Quantification of Metallothionein-like Proteins in the Mussel Mytilus

thioneins and their isoforms from the Moroccan mussel Mytilus galloprovincialis. ... evaluate the amount of metallothionein in the mussels either expo...
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Environ. Sci. Technol. 2003, 37, 5739-5744

Quantification of Metallothionein-like Proteins in the Mussel Mytilus galloprovincialis Using RP-HPLC Fluorescence Detection I S S A M E L G H A Z I , †,‡ S I E G L I N D E M E N G E , † JUERGEN MIERSCH,† ABDELGHANI CHAFIK,§ ALI BENHRA,§ M. KHALID ELAMRANI,‡ AND G E R D - J O A C H I M K R A U S S * ,† Martin-Luther-University Halle-Wittenberg, Department Biochemistry/Biotechnology, Division of Ecology and Plant-Biochemistry, Kurt-Mothes-Strasse 3, 06120 Halle, Germany, and Hassan II University, Faculte´ des Sciences, BP 5366, Maˆarif, Casablanca, Morocco, and Institut National de Recherche Halieutique, 2 Rue Tiznit, Casablanca, Morocco

A HPLC-fluorescence method, using the fluorophore SBD-F (ammonium-7-fluorobenz-2-oxa-1,3-diazole-4sulfonate), was adapted for the quantification of metallothioneins and their isoforms from the Moroccan mussel Mytilus galloprovincialis. The method was first optimized using a rabbit liver metallothionein. The effects of EDTA, tris(2carboxyethyl)phosphine, and SBD-F on the labeling efficiency were studied. The optimized method was then applied to evaluate the amount of metallothionein in the mussels either exposed to cadmium in the laboratory or collected from the Casablanca coast, Morocco. The concentrations of metallothioneins measured in the field samples describe the degree of contamination of the sites and are reflected by distinct isoform patterns.

Introduction Aquatic ecosystems are under increasing attack from all types of environmentally hazardous substances. Although chemical analysis is necessary, it is still not sufficient to evaluate the threat of these pollutants. Chemical analysis only offers insight into the concentrations of chemical hazards in different parts of the aquatic ecosystems (sediment, water, and biota), but it does not provide any insight into the effects of pollution on these ecosystems. Pollution is recognized as expressing biological features of the response of the ecosystems to contamination (1). It is therefore essential to incorporate tools that allow the measurement of such biological effects in monitoring programs (2). These tools are called biological markers or “biomarkers”. A biomarker represents a measurable event in a biological system; it is an indicator that an early change has occurred which could lead later to a disease (3). It can be defined as a biochemical, cellular, physiological, or behavioral variation measured in tissue or in the whole organism that provides evidence of * Corresponding author phone: +49-345-5524831; fax: +49-3455527012; e-mail: [email protected]. † Martin-Luther-University Halle-Wittenberg. ‡ Hassan II University. § Institut National de Recherche Halieutique. 10.1021/es035093+ CCC: $25.00 Published on Web 11/15/2003

 2003 American Chemical Society

exposure to one or more chemical pollutants (4). The most frequently used biomarkers in environmental monitoring of aquatic systems include the enzymes CYP-450 in the sea star Asterius rubens (5), acetylcholin-esterase, glutathione-Stransferase in the mussels Perna perna and Mytilus galloprovincialis (6, 7), as well as metallothioneins in the mussel M. galloprovincialis and in the clam Ruditapes decussatus (8, 9). Metallothioneins (MTs) are low molecular weight proteins (6-7 kDa) representing an interesting class of metalloproteins. Because of their unique primary sequence with up to 30% cysteine, they are able to bind heavy metals of groups 11 and 12 (up to 7 g-atom/mol in vivo and 18 g-atom/mol in vitro) (10). Several studies have demonstrated their inducibility by heavy metals such as Cd, Cu, and Hg (11, 12, 13, 14) and also by hydrogen peroxide (15) and glucocorticoids (16). Since their discovery in 1957 by Margoshes and Vallee, who identified a cadmium-binding protein allowing the natural accumulation of this metal in the equine kidney cortex (17), there is still no evidence of their correct biological functions. It is theorized that they are involved in three functions: heavy metal metabolism (18), protection against oxidative stress (15), and embryonic development (19, 20). Because they are inducible by metal ions, MTs are understood to be involved in controlling the availability of essentials metals (Cu, Zn) and the detoxification of nonessential metals such as cadmium. Brown et al. (21) first proposed the use of the variation of MT concentrations as a biomarker for heavy metal pollution. Since the discovery of MTs in mussels (22), the idea to use them as an early warning system for heavy metal pollution is now well-established. Several laboratory studies have proven the inducible nature of the MTs by heavy metals in marine invertebrates. Among them, the mussels Mytilus edulis (23) and M. galloprovincialis (24), the oyster Crassostrea virginica (25), the crabs Carcinus maenas (13) and Callinectes sapidus (26), and the gastropod Littorina littorea (27). In his review, Cosson (28) pointed out that there are some reservations to using MTs as biomarkers. These reservations concern mostly the analytical protocols developed to quantify the MT. The most often used isolation and quantification methods for MTs include solvent precipitation (with an eventual loss of isoforms) and heat denaturation (29), Hgsaturation assay (30), as well as the spectrophotometric method based on the Ellman reaction for the thiol quantification, which was adapted for MTs (31), and the polarographic method (32). Miyairi et al. (33) developed a protocol based on the derivation of MTs using the sulfhydryl labeling fluorophore SBD-F (ammonium 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonate) followed by a tandem column HPLC-fluorescence quantification. In the same way, Yang et al. (34) used the ABD-F (7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide) for the quantification of MTs and its metal-free protein (apothionein). Recently, new techniques have been developed and optimized for the MT quantification. Most attractive are the hyphenated techniques based on the coupling of the capillary electrophoresis and ICP-MS (35, 36). The advantage of using these techniques is their determination not only of the total level of MT, but also of the levels of different isoforms. In fact, in mammals four isoforms are so far known (37). These high isoform numbers are thought to be attributable to different biological functions of MT. In mammals, MT-1 and MT-2, which are expressed in all organs, are more involved in heavy metals metabolism. While MT-3, which is expressed mainly in the brain, is involved in Alzheimer disease (18). VOL. 37, NO. 24, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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soft bodies were used for the metallothionein determination. The soft bodies were stored at -80 °C until analysis.

FIGURE 1. Geographical location of the studied sites (1-4), Bay Casablanca-Mohammadia, Morocco. Dallinger et al. (38) reported the presence of two different isoforms which bind specifically Cu and Cd in snails. It seems therefore of great importance to evaluate not only the total levels of MT, but also the levels of the isoforms. The aim of this work was to optimize the thiol-specific SBD-F derivatization method for a highly sensitive quantification of MT isoforms. The method was then applied to evaluate the isoform concentrations in the mussels, M. galloprovincialis, exposed to cadmium in the laboratory. Thereafter, the method was used to screen the Casablanca coast, Morocco, using the same species considering distinct polluted coast areas. This organism was chosen because it has been used in the Moroccan national monitoring program and is commonly found along the Moroccan coast.

Materials and Methods Chemicals. Tris(2-carboxyethyl)phosphine (TCEP) was purchased from Molecular Probes (Eugene, OR). Ammonium 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonate (SBD-F) was purchased from Dojindo (Kumamoto, Japan). Rabbit liver metallothionein 2 (MT-2, Lot 80k7013, Cd content 4.5%) was purchased from Sigma (St Louis, MO). Cadmium was supplied as CdCl2‚H2O (Merck, Darmstadt, Germany). All other reagents were of analytical grade. Mussels. Laboratory Exposure. Mussels, M. galloprovincialis Lamarck, 1819, were collected from a noncontaminated site (site 1 ) Dar Bouazza, see Figure 1) 20 km in the south of Casablanca. The mussels were of comparable size (5.9 ( 0.9 cm). They were scrubbed to remove the adhering organisms and then acclimated in aerated natural seawater (salinity 31‰, 20 °C) for 1 week in the laboratory. The marine water, used for the acclimatization and for the cadmium exposure experiments, was taken from the collection site. The organisms were not fed during the whole experimental period. The water was renewed and contaminated daily. The mussels were exposed to a nominal concentration of 1 mg/L of cadmium for 1 week. Field Study. Four sites (site 1, Dar Bouazza; site 2, Ain Sebaaˆ; site 3, Hamimmou; site 4, Oued Mellah) were chosen for the field study covering the coast of CasablancaMohammadia (see Figure 1). The south site (site 1) represents a noncontaminated site, whereas the three in the north of Casablanca represent a gradient of pollution. Sites 2 and 3 are characterized by organic pollution (hydrocarbon and PCBs) and inorganic pollution: Hg, Pb for site 2 and Cd, Pb, Hg, Cu for site 3. Site 4 is characterized by urban pollution (39). Using an embryotoxicity test with a sea urchin Paracentrotus lividus, the sites were classified in an increasing gradient of toxicity: site 3 > site 2 > site 4 > site 1 (Benhra, manuscript in preparation). The mussels with a shell size of 5 ( 0.4 cm were collected from the four sites. Groups of 10 individuals and the whole 5740

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Metallothionein Purification. MTs were isolated and purified according to Pedersen et al. (13). The whole soft bodies were homogenized with an Ultra-Turrax in 2 vol (w/v) of ice-cold buffer 25 mM Tris-HCl pH 7.4, containing 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride. The homogenate was centrifuged for 40 min at 30 000g at 4 °C using Sorvall RC5. To avoid oxidation of MTs, the buffers were saturated with helium. The supernatant was then fractionated by a two-step acetone precipitation (45% and 80%). Acetone was dropped while stirring and bubbling helium. Between each addition the mixture was centrifuged for 15 min at 10 000g at 4 °C. The 45-80% pellet was then washed with 80% acetone, centrifuged again for 15 min at 10 000g at 4 °C, resuspended in deionized water, and ultrafiltrated using a membrane (cut off of 3 kDa, Microseparation, Northborough, MA). Size-Exclusion Chromatography (SEC). The resuspended 45-80% pellet was subjected to SEC analysis using a Superdex 75, 30/10 H/R, Pharmacia. For elution, 50 mM NH4HCO3, pH 8, was applied at a flow rate of 0.3 mL/min. The column was calibrated using HSA 69 kDa, ovalbumin 43 kDa, myoglobin 17 kDa, cytochrome c 12.3 kDa, aprotinin 6.5 kDa, insulin B 3.5 kDa. A 300 µg amount of protein was injected. The eluate was monitored at 254 nm, which is the characteristic absorption wavelength for the Cd-sulfide bond. Fractions were collected, and the metal contents were analyzed by graphite-furnace absorption atomic spectroscopy (GF-AAS), AAnalyst 800, Perkin-Elmer. Derivatization Procedure. MT derivatization was accomplished according to Miyairi et al. (33); however, some modifications were introduced. According to the optimized ratios of the assay components (compare to Results and Discussions) calculated volumes of EDTA (0.25 M) and TCEP (300 mM) were added to the protein suspension of the standard rabbit liver MT-2 in the range of 5-20 µg and the mussel metallothionein-like protein MLP (10 µg), respectively. The protein concentration was determined by Lowry’s method. For example, 20 µL of EDTA (0.25 M) and 11 µL of TCEP (300 mM) were added to 10 µL of MT-2 (0.5 mg/mL). Then 300 µL of 0.6 M borate buffer, pH 9.6, and 40 µL of SBD-F (0.5%) were added. The amounts correspond to the optimized ratios of 6000:4:1000:1 for EDTA:TCEP:SBD-F: protein. The total reaction volume did not exceed 600 µL and was always in a basic pH range. The mixture was heated for 30 min at 50 °C. A 50 µL amount of HCl (4 M) was subsequently added to stop the reaction. The labeled proteins were immediately transferred to HPLC analysis. Chromatographic Analysis. The RP-HPLC analyses of SBD-F labeled standard MTs and MLPs were performed using a LaChrom system (Merck-Hitachi), equipped with a pump L-7420, a fluorescence detector L-7480, a diode-array-detector L-7450, and an interface L-7000. The separation column used was a Eurosil Bios 300, C18, 5 µm, 250 × 4 mm, (Knauer, Germany). A gradient of acetonitrile (B) in 20 mM phosphate buffer pH 7 (A) was applied: 0-12 min, 2-5% B; 12-16 min, 5-10% B; 16-26 min, 10% B; 26-35 min, 10-50% B. The elution was carried out at a flow rate of 1 mL/min. The derivatized proteins were monitored with λex 384 nm and λem 510 nm. Gel Electrophoresis. One-dimensional SDS-polyacrylamide gel electrophoresis of SBD-F labeled standard MTs and MLPs was carried out in a 5% (w/v) stacking gel and 12% separating gel in the buffer system of Laemmli (40). After electrophoresis, the fluorescence was evidenced using an UV-transilluminator. Silver staining was used according to Heueskoven and Dernick (41).

FIGURE 2. Effect of EDTA on the efficiency of MT-2 labeling. FIGURE 5. Fractionation of the 45-80% acetone pellet extracted from the mussels exposed to 1 mg of cadmium L-1 for 1 week. The size-exclusion chromatography was done as described in Materials and Methods.

FIGURE 3. Effect of TCEP on the derivatization of MT-2.

FIGURE 4. Effect of the molar ratio SBD-F to MT-2 derivatization.

the apothionein. Gan et al. (43) studied the reaction between Cd7-MT-2 isolated from rabbit liver and EDTA. They found that the reaction has a biphasic kinetic. According to the authors, EDTA was able to release only 76% of cadmium bound to MT-2 within 3 h when using a ratio of 10:1 EDTA to Cd. Therefore, we checked the effect of EDTA on the labeling rate with high molar ratio of EDTA to protein. Figure 2 represents the effect of the variation of the EDTA: protein molar ratio on the labeling efficiency. Higher molar ratios of EDTA to protein enhanced the efficiency of the labeling reaction. This effect reaches a steady state at a EDTA to protein ratio of 6000:1. However, the EDTA is not able to release the copper from the MTs as reported by Myairi et al. (33). The subsequent use of a stronger chelator, bathocuproinedisulfonic acid (BCS), was necessary to improve the efficiency derivatization of mussel MT. TCEP Effect on the Labeling Efficiency. The oxidation state of the thiol functions of MTs is also important. The SBD-F reacts only with the reduced thiol function. The most frequently used reducing agents, DTT and 2-β-mercaptoethanol, have thiol functions themselves and, therefore, are not suitable because of possible competition with the protein. Trialkylphosphine compounds such as the water-soluble TCEP are able to reduce the disulfide functions according to the following reaction

Results and Discussion

(CH2CH2COOH)3P: + H2O + RSSR f (CH2CH2COOH)3P-O + 2 RSH

The TCEP, EDTA, and SBD-F ratio to protein concentrations were optimized using the rabbit liver MT-2 (Figures 2-4). Because standard mussel MTs are not available, we assumed that the derivatization procedure would be as effective for mussel MTs as for rabbit liver MT-2. The commercially available rabbit liver MT-2 was chosen as standard protein due to sequence similarities concerning the cystein content and clustering of the known subisoforms of rabbit liver MT-2 to the only known MT sequence of M. galloprovincialis (42). Although SBD-F is a specific thiol-function-labeling fluorophore, the labeling of the protein depends on a large number of factors. One of them is the labeling ratio of fluorophore to protein with regard to a target function. In the case of MT, the labeled functional group is the thiol, which represents up to 20 mol per mol of protein. Therefore, it is important to take into account not the number of moles of the MTs, but rather the amount of thiol groups. Effect of EDTA Concentration. At certain molar ratios, EDTA is able to release Cd and Zn bound to MT, generating

This compound, showing no thiol function, is more stable at pH values above 7.5 and does not compete with thiolspecific labeling of the proteins. Getz et al. (44), however, found in the case of myosin that the labeling with iodoacetamide (another thiol-selective fluorophore) was unaffected neither by the TCEP nor by DTT. Figure 3 shows the variation of the peak area of derivatized rabbit liver MT-2 by SBD-F. A steady state was achieved with a molar ratio of 4:1 TCEP to protein. Using this molar ratio, the efficiency of the peak area of the labeled MT was 1.5 times higher. According to Yang et al. (34), the presence of TCEP was necessary for the labeling of Zn7-MT with the ABDF. The authors reported that the TCEP presence is necessary not only for reducing the thiol functions, but as a possible catalyst for the reaction of ABD-F with MT. On the other hand, Miyairi et al. (33) reported that the presence of TBP (tri-n-butylphosphine), another trialkylphosphine, was necessary as a reducing agent for the labeling of the MT with VOL. 37, NO. 24, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 6. RP-HPLC chromatograms of the SBD-MT. The real samples (from M. galloprovincilias) were first prepurified using a twostep acetone precipitation. The column used was a Eurosil Bios 300, 5 µm, 250 × 4 mm. The elution was performed as described in Materials and Methods. Excitation and emission wavelengths were at 384 and 510 nm, respectively: (a) blank without any proteins, (b) derivatized rabbit liver MT2 (138 pmol), and (c) real samples (1.36 µg total protein).

FIGURE 7. Standard curve obtained by derivatizating different amounts of MT-2 (using optimized conditions; n ) 3; R 2 ) 0.99). SBD-F. Thus, the role of these compounds (TBP, TCEP) is still not clear. However, we recorded that by using TCEP at a higher molar ratio, a competition reaction occurs between the TCEP and MT-2. In this case, we observed a new fluorescent peak which eluted at a different retention time than SBD-MT. The emergence of a new reaction product

between TCEP and SBD-F was confirmed by a negative control (data not shown). Effect of SBD-F Ratio on the Labeling. As shown in the Figure 4, the optimal molar ratio SBD-F to protein is around 1000. At this ratio the peak area increased at around 8-fold. Real Samples. Size-Exclusion Chromatography. The sizeexclusion chromatography was carried out in order to characterize the 45-80% acetone pellet. The cadmium analysis of collected fractions revealed the presence of a major cadmium peak bound to two proteins with apparent molecular weights of 21 and 14 kDa (Figure 5). Copper was bound to this fraction at very low levels. Several studies showed the presence in mussels of a monomeric and dimeric form called, respectively, MT-10 and MT-20 (45, 11). For the derivatization, the 45-80% acetone pellet was used without further purification. Derivatization of MT. To release the copper bound to the MT fraction (metallothionein-like protein, MLP), 1 mM BCS was added to the labeling buffer as recommended by Myairi et al. (33). The 45-80% pellet enriched from the mussels, M. galloprovincialis, was derivatized using the optimal molar ratio for EDTA:TCEP:SBD-F:protein of 6000:4:1000:1. Figure 6 shows a typical HPLC chromatogram. The quantification was done according to a calibration obtained using rabbit liver MT-2 (Figure 7). To check whether the peaks detected were only labeled MLPs or other proteins, we subjected the labeled protein suspension (45-80% acetone pellet) to an SDS-PAGE (Figure 9). Only one fluorescent band was observed in all cases of real samples, which co-migrates with the band of SBD-MT2. Therefore, we concluded that the peaks represent the SBDMLPs purified from the mussels and that the-two step acetone precipitation was effective enough for MLP isolation. Laboratory Assay. The MLP concentrations in the control (site 1) and in the mussels exposed to 1 mg of cadmium L-1 for 1 week are shown in Figure 8a. In the exposed mussels the concentration of the first peak (noted MLP 1) reached a value of 1324.72 µg/g ww, which represents a 7-fold increase compared to a nonexposed sample from site 1 (205 µg/g ww). The second peak (noted MLP 2) reached a value of 389 µg/g ww in the mussels exposed to 1 mg of cadmium L-1 representing a 5-fold increase of the concentration than that found in mussels collected from site 1 (66 µg/g ww). Field Study. The concentrations of MLP in the mussels collected from the field are shown in the Figure 8b. The most

FIGURE 8. Metallothionein concentration (µg/g ww) in the mussel M. galloprovincialis: (a) exposed to 1 mg of cadmium L-1 for 1 week, (b) collected from different sites (n ) 3). (ns: not significantly different from site 1 (P > 0.05), *: significantly different from site 1 (P < 0.05), **: significantly different from site 1 (P < 0.01), ***: significantly different from site 1 (P < 0.001)). 5742

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FIGURE 9. One-dimensional electrophoresis of SBD-MT proteins using 12% SDS-PAGE. Fluorescence detection with UV-transilluminator (left) and silver nitrate staining (right). Lanes 1, 2, and 3: 0.32, 0.64, and 1.32 µg rabbit liver MT-2, respectively. Lane 4: site 2. Lane 5: site 3. Lane 6: site 4. Lane 7: site 1. Lane 8: exposed mussels. Lane 9: Aprotinin 6.5 kDa. polluted site, site 3, had the highest concentration with a value of 367.05 µg/g ww for MLP 1 and 181.35 µg/g ww for MLP 2. The concentrations seem to rise from site 1 (the less polluted), reaching a high value in site 3 (the most polluted), followed then by a diminution in site 4. These differences between the sites were much more pronounced for MLP 1 than for MLP 2. The concentrations of MLPs in the four sites were as follows: site 1 ≈ site 4 < site 2 < site 3. The use of MTs or MLP as a tool in biomonitoring could be more efficient if the analytical protocol is able to differentiate and evaluate different isoforms. Using the SBD-F derivatization method, we were able to quantify two isoforms of the MLP fraction from M. galloprovincialis. When exposed for 1 week to cadmium (1 mg/L), the induction was higher for the first isoform MLP 1 than for MLP 2. For the field samples, we found that the two isoforms were present at different levels depending on the location. The advantage of using the levels of the isoforms as biomarkers is based on the fact that these proteins have multiple functions. It is admitted that the function of MTs is the homeostasis of essential metals (Zn, Cu) and the detoxification of nonessential metals such as cadmium (46). Even if this is still elusive (47), the recent studies tend to confirm this hypothesis. Dallinger et al. (38) isolated two different isoforms in the invertebrate Helix pomatia: the first in the mantle and the second in the midgut gland. The midgut gland isoform binds cadmium and seems to be inducible only by this metal, whereas the mantle isoform binds copper. Geret and Cosson (11) observed a 4-fold increase in the concentration of MTs in the gills when exposing the mussels, Mytilus edulis, to 200 µg of cadmium.L-1 for 21 days. This increase was due to the induction of tissuespecific isoforms. Studying the metallothionein profile in oyster embryos Crassostrea virginica, Ringwood and Brouwer (19) were able to isolate four putative metallothioneins isoforms which bound most of Zn and Cu in contrast to adults where these metals are bound to high molecular weight proteins (enzymes and metalloproteins). Furthermore, during exposure of embryos to Cd and/or Cu, two new inducible isoforms (other than putative ones) were detected which bind almost only Cd and/or Cu. The RP-HPLC-fluorescence quantification method showed that MLP isoforms are differently expressed. The use of specific isoforms as biomarkers might be more relevant for a suitable biomonitoring program. However, the basal level fluctuation of the metallothioneins should first be determined to be able to differentiate between the physiological fluctuation which might be generated by different biotic and abiotic factors (water, temperature, age, etc.) and the fluctuation due to the anthropogenic pollution. This study is currently underway in our laboratory.

Acknowledgments This work was supported through a scholarship (I.E.G.) from the German Academic Exchange Service (DAAD) (A/ 00/21360).

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Received for review October 2, 2003. Accepted October 6, 2003. ES035093+