expression among control and Cd2+-exposed mussels of different ages. The ecotoxicological significance of MT utilization in biomonitoring of seawater for trace metals has been considered in light of these results.
Introduction Mussels are often selected as a sentinel organism to monitor marine pollution for a variety of reasons: they are sessile, filter feeding, distributed worldwide, and also cultivated for human food consumption. Their ability to sequester metals is the basis for the use of metallothioneins (MTs) as a biomarker of metal pollution in the aquatic environment. Cd is a toxic element for the cell and affects various cellular components, causing the inhibition of amino acid transport and of Na+/K+-ATPases and alterations in mitochondrial metabolism (1). In contrast to many proteins, MT quantity does not decrease with metal exposure; instead the metal induces MT synthesis, providing a protective mechanism against metal toxicity (2). MTs are a class of heat-stable, low molecular weight metal-binding proteins of a nonenzymatic nature that are characterized by an amino acid composition that has a high cysteine content (22-23 mol %) (3). The mussel MT amino acid sequences exhibit more similarity to those of vertebrate MTs than to those of * Corresponding author phone: +44 1273 872862; fax: +44 1273 677196; e-mail:
[email protected]. ‡ University of Sussex.§ National Institute for Marine Research and Development “Grigore Antipa”. 10.1021/es030110g CCC: $27.50 Published on Web 01/17/2004
2004 American Chemical Society
nonmolluscan invertebrate MTs, thus suggesting that the mussel MTs are class I MTs (4, 5). Cd-induced MTs from Mytilus edulis comprise two groups of isoforms having apparent molecular masses of 10 and 20 kDa (6). MT10 includes four isoforms (72 amino acids), and the dimeric MT20 variant can be resolved into five isoforms (monomer containing 71 amino acids). It has been suggested that the existence of nine isoforms in mussels probably reflects different metal-binding affinities and specificities of the gene promoter (7). Polymorphism of the MT family is a well-known phenomenon frequently observed in marine invertebrate species (4, 5, 8, 9). Although the biological role of such inducible, low molecular mass, cysteine-rich, metal-binding proteins has not yet been entirely defined, the multiplicity of MT isoforms may be attributed to several proposed physiological and protective functions particularly dealing with trace metal metabolism (1, 10, 11). Internal storage and detoxification of metal by MT has also been related to increased metal tolerance as well as genetic resistance (12). To date, among all invertebrate species investigated, the blue mussel, M. edulis, possesses the largest number of MT isoforms: MT10Ia, GenBank Accession No. AJ005451; MT10Ib, AJ005452; MT10II, AJ005453; MT10III, AJ005454; MT10IV, AJ005455; MT20II, AJ005456 (4, 5). Five protein subtypes of the MT20 isoform have been identified by N-terminal sequence determination on pooled mussel tissues from different individuals (13). Barsyte et al. (1999) reported that the exposure of a mussel to 400 µg L-1 Cd resulted in a marked increase in MT20, a primarily inducible isoform that is expressed at a low level in the absence of heavy metals (5). For this reason, MT20 can be considered an important biomarker in studies on marine heavy metal pollution. In contrast, the MT10 isoform is expressed at basal levels. Glutathione (GSH), along with cysteine-rich protein MT, has been suggested to play a cooperative role in protection against metal toxicity. GSH is capable of complexing and detoxifying heavy metal cations after they have entered the cell, thus representing the first line of defense against heavy metal toxicity (14). Heavy metal accumulation in the cells can therefore result in a decreased level of reduced GSH, stimulation of GST (glutathione S-transferase, which conjugates glutathione to xenobiotics) activity, and inhibition of GSH synthetase (15). It is established that deficiencies of heterozygous genotypes (the presence of different alleles at one or more loci on homologous chromosomes) are commonly reported for bivalve populations, particularly for early juvenile stages (16). M. edulis is reported to have high genetic variation due to the range of environments occupied by this species and an extended dispersal stage in the life cycle (5). Also, it has been demonstrated that the ability of organisms to respond to oxidative stress by increasing their rate of GSH synthesis decreases significantly with age (17). In this study, juvenile and adult mussels were used to investigate whether natural factors (such as size/age) may influence MT and GSTpi levels concurrently with contamination factors. Most studies on MT isoforms in mussels involved their isolation from pooled tissue from a large number of individuals. Consequently, there is a possibility that differentially expressed MT alloforms occurring in different individuals could contribute to the final expression of MT from a composite sample. Therefore, we isolated Cd-MT RNAs from each mussel digestive gland to analyze the individual variation in the expression of these metal-induced genes. VOL. 38, NO. 4, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Mortality (%) of Mussels in the Control Group and after Exposure to Cd in Solution for 20 Days life stage
control
exposure 1 (200 µg L-1)
exposure 2 (400 µg L-1)
adults juveniles
3.3 0
16.6 3.3
16.6 13.3
Materials and Methods Experimental Exposure. Specimens of M. edulis, adults (6.3 ( 0.4 cm shell length) and juveniles (4.05 ( 0.45 cm shell length), were collected in the English Channel near Brighton (Hope Gap) in November 2002. A total of 180 individuals were acclimatized for 7 days in three 90 L aquariums containing aerated seawater; subsequently, mussels were divided into three groups (two exposed groups and one control group) and transferred to separate aerated 90 L aquariums. Each aquarium contained 30 juveniles and 30 adults. Without addition of food, mussels were exposed to concentrations of 200 µg L-1 (exposure 1, E1) and 400 µg L-1 (exposure 2, E2) Cd, added as CdCl2 solution. After 20 days of treatment, 18 individuals (9 adults, 9 juveniles) randomly selected from each tank were removed and measured, and the soft parts (digestive gland and gonads) were dissected. Tissues obtained from each mussel were frozen in liquid nitrogen and stored separately. Throughout the exposure regime, seawater from each tank was changed every other day; Cd concentration from exposure tanks was analyzed daily by atomic absorption spectrophometry and reestablished. For the 20-day exposure, the mussel mortalities are presented in Table 1. Sample Preparation for RT-PCR Analysis. The total RNA from the digestive gland from each mussel was extracted according to the manufacturer’s instructions (Qiagen Ltd., U.K.). First strand cDNAs were synthesized using approximately 1 µg of total RNA from each sample. After cDNA synthesis 3 µL of the reaction mixture was used as a template for subsequent PCR. Sequences for the primers used were as follows: GSTpi sense, 5′-CGG TTT CCA ACT GGT GCA GTC-3′/antisense, 5′-CCT GGT CTT GCC AAC ACT CGC T-3′, 381 base pairs (bp); MT10 IV sense, 5′-AAT CAG AAA GCC GAG CGC CAA3′/antisense, 5′-TTC ACT TGC AGG AAC AGC CAG G-3′, 301 bp; MT20 II sense, 5′-CGA CAT ACT ACC CAG ATA CCA CCC3′/antisense, 5′-CCA GTG CAG TCA CAT CCA CAC GC-3′, 259 bp (synthesized by Invitrogen). Semiquantitative RT-PCR. To perform semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we measured the amount of isolated RNA by UV spectroscopy at 260 nm. Subsequently, for the reverse transcription phase we used only 1 µg of total RNA from each sample. To normalize differences in efficiency during amplification, we have used actin primers, to amplify actin as internal standard: sense, 5′-GCA CTT CCT CAC GCT ATC CTC C-3′/ antisense, 5′-TGT CCA TCT GGC AGT TCG TAG C-3′, 230 bp. Amplification was performed with a BioRad iCycler in 50 µL reaction volumes, for 35 sequential cycles at 94 °C for 60 s, 55 °C for 50 s, and 72 °C for 60 s, followed by a final 2 min extension at 72 °C. An 18 µL sample of each PCR product was taken for agarose gel electrophoresis (0.8% and 2.4% agarose, TBE buffer). Metal Determination. Heavy metal concentration (Cd) was performed with a Perkin-Elmer AAnalyst 100 spectrophotometer (18). Mass fractions of metals were calculated as micrograms of metal per gram of dry weight tissue (dwt). The analytical methods were validated using an internal standard CRM 278R mussel (M. edulis) tissue (Promochem). Cd analyses were performed on freeze-dried tissues from each mussel, and the data were analyzed by analysis of 1074
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FIGURE 1. (a, top) Bioaccumulation of Cd in juvenile mussel digestive gland and gonad tissues. (b, bottom) Bioaccumulation of Cd in adult mussel digestive gland and gonad tissues.
variance (Bartlett’s test, ANOVA) and the t-test. The level of significance was set at 0.05.
Results Metal Bioaccumulation. In digestive gland and gonad, Cd bioaccumulation was dependent on both contamination level and the size/age of the mussels (Figure 1). After 20 days of exposure, Cd had bioaccumulated in both digestive gland and gonads (P < 0.05). Cd concentrations in gonads of adults were similar at the two exposure treatments (E1, 82.67 ( 49.89 µg of Cd g-1 of dwt; E2, 97.72 ( 37.8 µg of Cd g-1 of dwt) (Figure 1b), but it was higher at the E2 treatment for juveniles (E1, 9.98 ( 10.7 µg of Cd g-1 of dwt; E2, 49.86 ( 38.03 µg of Cd g-1 of dwt) (Figure 1a). Metal concentrations in the digestive gland (juveniles, E1, 322.42 ( 140.30 µg of Cd g-1 of dwt; E2, 430.24 ( 85.47 µg of Cd g-1 of dwt (Figure 1a); adults, E1, 210.08 ( 53.72 µg of Cd g-1 of dwt; E2, 448.95 ( 180.10 µg of Cd g-1 of dwt (Figure 1b)) were significantly higher (P < 0.05) than in gonads. Consequently, the subsequent RT-PCR analyses were carried out using the digestive gland. The mortality data suggest that adults were more sensitive to Cd than juveniles; 16.6% of juveniles died during the experiment compared with 36.5% of adults (Table 1). In the E2 exposure aquaria the percentage of mortality of Cdexposed mussels was increased in juveniles only (13.3% mortalities in E2 compared with 3.3% in E1) (Table 1). MT10IV Isoform Expression. RT-PCR was performed using M. edulis specific primers that amplified a product of 300 bp, the MT10IV isoform. An actin amplification (of a 230 bp product), performed using control samples, showed that slight variation between adult and juvenile samples exists in the general level of expression before exposure. The MT10IV
FIGURE 2. Expression of MT10IV isoform in mussel digestive gland following Cd exposure. Products were resolved and visualized on ethidium bromide stained agarose gel. Key: C, control; E1, exposure 1 (200 µg L-1); E2, exposure 2 (400 µg L-1); M, molecular weight marker (100 bp Invitrogen ladder); A1-A9, adult mussels; J1-J9, juvenile mussels; N, negative sample.
FIGURE 3. Expression of MT20II isoform in mussel digestive gland following Cd exposure. Products were resolved and visualized on ethidium bromide stained agarose gel. Key: C, control; E1, exposure 1 (200 µg L-1); E2, exposure 2 (400 µg L-1); M, molecular weight marker (100 bp Invitrogen ladder); A1-A9, adult mussels; J1-J9, juvenile mussels; N, negative sample. Additional bands: a, approximately 400 bp; b, approximately 500 bp; d, approximately 650 bp. expression levels in adult control samples were slightly higher than those in juvenile control samples, though this may be related to the observed difference in actin expression. After 20 days of Cd exposure, an increase of MT10IV expression, compared with that of control samples, was observed for E1 adult and juvenile samples (Figure 2). The increase in expression was greatest in juveniles (E1, J1, J5J9). In contrast, a decrease of MT10IV expression, compared with that of control samples, was observed in adult and juvenile samples following the E2 exposure. In E2 samples, only two individuals (adult A8 and juvenile J1) showed any evidence of expression of the monomeric form of MT (Figure 2). MT20II Isoform Expression. The dimeric form of MT, MT20II, was amplified using specific primers designed for a 260 bp product. A minority (5/18) of control samples expressed the dimeric form, with no expression detected in control juveniles (Figure 3). MT20II expression was higher in Cd-exposed mussels than in control samples, particularly E1 and E2 juvenile samples (Figure 3, E1, J1, J3, J5, J7-J9; E2,
J1, J5, J7, J9), but also in a number of adult samples (Figure 3, E1, A5, A6; E2, A2-A5, A9). The MT20II primers also amplified additional bands, of approximately 400, 500, 650, and 750 bp, the significance of which is discussed later. GSTpi Response in Mussel Digestive Gland. In the digestive gland of unexposed mussels, GSTpi was expressed at a lower level in juvenile samples compared with adults. A significant increase of GSTpi expression was detected in E1 juvenile samples as well as in E2, for both adults and juveniles, while GSTpi synthesis in E1 adults appeared to be decreased compared with that in unexposed adults. All of the E2 adult and juvenile mussels expressed GSTpi (Figure 4). Actin Internal Standard Expression. RT-PCR was performed using M. edulis specific primers to amplify a product of 230 bp of the actin gene simultaneously with primer pairs for MT isoforms and GSTpi. Individual expression was investigated using the digestive gland of mussels under normal conditions (Figure 2, section C) and following Cd exposure (Figure 3, section E1; Figure 4, section E2). RTVOL. 38, NO. 4, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4. Expression of M. edulis GSTpi in digestive gland following Cd exposure. Products were resolved and visualized on ethidium bromide stained agarose gel. Key: C, control; E1, exposure 1 (200 µg L-1); E2, exposure 2 (400 µg L-1); M, molecular weight marker (123 bp Invitrogen ladder); A1-A9, adult mussels; J1-J9, juvenile mussels; N, negative sample. PCR from the same tissue and different individuals showed a stable expression profile for adults, but not for juveniles (Figure 2, J1 and J5). Following Cd exposure, RT-PCR from the same tissue and different individuals showed a relatively stable expression profile for both adults and juveniles compared with the control samples (Figures 3 and 4). This experiment suggests that actin has potential as a reliable internal standard for monitoring natural versus experimentally induced changes in MT and GSTpi mRNA levels.
Discussion In the past, studies of MT isoforms have involved the quantification of total MT using liquid chromatographymass spectrometry (8, 19-21), capillary electrophoresis (11), differential pulse polarography (10, 22, 23), or immunological techniques (24). The recent introduction of modern molecular biology techniques has led to the analysis of MT mRNA by means of polymerase chain reaction (25). Using RT-PCR and mussel specific primers for MT isoforms, MT10IV and MT20II, and GSTpi, we have investigated the previously uncharacterized individual variation in detoxification response to Cd exposure. In mussels, the digestive gland functions both as a site for metal uptake and as an important reservoir for metal storage. In this investigation, Cd accumulation was observed in the digestive gland of mussels in a dose-dependent manner over a period of 20 days. A survey of the literature on the assessment of Cd accumulation in different tissues of the marine bivalves (Table 2) shows that the values presented herein are consistent with the values reported. The Cd doses selected in this study, though too high to be environmentally realistic, have allowed an investigation of the bioaccumulative effects and corresponding molecular level changes in metal detoxification responses. At these artificially high Cd levels, the mortality results suggested that the adults were more sensitive to Cd exposure than juveniles. Increased susceptibility of aging organisms to a particular kind of stress is thought to be due to a general lowering of cell metabolism, which may account for the apparent difference in sensitivity to Cd exposure observed (17). The bioaccumulation results presented indicate that the level of Cd uptake is tissue dependent at the doses used: Cd bioconcentration in the digestive gland was significantly higher (ANOVA, P < 0.05) than in gonads. Several studies on mussels have evidenced the hepatopancreas as the preferential organ for the accumulation of the different metals: Adami et al. (2002) reported a high affinity of the Cd for hepatopancreatic tissue in Mytilus galloprovincialis (29), and Geret and Cosson (2000) underlined the preponderant role of the digestive gland in metal metabolism (2), while 1076
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Cardellicchio et al. (1998) observed that the accumulation in the hepatopancreas increases as the mussel grows (30). Bioaccumulation of metals may be influenced by many variables, both environmental and physiological (19). Several studies have reported environmentally induced variation, but few have investigated the influence of age/size on Cd uptake in mussels. Wallace et al. (2003) reported that after 14 days of exposure to 3.5 µg L-1 Cd, smaller clams accumulated roughly twice as much metal as the larger ones (12). The authors suggest that a higher metabolic rate, and as a result the importance of MTs for metal storage, is increased in smaller individuals. As a clam ages, these authors suggest that the role of metal-rich granules becomes more important than MT in detoxifying metals (12). In contrast, Cd concentrations have been positively correlated with body size (expressed as shell length) in the limpets Patella intemedia and Patella vulgata (3). Geret and Cosson (2000), on the other hand, emphasize the effect of dilution that occurs with the increase of tissue mass in aged bivalves (2). However, our observations showed that after 20 days of exposure to 200 and 400 µg L-1 Cd, the levels of toxic metal in both mussels’ digestive gland and gonads are not significantly different (ANOVA, P < 0.05) in adults compared with juveniles. Lack of size dependence in metal concentrations is a common observation. For instance, studies on Potamocorbula amurensis have shown that, after experimental exposure, Cd and Zn concentrations do not increase with size. The complexity of the tissue metal concentration and animal size relationship is well acknowledged (12). Our study supports that of Barsyte et al. (1999) in that the MT20 isoform represents a Cd-inducible form of MT, whereas MT10 is basally expressed (5). Differential expression of the two MT isoforms MT10 and MT20 is thought to relate to a different involvement in metal regulation (31). The MT20 isoforms are one amino acid shorter than the MT10 isoforms and are characterized by two additional cysteine residues. Such residues may be responsible for additional chelation of metals or the formation of intermolecular linkages between monomeric units of the MT20, through either disulfide bonds or S-Cd-S bridging (32). MT10IV PCR amplified fragments in both adults’ and juveniles’ digestive gland showed that this MT isoform is present in unexposed mussels, probably as a result of its involvement in the metabolism of essential trace metals. The response of MT10IV, measured as expression following Cd exposure, differs in adults compared with juveniles. The latter respond with an increase in MT10IV expression at the E1 dose, but the expression is subsequently decreased following Cd exposure at the E2 dose. In contrast, adult expression of MT10IV is seemingly less affected by Cd exposure. Similarly,
28 AA spec ∼700 µg g-1 of dwt Ruditapes decussatus
wwt, wet weight per gram of tissue; dwt, dry weight per gram of tissue.
Gill a
10 Ciocan and Rotchell 19 26 27 11 polarized Zeeman AA AA spec IL-S11 flame AA spec flame AA spec AA spec AA spec M. edulis M. edulis M. galloprovin cialis M. galloprovin cialis Scapharca inaequival vis Mercenaria mercenaria
ref analysis
56.78 ( 4.75 µg of wwt 448.94 ( 180.09 µg g-1 of dwt -1 945 µg g of dwt ∼1200 µg g-1 of dwt 725.7 µg g-1 of dwt 121.80 ( 090 µg g-1 of dwt
200 µg 21 days 400 µg L-1; 20 days -1 0.5 µg mL ; 7 days 100 µg L-1; 34 days 0.5 µg mL-1; 28 days 0.1 mL of 500 µg L-1 Cd, injected into foot; 4 days 100 µg L-1; 40 days
digestive gland digestive gland gill gill kidney red gland
g-1 L-1;
max values reporteda organ Cd exposure conditions organism
TABLE 2. A Summary of Cd Bioaccumulation Data in Several Bivalve Species Reported in the Literature
Lemoine et al. (2000) reported an increase of MT10 isoform after a relatively short exposure (4 days) of mussels to Cd; for longer exposure (21 days), MT10 mRNA levels decreased (7). While the MT10IV isoform appeared to be almost unexpressed in the E2 group, MT20II was greatly enhanced by metal ions. Juveniles from the control group exhibited no expression of the dimeric MT isoform, but after exposure to E1 and E2 doses, MT20II transcription increased. The induction of the MT20 gene in response to Cd exposure indicates that these MT20 isoforms are involved in the sequestration and detoxification of intracellular Cd. Previous studies have shown the absence of MT20 isoforms from unexposed mussels, probably as a result of their lack of involvement in bioessential metal metabolism (7). In our study, the presence of MT20II isoform in untreated adults suggested that the animals were potentially previously exposed in the environment to substances that induce MT synthesis, whereas juveniles, with a much shorter life history, have been able to utilize the basally expressed isoform to sequester efficiently the toxic compounds to which they have been exposed. A number of additional PCR products, of a larger size, were also obtained for the adult control samples using the MT20II primers, the significance of which is not known. Season, sex, and age/size are natural factors that influence MT levels in bivalves (3, 26, 33). Such factors play a fundamental role in the buffering of intracellular metals (34). Recently, it demonstrated that total MT levels (quantified by DPP) increase exponentially with shell length or dry weight of the soft tissues in a population of limpets (3). In another study, Serafim et al. (2002) found that Cd induced synthesis of total MT in the gills of both small and large mussels, though the gills of large mussels displayed significantly higher increases in MT than small organisms (26). As a result of these findings, the authors pointed out that the effect of size in MT concentrations could be minimized if the organisms studied are as uniform as possible. It is also suggested that, although sex and size affect MT levels to some extent, it is less important than metal exposure (34). In light of the results presented herein, we can add that size-related differential expression of MT isoforms must also be taken into account to avoid erroneous interpretation in biomonitoring analysis. Cd has a high affinity for GSH, a primary intracellular antioxidant agent. It can also bind to GSH, causing the irreversible excretion and consequent depletion of GSH (10). Zaroogian and Jackim (2000) suggested that when the intrinsic GSH concentrations are unable to compensate for high concentrations of free metal, MT synthesis is induced (11). In M. edulis GSTs, which conjugate glutathione to xenobiotics, play a major role in phase II detoxification reactions (35). In terms of natural variation in GST response to metals, Canesi and Viarengo (1997) reported a progressive decrease in GST activity in the gills of large mussels compared with small ones, possibly indicating a reduced capacity of metabolizing both endogenous and foreign compounds with aging (17). In contrast, Robillard et al. (2003) demonstrated an increased activity of GST with mussel length. The same study also reported that levels of GST activity were related to changes in abiotic factors such as pH, dissolved oxygen, and temperature (36). The results presented herein suggest that variation in GSTpi expression follows a more complex pattern than previously reported. Juveniles responded to the E1 dose by increasing GSTpi synthesis, which remained constant at the higher concentration of metal. In contrast, adults required an E2 dose before enhanced GSTpi transcription was observed. Variation in GST activity is therefore apparently related to natural factors such as age/size and season, as well as the level of metal concentration present in the environment. VOL. 38, NO. 4, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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In summary, the results suggest that the MT isoforms and GST isoform in M. edulis (selected for this investigation) are differentially expressed following experimental Cd exposure (at high concentration) at different stages in their life. This finding raises concerns regarding the use of MT expression as a biomarker for specific metal contamination in mussels. An investigation of this kind can however provide an insight into the molecular mechanisms that underpin the organisms’ defense against metal toxicity.
Acknowledgments This work was funded by an EU Marie Curie Training Site fellowship to C.M.C. We thank C. Dadswell, M. Henry, and K. Evans for help with sample collection, processing, and the AA analysis.
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Received for review August 8, 2003. Revised manuscript received November 12, 2003. Accepted November 13, 2003. ES030110G
