* Department of Biology, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium, and Department of Chemistry, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium
Received February 24, 2004; accepted April 12, 2004
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ABSTRACT |
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Key Words: Cyprinus carpio; metallothionein; copper; zinc; cadmium; HPLC ICP-TOFMS.
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INTRODUCTION |
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In fish, the expression and role of MTs have mostly been studied in organs that play a central role in metal uptake and accumulation, i.e., the liver, kidney, and gills. It has also been shown that significant differences can appear in the expression and induction of MT among different fish species (De Boeck et al., 2003) and among tissues within the same fish species (Olsson, 1993
). In fact, the MT pool often consists of different isoforms that contain certain amino acid substitutions. Generally, two major MT isoforms can be detected in carp liver and kidney (Kito et al., 1986
; Ren et al., 2000
), and isoform-specific induction and differences in metal-binding capacities of the different isoforms after metal exposure have been reported (Kammann et al., 1997
; Muto et al., 1999
).
To reveal the role of MT in metal complexation and buffering, information is required concerning the induction of the various MT isoforms and their metal binding patterns under different exposure scenarios. Metal accumulation may also cause changes in cellular metal binding and speciation patterns, disturbing essential metal homeostasis. Classical techniques, such as enzyme-linked immunosorbent assays, metal-saturation assays, and pulse polarography, provide information on total MT levels, but do not provide information on the induction of different MT isoforms and the differential binding of metals to these and other metal-binding proteins (Lobinski et al., 1998). A high selectivity and resolution in terms of metal binding proteins to analyze the differential complexation of metals can be achieved by the coupling of a high resolution chromatographic separation method to a fast multi-metal detection technique. The coupling of high performance liquid chromatography (HPLC) with inductively coupled plasma mass spectrometry (ICP-MS) is a powerful hybrid technique for speciation studies of metal-binding proteins (Mason and Storms, 1993
; Welz, 1998
). The use of an ICP-time of flight (TOF) MS instead of an ICP-MS system allows the on-line, very fast, and simultaneous quantification of many different metals in the protein fractions separated on the liquid chromatograph (Ferrarello et al., 2002
; Goenaga et al., 2002
).
In the current study we have exploited the advantages of HPLC-ICP-TOFMS to investigate the effect of Cd exposure on the binding of Cd, Cu, and Zn to MT and other metal-binding proteins in the cytoplasmic fractions of the most important metal accumulation organs of the common carp, Cyprinus carpio. Common carp were exposed in a short-term experiment to different Cd concentrations. Total tissue and cytosolic Cd, Cu, and Zn concentrations and (Cu, Cd, Zn)-MT levels were determined in liver, kidney, and gills. The cytosolic metal partitioning was determined by size exclusion (SE) and anion-exchange (AE) high performance liquid chromatography directly coupled to an ICP-TOFMS. The central questions addressed were (1) to what extent cadmium exposure resulted in a metal-dose related MT induction in the different organs, (2) to what extent other metal binding fractions were involved in cadmium sequestration, and (3) whether or not cadmium caused changes in metal-binding patterns of the essential metals copper and zinc.
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MATERIALS AND METHODS |
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Two weeks before the experiments carp (body weight 2034 g, length 90120 mm) were transferred to 80 liter glass aquaria filled with reconstituted fresh water according to the OECD test guidelines (CaCl2.2H2O: 2 mM, MgSO4.7H2O: 500 µM, NaHCO3: 771 µM, KCl: 77.1 µM; OECD, 1993). All salts used were of analytical grade and supplied by Merck (Darmstadt, Germany).
Waterborne Cd exposure experiment. Carp (n = 5 for each concentration) were exposed to (actual concentrations in parenthesis) concentrations of 0 (0.0038 ± 0.0025), 0.1 (0.082 ± 0.013), 1 (0.82 ± 0.09), 2.5 (2.05 ± 0.20), 10 (8.97 ± 0.69), and 20 (19.13 ± 0.69) µM Cd for 96 h at 25 ± 0.5°C by addition of Cd from a 1 g/l Cd stock prepared in deionized water (CdCl2.H2O, Extra Pure, Merck). Exposure to 20 µM resulted in high mortality (80%) and these fish were not considered further in the analysis. The water was replaced every day and the Cd, Cu, and Zn concentrations in the water were determined by ICP MS (Varian Ultra Mass 700, Victoria, Australia). The Cu and Zn concentrations remained nearly constant during the exposure time and the actual Cu and Zn concentrations measured in the different exposure tanks (i.e., 0, 0.1, 1, 2.5, 10 µM Cd) were 0.014 ± 0.009, 0.032 ± 0.029, 0.025 ± 0.022, 0.020 ± 0.022, and 0.027 ± 0.011 µM for Cu and 0.051 ± 0.025, 0.074 ± 0.023, 0.094 ± 0.054, 0.083 ± 0.043, and 0.074 ± 0.057 µM for Zn. The water hardness was 250 mg/l expressed as mg/l CaCO3 and the water pH varied between 7.32 and 7.53. The carp were not fed during the experiment and starved 24 h before the experiment.
After the 96 h-experiment the carp were killed by a single blow on the head, and liver, kidney, and gill samples were dissected on ice. The samples were divided in two parts, weighed, immediately frozen in liquid nitrogen, and stored at 80°C for further processing.
Metal analysis. After thawing, the tissue samples were dried for 48 h at 60°C. The dried samples were weighed (approximately 20 µg) and 150 µl of sub-boiled nitric acid (70%, Pro Analysis, Merck) and 15 µl of hydrogen peroxide (30%, Pro Analysis, Merck) were added. Sample digestion was performed in a microwave oven (Blust et al., 1988). After cooling down, 1 ml of Milli-Q water (Millipore, Bedford, MA) was added and the samples were weighed again. The samples were diluted 50 times with 1% nitric acid. The total Cd, Cu, and Zn concentrations were measured using ICP MS. All tissue concentrations are expressed on a fresh weight basis. Dry weight/fresh weight ratios for liver, kidney, and gills were respectively: 0.246 ± 0.072, 0.241 ± 0.052, 0.119 ± 0.026 (means ± SD).
Ten µl of each cytosolic sample was incubated with 200 µl of 70% HNO3 until total digestion and further diluted to 2 ml with 1% nitric acid. The cytosolic Cd, Cu, and Zn contents were measured using ICP MS. Preparation of the cytosolic fraction is described further in the text. Total Cd, Cu, and Zn concentrations in the exposure water were also measured with ICP MS after acidification with nitric acid to a 1% HNO3 solution. The accuracy of the metal analysis was verified using certified mussel tissue reference material (CRM 278) of the Community Bureau of Reference (EU). Recoveries were within 10% of the certified values.
MT quantification. After thawing, the tissue samples were homogenized using an Ultra-turrax T8 (IKA, Labortechnik, Staufen, Germany) in 3 volumes (v/w) of 10 mM Tris-HCl buffer (pH 7.4) containing 5 mM ß-mercaptoethanol (to prevent oxidation), and 0.1 mM phenylmethanesulfonylfluoride (PMSF, protease inhibitor). Tris-(hydroxylmethyl)-aminomethane (Tris), PMSF, and ß-mercaptoethanol were obtained from Sigma (Sigma-Aldrich, St. Louis, MO). The samples were centrifuged at 9000 x g at 4°C for 10 min. (Eppendorf Centrifuge 5804R, Hamburg, Germany) and ultra centrifuged at 100 000 x g for 60 min. at 4°C (Sorval Discovery TM 90 Ultra speed centrifuge, Newton, CT). Supernatant aliquots (cytosolic fractions) were stored at 80°C for a period not longer than 24 h before use. Total cytosolic MT concentrations were measured using the cadmium thiomolybdate saturation assay from Klein et al. (1994). Before use, the ion exchangers (CM-Sephadex, DEAE-Sephacel, Chelex 100) were washed with 30 vol of 10 mM Tris-HCl, 1 M NaCl, pH 7.4, and equilibrated with 30 vol of 10 mM Tris-HCl, 85 mM NaCl, pH 7.4 (buffer A). Fifty µl of the cytosol was mixed with 10 µl of 300 mM ZnSO4.7H2O (Merck) in a 1.5 ml vial and subsequently incubated with 10 µl of 140 mM beta-ME at room temperature for 30 min. After incubation with 70 µl acetonitrile (Merck) for 3 min 500 µl of buffer A and 100 µl of CM-Sephadex (66 % [v/v] suspension in buffer A) were added. The mixture was shaken during 3 min and incubated with first 50 µl of bovine serum albumin (30 mg/l, freshly prepared) and subsequently with 20 µl of ammonium tetrathiomolybdaat (500 µM freshly prepared in buffer A) both for 2 min. The ammonium tetrathiomolybdate was added to complex and remove the Cu(I) molecules bound to MT. After shaking with 100 µl of DEAE-Sephacel (66% [v/v] suspension in buffer A) for 3 min the precipitate was removed by centrifugation at 8000 x g for 5 min. An aliquot of 600 µl of the supernatant was saturated with 10 µl of 109Cd-labeled CdCl2 (1 mM, 740 kBq/ml, specific activity) for 5 min, thereby exchanging the endogeneous Cd and Zn. The excessive 109Cd(II) was complexed by Chelex-100. CdCl2 was supplied by Merck and 109Cd (37 MBq/µg Cd) was obtained from Amersham Biosciences (Piscataway, NJ).
Following a centrifugation step at 8000 x g for 5 min, 500 µl of the supernatant was incubated with 500 µl of acetonitrile for 3 min. The precipitate was removed by centrifugation, and the 109Cd(II) bound to MT in the supernatant solution was measured with a Minaxi-Autogamma 5530 counter (Canberra Packard, Boston, MA). The MT concentrations were calculated on the basis of a molar ratio of Cd/MT of seven (Kito et al., 1982). It has been shown that this method compares very well with other established techniques such as Elisa or the Ag-saturation method (Bienengräber et al., 1995
).
HPLC analysis. For the separation and detection of the cytosolic metalloprotein fractions and the MT isoform fractions a chromatographic system consisting of a Shimadzu (Kyoto, Japan) PEEK Solvent Delivery module LC-Ai equipped with an FCV-10AL quaternary valve, a Rheodyne Model 7125 PEEK sample injector (Rohnert, CA) fitted with a 50-µl loop was used. The size-exclusion chromatography was performed on a SUPELCO (Bornem, Belgium) TSK gel G 3000 PWXL gel-filtration column (7.8 mm i.d.: 30 cm, 6 µm particle size). Anion-exchange HPLC was performed using a polymeric Protein-PAK DEAE-5PW anion-exchange column (75: 7.5 mm i.d., 10 µm particle size) (Waters, Mildford, MA). The HPLC system was directly coupled to the nebulizer of the ICP-TOFMS (Renaissance, LECO, St. Joseph, MI) via peek tubing (0.5 mm i.d.: 7.0 cm). A detailed description of the system is given elsewhere (Tian et al., 1999).
The cytosolic fractions were filtered on 0.45 µm cellulose acetate filters (Alltech, Nicholasville, KY) just before application on the column. Cytosol samples of 50 µl, or the corresponding gel-filtration and MT standard solutions used for calibrating the column, were injected. The separation and elution of the different size fractions was achieved by SE using 30 mM Tris-HCl (pH 7.4, flow 0.8 ml/min) as mobile phase. The separation and elution of the MT isoforms, after partial purification of the MT fraction by SE HPLC, was achieved by AE using a concentration gradient of 2200 mM Tris-HCl buffer. The chromatographic conditions were as described by Goenaga et al. (2002).
Theoretical concentration of MT. The calculation of the theoretical concentration of MT (MTtheor.) taking into account the fraction of the cytosolic metals bound to the MT fraction for the different exposure groups was done using the following equation:
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The exact amount of metal bound to the MT peak was determined on two pooled liver and kidney cytosols for every exposure group separately and measured by external calibration using commercial rabbit liver MT I (Sigma). Recovery studies of our cytosols spiked with the MT 1 rabbit liver standards revealed very good results: 103% for Cd, 98% for Zn, and 92% for Cu.
To quantify the amount of metal bound to the MT peak for the individual samples the parameter Frmetal was calculated. The Frmetal is the concentration of Cd, Cu, or Zn bound to the MT peak measured on the pooled samples, divided by the total concentrations of the metals present in the individual cytosols. In other words, MT theor. is the MT equivalent of the amount of Cd, Cu, and Zn associated with the MT peak taking into account the maximum theoretical metal binding capacity of the MT molecules.
Statistical analysis. All values are given as mean ± SD. Statistical analyses were performed with Statistica (version 5.1, Statsoft, Tulsa, OK) using Pearson correlation and one-way analysis of variance (ANOVA). Normality of data was verified using the Kolmogorov Smirnov test, and the homogeneity of variances of the data was checked by the Levene's test. Post hoc comparisons were done using Dunnett's test for comparing the exposure groups to the control group. Significance levels of tests are indicated by asterisks according to the following probability ranges: *0.05 p > 0.01, **0.01
p > 0.001 and ***p
0.001.
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RESULTS |
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DISCUSSION |
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Liver and kidney are clearly the main Cd accumulating organs. These high Cd concentrations contributed for an important part to the presence of high MT levels, since at the highest Cd exposure concentration nearly 80% of the hepatic and renal cytosolic Cd was found in the MT pool. The highest Cu concentrations were found in the liver followed by the kidney and the gills. The high hepatic Cu load was also reflected in high Cu-MT concentrations, which may be caused by relative high background Cu exposure prior to the Cd exposure. In comparison to other fish species very high concentrations of Zn were found in the liver and gill tissues. This has also been reported by Hogstrand et al. (1996), Sun and Jeng (1998)
, and Jeng et al. (1999)
who found that tissues of the squirrelfish and the common carp, respectively, contained extraordinarily high zinc concentrations in comparison to other freshwater fish species. In our study, the cytosolic renal Zn concentrations increased significantly in the highest Cd exposure group. This was also found by Kuroshima (1992)
, who exposed carp to 0.3 mg/l Cd for 96 h. Also, a positive correlation between the renal cytosolic concentrations of Cd and Zn was found (n = 23, r = 0.73, p < 0.01). In the liver and gill tissues no significant correlation between the cytosolic Cd and Zn concentrations was found. This is in correspondence with the findings of Roméo et al. (2000)
who reported increasing renal Zn concentrations with increasing renal Cd concentrations after ip injections of Cd. Since only the cytosolic Zn concentration increased and not the Zn concentration in the total tissue, a redistribution of Zn among different cellular compartments appears to occur. Jeng et al. (1999)
demonstrated that 1040% of the total cellular zinc is found in the cytosol, which is well corresponding with our data since 2030% of the Zn was found in the cytosol. According to the same authors 6090% can be found in the nuclei/cell debris fraction. They also showed that a citrate buffer could easily release the zinc bound to these fractions, suggesting that these zinc pools may serve as a zinc reservoir able to receive or donate Zn to the cytosolic fraction whenever needed. Yang et al. (2000)
suggested that the increase of Zn, which was mostly associated with the HMW fractions, could be related to an increase in HMW Zn-binding proteins or a binding of Zn to proteins that normally do not contain Zn. Indeed, HMW proteins play a much more important role in renal cytosolic Zn speciation as only 2% of the cytosolic Zn was found to be bound to MT. Likewise Jeng et al. (1999)
concluded that MT could not account for the high concentrations of zinc normally found in common carp tissues.
The results show a clear hepatic and renal MT induction with increasing Cd exposure concentrations leading to more or less clear dose-response relationships between the tissue Cd concentrations and the tissue MT levels. Also, clear tissue-specific differences in response to Cd exposure were observed. In the liver a pronounced Cd-MT induction was only observed in the highest exposure group, reflecting an apparently low capacity of the liver tissue for Cd-MT induction after a short waterborne Cd exposure. Indeed, the endogenous liver Cu-MT levels were already high in comparison to the Cd- and Zn-MT present. Changes in (Cu, Cd, Zn)-MT levels due to small increases of Cd-MT levels will consequently not be detected unless this Cd-MT induction is highly significant as for the 10 µM Cd exposure concentration. A much clearer dose-response induction of the Cd-MT peak was found for the kidneys. Cadmium also was associated to high-molecular weight proteins in the fish exposed to 10 µM Cd. This spill-over of Cd from the Cd-MT pool to other cytosolic pools may result in cellular toxicity by the inhibition of essential functions (Cherian and Nordberg 1983; Klaassen et al., 1999
). Such effects may explain the high mortality in the 20 µM-exposure group that was not observed in the 10 µM exposure group over the short exposure period. The tissue-specific differences were also expressed in the percentage of metals bound to the MT fraction. Whereas the proportion of Cd bound to MT in the liver decreased with increasing Cd exposure concentrations and only increased again at the 10 µM Cd exposure because of the significant Cd-MT induction, the renal MT-bound Cd-fraction increased with increasing Cd exposure concentrations over the entire Cd exposure range. Normally, it is expected that the hepatic Cd-MT increases first before the renal since the liver has been shown to be the first organ for MT-induction (Klaassen et al., 1999
). However, it has been shown that under acute and highly toxic circumstances, Cd can be directly transported via the blood to the kidneys, where it can induce MT-synthesis. Similar results have been found by De Smet et al. (2001b)
and Hollis et al. (2001)
, who found that the most important MT increase during cadmium exposure, occurred in the kidney. Moreover, it was observed that in the kidney the binding of Cd to the HMW fraction occurred at lower cytosolic Cd concentrations and lower Cd-MT and total MT levels than in the liver. At higher renal Cd concentrations the rate of MT induction in the kidneys did not appear to be high enough to keep track with the high Cd accumulation rates in order to bind all newly incoming Cd.
The (Cd, Cu, Zn)-MT concentrations measured by the saturation assay were in reasonably good agreement with the MT levels estimated on the basis of the percentages of cytosolic Cd, Cu, and Zn bound to the MT fraction and the specific binding capacities of MT. The theoretical MT concentrations based on the -SH content of MT and stoichiometry of Cd, Zn, and Cu for -SH in MT (Roesijadi, 1994b) resulted in comparable values of MTtheor. as presented in the article. The differences between the thiomolybdate and theoretical MT levels can be due to the fact that the theoretical estimates assume certain stoichiometric binding characteristics. In addition, the thiomolybdate Cd saturation method also measures the apothioneine pool, if present. Langston et al. (2002)
found similar results correlating MT levels obtained by differential pulse polarography and MT levels estimated from metal binding profiles in the liver of eel.
Only few studies report on the multi-metal speciation of the MT-isoform fractions (Dallinger et al., 1997; Lacorn et al., 2001
; Muto et al., 1999
). Some authors reported differences in inducibility and binding properties of the different MT isoforms (Brouwer et al., 1992
; Kammann et al., 1997
). Our data confirmed the induction of two Cd-MT isoforms by Cd exposure in common carp in kidney and liver tissue. In nonexposed carp Cu- and Zn-MT I and -MT II were present in the liver, but the MT I-isoform fraction was predominant. In the kidney only Cu-MT I was detected and no Zn-MT I and II was found. After Cd-exposure, a clear induction of Cd-MT I and II was found in the liver and kidney cytosols. In the renal cytosols of the carp exposed to 10 µM Cd, both Zn-MT I and II could be detected. These data support the view that MT I plays a role in normal metal homeostasis. Muto et al. (1999)
and Hermesz et al. (2001)
found that Cd exposure prominently induced the synthesis of MT II-mRNA and the MT II protein in carp. MT I-mRNA and MT I protein were more efficiently induced by other stressors such as dexamethasone. According to Hermesz et al. (2001)
the MT protein isoforms, previously reported by Kito et al. (1986)
and Muto et al. (1999)
are not in agreement with the classification made on the basis of the MT I and MT II genes. Therefore they suggested that the common carp most likely has more than two genes for MTs and that MT I and/or MT II protein isoforms are a mixture of different proteins and can not be directly linked to the MT I and MT II mRNA levels. They also found that specific motifs in the 3'-untranslated ends may result in different turnover rates for the mRNA isoforms in different tissues and the cell-type specific translation of the different MT isoforms. These findings indicate that the different MT isoforms have different physiological roles and different ways of induction, depending on the cell types and tissues. As demonstrated, the SE-HPLC profiles showed significantly increased levels of Zn associated to the MT- and HMW fractions after exposure to 10 µM Cd for 96 h. It has also been shown that Zn plays a key role in MT induction (Klaassen et al., 1999
; Roesijadi, 1994
). Briefly, the binding of Zn to the metal transcription factor (MTF-1) allows this protein to bind to a metal response elements (MREs) in the promotor region of the MT gene which, in turn, initiates the MT-gene transcription. It has been proposed that MTF-1 regulates the labile or free Zn concentration by controlling the expression of MT (Coyle et al., 2002
).
In conclusion, the tissue specific cytosolic speciation of Cu, Zn, and Cd, with special emphasis on MT, was studied in common carp under control and different Cd exposure levels. Both hepatic as well as renal MT were induced by Cd exposure, but in the kidney this MT induction was found to occur at lower cytosolic Cd concentrations. In the gills Cd accumulation and MT induction were much less. On the level of cytosolic metal speciation, the kidney was found to be more responsive to Cd exposure. Cd-MT levels in the kidneys showed a steady increase with increasing Cd exposure, while in the liver only for the 10-µM Cd exposure group a clear Cd-MT induction was detected. At this concentration also a small amount of Cd appeared in the HMW pool. In parallel with the Cd-MT induction, a clear increase of Zn bound to the HMW- and MT-fractions was found in the kidney but not the liver.
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NOTES |
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3 Present address: LGC LTD, Queens Road, Teddington, Middlesex, TW11 OLY, UK.
1 To whom correspondence should be addressed at Ecophysiology, Biochemistry and Toxicology Group, Department of Biology, Campus Middelheim, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium. Fax: 00 32 3 265 34 97. E-mail: karen.vancampenhout{at}ua.ac.be.
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