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Induction of two major isoforms of metallothionein in crucian carp (Carassius cuvieri) by air-pumping stress, dexamethasone, and metals
Comparative Biochemistry and Physiology Part C 122 (1999) 75 – 82
Induction of two major isoforms of metallothionein in crucian carp (Carassius cu6ieri ) by air-pumping stress, dexamethasone, and metals Norio Muto 1, Hong-Wei Ren, Gab-Soo Hwang 2, Shinpei Tominaga, Norio Itoh, Keiichi Tanaka * Laboratory of Toxicology, Graduate School of Pharmaceutical Sciences, Osaka Uni6ersity, Suita, Osaka 565 -0871, Japan Received 6 January 1998; received in revised form 16 May 1998; accepted 11 June 1998
Abstract The induction of metallothionein (MT) by physical and chemical stress was assessed using the fresh-water fish, crucian carp (Carassius cu6ieri Temminck et Schlegel). The fish exposed to violent air-pumping stress for 6 days revealed time-dependent induction of MT-like metal-binding proteins in both their livers and kidneys. Their hepatic contents after exposure to stress were elevated to twice the basal level with 24 h, resulting in more than a 3-fold increase at 144 h, whereas their renal contents gradually increased after 24 h and reached the same level as that in the liver around 96 h. Two major inducible proteins were purified from livers of fish exposed to stress and were shown to be MT based upon their chromatographic behavior, UV absorption spectra and their molecular weights. Consequently, they were termed ccMT-1 and ccMT-2, according to their elution sequence upon anion-exchange chromatography. Both proteins mainly bound zinc in their endogenous forms and showed different immunogenicity to rat and rabbit MTs. Dexamethasone, a potent inducer for MT synthesis in mammals, induced the production of both isoforms in crucian carp, whereas cadmium and zinc ions prominently induced the synthesis of ccMT-2. These results indicate that crucian carp have the ability to produce MTs in response to various kinds of environmental stress and that violent air-pumping stress in crucian carp may induce MT synthesis, in part, via the release of endogenous factor(s), such as glucocorticoids. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Air-pumping stress; Crucian carp; Dexamethasone; Induction; Isoform; Metal; Metallothionein
1. Introduction Metallothionein (MT) is a low-molecular weight protein that binds heavy metals such as cadmium, mercury, zinc, and copper with high affinity and it is also characterized by unique biochemical properties, such as the lack of aromatic amino acids and a high content of cysteine residues in the molecule [17,18,22].
* Corresponding author. Fax: +81-6-8798234; e-mail: [email protected] 1 Present address: Graduate School of Applied Biosciences, Hiroshima Prefectural University, Shobara, Hiroshima 727-0023, Japan. 2 Present address: Department of Environmental Engineering, Kunsan National University, Kunsan, Korea.
Mammalian MT is known to occur in multiple forms in the liver and some other tissues. They are inducible in response to certain heavy metals in addition to a great variety of pathophysiological factors, including restriction of food intake, bacterial infection, and exposure to physical and inflammatory stress [3]. Therefore, MT has been considered to be an important factor in the detoxification of heavy metals, in the metabolism of intracellular zinc and copper, and/or in the scavenging of reactive oxygen species. In fish, it has been known that metal-binding proteins exist mainly in the livers and kidneys and they are easily induced by the administration of heavy metals [37]. Fish MTs have been isolated from several fish species including rainbow trout (Salmo gairdneri ), dogfish (Scyliorhinus canicula), and catfish (Heterop-
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neustes fossilis) [9,13,21], and their gene structures have also been revealed [7,15,20,38]. As well as mammalian MTs, fish MTs have also been investigated from the view point of not only their physiological function in maintaining homeostasis but also in the assessment of aquatic environmental pollution, although both aspects still remain to be investigated. Therefore, it is very important to clarify the induction kinetics of MT in fish which were exposed to various environmental variables, such as temperature, light, sound, vibration, solubilized oxygen and nitrogen, chemical substances, infection, starvation, and so on [1,6,23,24,29,35]. In addition to these exogenous inducers, cytokines and glucocorticoids are known to be representative endogenous inducers of MT synthesis in mammals. There have only been a few reports examining the inducibility of MTs by stress and glucocorticoids in fish species in vivo [8,29,34,35]. However, there is not a clear pattern of response between different fish species. Therefore, to accumulate more data for drawing general conclusions regarding the effect of stress on fish MT, the responses to stress, glucocorticoids, and heavy metals were compared using the fresh-water fish, crucian carp (Carassius cu6ieri Temminck et Schlegel), widely distributed in Japan. We report here that violent aeration stress induces the synthesis of two major isoforms of MTs in the liver and kidney of fish. In addition, the induction profile of MT isoforms are compared with those with dexamethasone and metals.
2. Materials and methods
2.1. Chemicals Dexamethasone, cadmium chloride, zinc chloride, and 2,2%-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) were obtained from Nacalai Tesque (Kyoto, Japan). Dexamethasone 21phosphate disodium salt and rabbit MT-I and II were purchased from Sigma (St. Louis, MO). Peroxidaseconjugated goat IgG fraction to rabbit IgG was purchased from Organon (Durham, NC). All other chemicals employed were of the highest purity available from the commercial sources. Rat MT-II was purified from cadmium-treated rat liver by the standard chromatographic procedure described below.
2.2. Animals and treatment Japanese crucian carp (Carassius cu6ieri Temminck et Schlegel) of 15–20 cm length were obtained from Yamaguchi Fish Farm (Higashi-osaka, Japan). They were maintained in 500-l tanks (200 cm length×50 cm width ×50 cm depth) supplied with fresh water under mild air-pumping conditions (air pump model, NS-
Pump mini-w; air output rate, 1.0 l min − 1) for several days. To evaluate the effect of violent air-pumping stress, the fish were subsequently cultured in the same water tank bubbled vigorously with a high-output pump (air pump model, Hiblow SPP-3GA; air output rate, 4.5 l min − 1) for up to 6 days. Groups of three to six fish were sacrificed on the indicated days. In another experiment, the fish maintained under mild air-pumping conditions were treated with an intraperitoneal injection of dexamethasone (0.4, 4, 20, and 40 mg kg − 1) suspended in 5% arabic gum, dexamethasone 21-phosphate (4 and 40 mg kg − 1), cadmium chloride (1 mg kg − 1) or zinc chloride (10 mg kg − 1) dissolved in saline. A group of three fish were sacrificed 1 day after injection. In every experiment, control fish were treated in the same way and given each of the corresponding vehicles.
2.3. MT and metal assays Liver and kidney were dissected out from each fish, weighed, and homogenized in 4 vol of 0.25 M sucrose with a Potter–Elvehjem homogenizer. After centrifugation at 12000× g for 20 min, the supernatants obtained were assayed for cadmium concentration bound to MTs according to the cadmium saturation assay method [28]. In brief, to an aliquot (0.25 ml) of sample 0.2 ml of 0.1 M Tris–HCl buffer (pH 8.0) and 0.5 ml of 10 mg/ml cadmium solution were added. Next, 0.1 ml of 9% hemolysate, which was prepared from washed rabbit red blood cells, was added to the mixture to adsorb excess amounts of cadmium, followed by heat treatment at 100°C for 3 min. The mixture was centrifuged at 6000× g for 5 min and the hemolysate treatment was repeated in the same tube two more times. Then, the cadmium concentrations of the final supernatants were determined in an atomic absorption spectrophotometer (Shimadzu) and MT concentrations were calculated by assuming 7 mol of cadmium per mol of MT and a molecular weight of 6100 for MT. Cadmium and zinc levels of the chromatographic fractions were also determined by atomic absorption spectrophotometry.
2.4. Chromatography of MTS The supernatants of fish liver homogenates were first mixed with cadmium solution at a final concentration of 10 mg cadmium ml − 1, since mammalian MTs are known to become resistant to oxidation by this treatment. All buffers used were also saturated with nitrogen gas to avoid spontaneous oxidation of thiol-rich proteins during the course of purification. They were subjected to gel filtration on a Sephadex G-75 column (2.6× 80 cm) equilibrated with 10 mM Tris–HCl buffer (pH 8.0). Each fraction was monitored for cadmium
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concentration and the peak fractions with an intermediate molecular weight were pooled. The fractions were diluted twice with distilled water and loaded on to a DEAE-Sepharose column (2.6×15 cm) equilibrated with 5 mM Tris–HCl buffer (pH 8.0). Cadmium-binding MTs were eluted with a linear gradient of Tris, ranging from 5 to 300 mM, and monitored by measuring both the absorbance at 254 nm and the cadmium concentration.
2.5. Electrophoretic and spectrophotometric analyses The purified proteins (15 – 20 mg per lane) were electrophoresed on a 10% polyacrylamide gel according to the method of Davis [11] and the gel was stained with 0.1% Coomassie brilliant blue R250. The purified samples were diluted to an appropriate concentration and scanned for their UV absorption spectra at neutral and acidic pHs in a spectrophotometer (Hitachi).
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The basal levels of MT-like metal-binding proteins in the liver and kidney of crucian carp maintained under mild air-pumping conditions were 29.39 5.7 and 18.69 4.3 mg MT equivalent/g tissue, respectively. When fish were cultured under violent air-pumping stress, the increases of MT-like metal-binding proteins were apparently observed in both the liver and kidney (Fig. 1). Both levels gradually increased with the period of exposure to stress, reaching the highest value of 100–110 mg MT equivalent/g tissue. However, there was a difference in the time-dependent induction profiles in these tissues. Hepatic MT-like proteins were rapidly and significantly induced within 12 h and the level had doubled 24 h after exposure. On the contrary, the renal MT-like proteins were hardly induced within 24 h, but reached a level similar to hepatic ones after around 4 days.
3.2. Purification of MT-like metal-binding proteins in crucian carp exposed to stress
2.6. Immunological analysis A major isoform of MT purified from the livers of cadmium-injected fish was polymerized with glutaraldehyde and subcutaneously injected into several sites on the backs of rabbits with Freund’s complete adjuvant. Boost injections of the protein with Freund’s incomplete adjuvant were repeated twice at an interval of 3 weeks. Blood was collected through the carotid artery after 1 week of the final injection and antisera were obtained. Immunological cross-reactivity of the antiserum containing the highest titer of antibody was examined by the enzyme-linked immunosorbent assay method. Various concentrations of each MT were adsorbed in 96-well immunoplates at 37°C for 2 h. After blocking the wells with 0.5% gelatin – 20 mM phosphate-buffered saline – 0.05% Tween 20, each well was treated with the antiserum diluted by 1:1000 at 37°C for 1 h, followed by incubation with peroxidase-conjugated goat anti-rabbit IgG antibody at 37°C for 1 h. The plates were finally incubated with an ABTS solution (pH 4.0) as a substrate and then monitored for absorbance at 415 nm in a plate reader (BioRad).
Hepatic extracts of crucian carp exposed to stress for 6 days were subjected to sequential chromatography on Sephadex G-75 and DEAE–Sepharose columns. Gel filtration gave a typical elution profile similar to those observed in the separation of mammalian MTs and a small UV absorption peak with an intermediate molecular weight (around 10 kDa) contained a high concentration of cadmium (data not shown). Ion-exchange chromatography of this fraction gave two peaks, which were detected by both absorbance at 254 nm and the cadmium concentration (Fig. 2).
2.7. Statistical analysis Data were analyzed by the Student’s t-test with significant probability levels of less than 0.05 or 0.01.
3. Results
3.1. Induction of MT-like metal-binding proteins in crucian carp by 6iolent air-pumping stress
Fig. 1. Time course of induction of MT-like metal-binding proteins in crucian carp by violent air-pumping stress. At the indicated periods of cultivation, fish were sacrificed and the contents of MT-like metal-binding proteins in the liver () and kidney () were determined by the cadmium saturation assay method. The contents were calculated as the amounts equivalent to MT. All data are means 9 SD from three to six fish at each point examined. Significantly different from the corresponding controls, *P B0.05, **PB 0.01.
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3.4. Induction of MT in crucian carp li6ers and kidneys by dexamethasone
Fig. 2. Detection of two MT-like metal-binding proteins in crucian carp liver by ion-exchange chromatography. Hepatic extracts of fish exposed to stress for 6 days were subjected to gel filtration on a Sephadex G-75 column after saturation with cadmium. The cadmium-rich intermediate-molecular weight fractions were then chromatographed using linear gradient Tris elution (——) on a DEAE – Sepharose column. The fractions were monitored for absorbance at 254 nm () and the cadmium concentration ().
Since glucocorticoid hormones physiologically participate in the homeostatic control following exposure to various kinds of stress in mammals, dexamethasone, a synthetic glucocorticoid, was evaluated for its potential to induce MT synthesis in crucian carp. A single injection of dexamethasone caused a dose-dependent rise in MT production in livers 24 h after its administration, resulting in 2- and 3-fold increases at doses of 4 and 40 mg kg − 1, respectively, compared with that of vehicleinjected fish (Fig. 5). The induction of renal MT was only weakly detected at a dose of 40 mg kg − 1, but its inducing effect was not detectable in the intestine. Similar induction was observed in both the liver and kidney 24 h after injection of the same doses of the water-soluble form, dexamethasone 21-phosphate, though their responses were a little lower than those in dexamethasone-treated fish (data not shown).
3.5. Comparison of induction profiles of MT isoforms by 6arious stimuli These two peaks (1 and 2) were homogeneous, as shown by native polyacrylamide gel electrophoresis and had the same mobilities as those of rabbit MT-I and MT-II, respectively (Fig. 3a). Both purified proteins showed no absorption at 280 nm and a weak absorption at around 240 – 250 nm at neutral pH, but such typical profiles diminished completely at acidic pH (Fig. 3b). These results indicate the presence of a metal –thiolate structure in their molecules. Judging from these biochemical characteristics, both proteins could be classified as MT and, therefore, they were designated ccMT-1 and ccMT-2, respectively. To identify the metal endogenously bound to these MTs in fish exposed to stress, the hepatic extracts were applied to gel filtration without cadmium saturation. An intermediate molecular weight peak containing MTs coincided with the peak of zinc concentration (data not shown). This indicates that these inducible MTs are mainly in zinc-bound forms.
A single intraperitoneal injection of cadmium chloride (1 mg kg − 1) or zinc chloride (10 mg kg − 1) induced a drastic rise (150–200 mg MT equivalent/g liver) in MT synthesis after 24 h. These levels were about 2 times higher than their maximum values in fish exposed to aeration stress and dexamethasone. The induction profile of MT isoforms in the livers of fish treated with these metal ions and dexamethasone (20 mg kg − 1) were compared with that by aeration stress. The soluble fractions of liver homogenates from crucian carp treated with these inducers were saturated with cadmium to stabilize MT molecules and then subjected to sequential chromatographies. Fig. 6 illustrates the elution profiles of major MT isoforms in ion-exchange chromatography. Dexamethasone was found to induce both MT isoforms effectively, while the metals induced to synthesize ccMT-2 predominantly. These results reveal that the induction profile of MT isoforms by violent air-pumping stress resembles that with dexamethasone.
3.3. Immunological characterization of crucian carp MT isoforms
4. Discussion
Immunological cross-reactivity of the antiserum raised against cadmium-containing ccMT-2 with crucian carp MTs, rabbit MT-I and rat MT-II was determined by the enzyme-linked immunosorbent assay method. The antiserum only exerted immunological specificity to the crucian carp MT isoforms and it was more sensitive to ccMT-2 than ccMT-1 (Fig. 4). The serum showed no reactivity with rat and rabbit MTs.
In this experiment, we first demonstrated the production of MT-like metal-binding proteins in the liver and kidney of crucian carp maintained under violent airpumping conditions for 6 days. These inducible proteins were purified from liver extracts by the same chromatographic procedure as mammalian MTs were. They possessed some typical biochemical similarities to mammalian MTs, such as a low molecular weight, a
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Fig. 3. Electrophoretic and spectroscopic characteristics of the purified MT-like metal-binding proteins of crucian carp. (a) Crucian carp and rabbit proteins (15 – 20 mg per lane) were electrophoresed on a 10% acrylamide gel under native conditions and stained with Coomassie brilliant blue. (b) Crucian carp proteins were dissolved at an appropriate concentration in 10 mM phosphate buffer (pH 7.0) and monitored for their UV spectra (——). The solutions were then acidified by adding 1/100 volume of 1 N HCl and their UV spectra were recorded (- - -).
high metal-binding capability, and a spectroscopic feature characteristic of a metal – thiolate complex. Consequently, these inducible proteins were concluded to be MT in crucian carp. Stress-induced synthesis of MTs was observed in a time-dependent manner in both the liver and kidney, but their induction kinetics were considerably different. Judging from their responses, the liver was found to be more sensitive to environmental variables than the kidney, resulting in the rapid de novo synthesis of MTs. Similar responses were observed in metal-injected fish, although the MT induction in livers was considerably high. These findings suggest that this protein has essential functions, especially in the liver, to maintain homeostasis. The culture
conditions used in this study are thought to continuously generate sound, vibration, and over-oxygenation in the tank, which may become physical stresses to induce the synthesis of some proteins. Recently, several papers have described the effect of physical stresses on the induction of MT synthesis in fish. Capture stress and handling stress increased the amount of MT-like proteins in mullet (Mugil cephalus L.) liver [1] and rainbow trout [34], respectively. Culture temperature affected MT synthesis especially in the liver of goldfish (Carassius auratus) [6]. In addition, restraint stress in largemouth bass (Micropterus salmoides) [35] and starvation stress in Atlantic cod (Gadus morhua) [23] also enhanced the levels of metal-binding proteins and MT
Fig. 4. Immunoreactivity of the antiserum raised to ccMT-2 with various kinds of MTs. Each MT isoform was adsorbed onto the wells of 96-well immunoplate at the indicated concentrations for 2 h at 37°C. After washing the plate, diluted antiserum raised to ccMT-2 was added to each well, followed by treatment with peroxidase-labeled second antibody. Bound enzyme activity was measured at 415 nm after incubation with the substrate.
Fig. 5. Induction of MT in crucian carp by dexamethasone. Fish were given an intraperitoneal injection of dexamethasone at the indicated doses. After 24 h, the liver, kidney and intestine were excised and the MT contents in these tissue homogenates were determined by the cadmium saturation assay method. All data are means 9SD from three fish. Significantly different from the corresponding controls, *P B0.05.
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Fig. 6. Chromatographic comparison of MT isoforms induced by various stimuli in crucian carp. Hepatic extracts from fish treated with dexamethasone (20 mg/kg) (a), cadmium chloride (1 mg/kg) (b) or zinc chloride (10 mg/kg) (c) were chromatographed according to the isolation procedure, and their ion-exchange chromatograms were demonstrated. Each fraction was monitored for absorbance at 254 nm () and the cadmium concentration ().
mRNA, respectively. This evidence indicates that the stress-induced MTs may play a role in self defense and/or adaptation to environmental changes. The induction of two isoforms of MT in crucian carp livers was confirmed by ion-exchange chromatography. This means that crucian carp have the potential to synthesize two major isoforms of MT. Among several fish species, rainbow trout [20,31] and gibel (Carassius auratus langsdorfi) [32] have been reported to synthesize two types of MT, while roach (Rutilus rutilus) and stone loach (Noemacheilus barbatulus) produce a single isoform of MT [4,19]. We also found that there was a marked difference in the induction profiles of crucian carp MTs by several inducers. The administration of dexamethasone resulted in the moderate induction of both isoforms, as did exposure to aeration stress, whereas the injection of cadmium or zinc induced synthesis of the MT-2 isoform more prominently than the
MT-1 isoform. This finding suggests that stress-induced MT synthesis may be partly mediated through the release of endogenous factors, such as glucocorticoid hormones. Although stress is known to release glucocorticoids in mammals, there are only a few reports dealing with their release and function on MT induction in fish. Tort et al. [34] have recently reported that handling stress increased the levels of liver MT and plasma cortisol in rainbow trout. Receptor-like binding of cortisol was demonstrated in hepatic cytosols of rainbow trout [30] and the elevation of serum cortisol levels in response to restraint stress was observed in largemouth bass [35]. In addition, in vitro studies have demonstrated the induction of MT synthesis in cultured fish cells by glucocorticoids [5,12,16,26]. However, opposite findings that stress (due to catching), dexamethasone, and cortisol failed to stimulate MT synthesis in plaice (Pleuronectes platessa) [29], and that dexamethasone did not induce an increase of MT mRNA in winter flounder (Pseudopleuronectes americanus) [8] have been reported. Thus, hormonal control of MT synthesis in different fish species has not been established. In order to clarify the induction mechanism of MT synthesis by glucocorticoids in crucian carp, it will be necessary to identify steroid regulatory elements in the 5%-flanking region of the MT-coding genes. Mammalian MT genes have been revealed to contain both a metal responsive element and a glucocorticoid responsive element within the 5%-flanking regions of the MT genes [33]. However, very little is known about regulatory elements flanking MT genes in non-mammalian vertebrates, such as fish. Only a limited analysis of cDNA clones which encode MTs for rainbow trout, pike, and stone loach demonstrated that the primary structures of the proteins were highly homologous among these three fish species [2,19,27]. Especially, in rainbow trout, sequences with similarity to glucocorticoid responsive element have been identified in the distal portion of the promoter sequence of MT-A gene, in addition to six metal responsive elements [27]. On the contrary, analyses of regulatory elements flanking the MT genes in pike and stone loach have shown that neither gene locus shows any homology with the glucocorticoid or interferon responsive elements [19]. We have observed MT induction in crucian carp by the administration of dexamethasone as well as metals. This study suggests the presence of glucocorticoid-dependent regulatory mechanism for MT synthesis in this fresh-water fish. Based on these facts, it is very important to clarify the dynamics of MT induction in response to various aquatic environmental variables. It has been shown that there is no immunological homology between fish and chicken or mammalian MTs, indicating apparent differences in their amino acid sequences [9,10,14,25]. Two antigenic determinant peptides which are distributed in the NH2-terminal
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regions (residues 1– 5 and residues 20 – 25) were identified in mammalian MT molecules [36]. However, fish (rainbow trout, pike and stone loach) MTs have been demonstrated to have distinct sequences in these two antigenic regions [20]. In this study, the antiserum raised against ccMT-2 exhibited preferential reactivity with ccMT-2 and moderate cross-reactivity with ccMT1, but absolutely no cross-reactivity with rat and rabbit MTs. This means that the antibody strictly recognizes NH2-terminal peptide characteristic of crucian carp MTs. Indeed, our preliminary study for analyzing the amino acid sequence of ccMT-2 suggests the deletion of asparagine at position 4 within the NH2-terminal region of mammalian MTs (unpublished results). In addition, their structural differences should be clarified to explain the difference between the two crucian carp MTs in their immunoreactivity with this antibody. Since the tissue levels of MTs in crucian carp reflect the extent and degree of physical and chemical stresses, the assay methods specific to each MT may be useful for assessment of the presence of environmental stressors in fish. Although a radioimmunoassay system has been established only for perch MT [14], it is necessary to develop a specific and sensitive immunoassay system applicable to crucian carp in the fresh-water environment. This approach may lead us to establish a reliable biological monitoring system for evaluating aquatic environmental contamination.
Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research (C) (No. 06680506) and Monbusho International Scientific Program for Joint Research (No. 07044145) from the Ministry of Education, Science, and Culture of Japan.
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[33] Stuart GW, Searle PF, Palmiter RD. Identification of multiple metal regulatory elements in mouse metallothionein-1 promoter by assaying synthetic sequences. Nature 1985;317:828 –31. [34] Tort L, Kargacin B, Torres P, Giralt M, Hidalgo J. The effect of cadmium exposure and stress on plasma cortisol, metallothionein levels and oxidative status in rainbow trout (Oncorhynchus mykiss) liver. Comp Biochem Physiol 1996;114C:29 – 34. [35] Weber DN, Eisch S, Spieler RE, Petering DH. Metal redistribution in largemouth bass (Micropterus salmoides) in response to restrainment stress and dietary cadmium, role of metallothionein and other metal binding proteins. Comp Biochem Physiol 1992;101C:255– 62. [36] Winge DR, Garvey JS. Antigenicity of metallothionein. Proc Natl Acad Sci USA 1983;80:2472 – 6. [37] Woodworth J, Evans AS, Pascoe D. The production of cadmium-binding protein in three species of fresh water fish. Toxicol Lett 1983;15:289 – 95. [38] Zafarullah M, Bonham K, Gedamu L. Structure of the rainbow trout metallothionein B gene and characterization of its metal-responsive region. Mol Cell Biol 1988;8:4469 – 76.
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Induction of two major isoforms of metallothionein in crucian carp (Carassius cu6ieri ) by air-pumping stress, dexamethasone, and metals Norio Muto 1, Hong-Wei Ren, Gab-Soo Hwang 2, Shinpei Tominaga, Norio Itoh, Keiichi Tanaka * Laboratory of Toxicology, Graduate School of Pharmaceutical Sciences, Osaka Uni6ersity, Suita, Osaka 565 -0871, Japan Received 6 January 1998; received in revised form 16 May 1998; accepted 11 June 1998
Abstract The induction of metallothionein (MT) by physical and chemical stress was assessed using the fresh-water fish, crucian carp (Carassius cu6ieri Temminck et Schlegel). The fish exposed to violent air-pumping stress for 6 days revealed time-dependent induction of MT-like metal-binding proteins in both their livers and kidneys. Their hepatic contents after exposure to stress were elevated to twice the basal level with 24 h, resulting in more than a 3-fold increase at 144 h, whereas their renal contents gradually increased after 24 h and reached the same level as that in the liver around 96 h. Two major inducible proteins were purified from livers of fish exposed to stress and were shown to be MT based upon their chromatographic behavior, UV absorption spectra and their molecular weights. Consequently, they were termed ccMT-1 and ccMT-2, according to their elution sequence upon anion-exchange chromatography. Both proteins mainly bound zinc in their endogenous forms and showed different immunogenicity to rat and rabbit MTs. Dexamethasone, a potent inducer for MT synthesis in mammals, induced the production of both isoforms in crucian carp, whereas cadmium and zinc ions prominently induced the synthesis of ccMT-2. These results indicate that crucian carp have the ability to produce MTs in response to various kinds of environmental stress and that violent air-pumping stress in crucian carp may induce MT synthesis, in part, via the release of endogenous factor(s), such as glucocorticoids. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Air-pumping stress; Crucian carp; Dexamethasone; Induction; Isoform; Metal; Metallothionein
1. Introduction Metallothionein (MT) is a low-molecular weight protein that binds heavy metals such as cadmium, mercury, zinc, and copper with high affinity and it is also characterized by unique biochemical properties, such as the lack of aromatic amino acids and a high content of cysteine residues in the molecule [17,18,22].
* Corresponding author. Fax: +81-6-8798234; e-mail: [email protected] 1 Present address: Graduate School of Applied Biosciences, Hiroshima Prefectural University, Shobara, Hiroshima 727-0023, Japan. 2 Present address: Department of Environmental Engineering, Kunsan National University, Kunsan, Korea.
Mammalian MT is known to occur in multiple forms in the liver and some other tissues. They are inducible in response to certain heavy metals in addition to a great variety of pathophysiological factors, including restriction of food intake, bacterial infection, and exposure to physical and inflammatory stress [3]. Therefore, MT has been considered to be an important factor in the detoxification of heavy metals, in the metabolism of intracellular zinc and copper, and/or in the scavenging of reactive oxygen species. In fish, it has been known that metal-binding proteins exist mainly in the livers and kidneys and they are easily induced by the administration of heavy metals [37]. Fish MTs have been isolated from several fish species including rainbow trout (Salmo gairdneri ), dogfish (Scyliorhinus canicula), and catfish (Heterop-
0742-8413/99/$ - see front matter © 1999 Elsevier Science Inc. All rights reserved. PII S0742-8413(98)10081-6
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neustes fossilis) [9,13,21], and their gene structures have also been revealed [7,15,20,38]. As well as mammalian MTs, fish MTs have also been investigated from the view point of not only their physiological function in maintaining homeostasis but also in the assessment of aquatic environmental pollution, although both aspects still remain to be investigated. Therefore, it is very important to clarify the induction kinetics of MT in fish which were exposed to various environmental variables, such as temperature, light, sound, vibration, solubilized oxygen and nitrogen, chemical substances, infection, starvation, and so on [1,6,23,24,29,35]. In addition to these exogenous inducers, cytokines and glucocorticoids are known to be representative endogenous inducers of MT synthesis in mammals. There have only been a few reports examining the inducibility of MTs by stress and glucocorticoids in fish species in vivo [8,29,34,35]. However, there is not a clear pattern of response between different fish species. Therefore, to accumulate more data for drawing general conclusions regarding the effect of stress on fish MT, the responses to stress, glucocorticoids, and heavy metals were compared using the fresh-water fish, crucian carp (Carassius cu6ieri Temminck et Schlegel), widely distributed in Japan. We report here that violent aeration stress induces the synthesis of two major isoforms of MTs in the liver and kidney of fish. In addition, the induction profile of MT isoforms are compared with those with dexamethasone and metals.
2. Materials and methods
2.1. Chemicals Dexamethasone, cadmium chloride, zinc chloride, and 2,2%-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) were obtained from Nacalai Tesque (Kyoto, Japan). Dexamethasone 21phosphate disodium salt and rabbit MT-I and II were purchased from Sigma (St. Louis, MO). Peroxidaseconjugated goat IgG fraction to rabbit IgG was purchased from Organon (Durham, NC). All other chemicals employed were of the highest purity available from the commercial sources. Rat MT-II was purified from cadmium-treated rat liver by the standard chromatographic procedure described below.
2.2. Animals and treatment Japanese crucian carp (Carassius cu6ieri Temminck et Schlegel) of 15–20 cm length were obtained from Yamaguchi Fish Farm (Higashi-osaka, Japan). They were maintained in 500-l tanks (200 cm length×50 cm width ×50 cm depth) supplied with fresh water under mild air-pumping conditions (air pump model, NS-
Pump mini-w; air output rate, 1.0 l min − 1) for several days. To evaluate the effect of violent air-pumping stress, the fish were subsequently cultured in the same water tank bubbled vigorously with a high-output pump (air pump model, Hiblow SPP-3GA; air output rate, 4.5 l min − 1) for up to 6 days. Groups of three to six fish were sacrificed on the indicated days. In another experiment, the fish maintained under mild air-pumping conditions were treated with an intraperitoneal injection of dexamethasone (0.4, 4, 20, and 40 mg kg − 1) suspended in 5% arabic gum, dexamethasone 21-phosphate (4 and 40 mg kg − 1), cadmium chloride (1 mg kg − 1) or zinc chloride (10 mg kg − 1) dissolved in saline. A group of three fish were sacrificed 1 day after injection. In every experiment, control fish were treated in the same way and given each of the corresponding vehicles.
2.3. MT and metal assays Liver and kidney were dissected out from each fish, weighed, and homogenized in 4 vol of 0.25 M sucrose with a Potter–Elvehjem homogenizer. After centrifugation at 12000× g for 20 min, the supernatants obtained were assayed for cadmium concentration bound to MTs according to the cadmium saturation assay method [28]. In brief, to an aliquot (0.25 ml) of sample 0.2 ml of 0.1 M Tris–HCl buffer (pH 8.0) and 0.5 ml of 10 mg/ml cadmium solution were added. Next, 0.1 ml of 9% hemolysate, which was prepared from washed rabbit red blood cells, was added to the mixture to adsorb excess amounts of cadmium, followed by heat treatment at 100°C for 3 min. The mixture was centrifuged at 6000× g for 5 min and the hemolysate treatment was repeated in the same tube two more times. Then, the cadmium concentrations of the final supernatants were determined in an atomic absorption spectrophotometer (Shimadzu) and MT concentrations were calculated by assuming 7 mol of cadmium per mol of MT and a molecular weight of 6100 for MT. Cadmium and zinc levels of the chromatographic fractions were also determined by atomic absorption spectrophotometry.
2.4. Chromatography of MTS The supernatants of fish liver homogenates were first mixed with cadmium solution at a final concentration of 10 mg cadmium ml − 1, since mammalian MTs are known to become resistant to oxidation by this treatment. All buffers used were also saturated with nitrogen gas to avoid spontaneous oxidation of thiol-rich proteins during the course of purification. They were subjected to gel filtration on a Sephadex G-75 column (2.6× 80 cm) equilibrated with 10 mM Tris–HCl buffer (pH 8.0). Each fraction was monitored for cadmium
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concentration and the peak fractions with an intermediate molecular weight were pooled. The fractions were diluted twice with distilled water and loaded on to a DEAE-Sepharose column (2.6×15 cm) equilibrated with 5 mM Tris–HCl buffer (pH 8.0). Cadmium-binding MTs were eluted with a linear gradient of Tris, ranging from 5 to 300 mM, and monitored by measuring both the absorbance at 254 nm and the cadmium concentration.
2.5. Electrophoretic and spectrophotometric analyses The purified proteins (15 – 20 mg per lane) were electrophoresed on a 10% polyacrylamide gel according to the method of Davis [11] and the gel was stained with 0.1% Coomassie brilliant blue R250. The purified samples were diluted to an appropriate concentration and scanned for their UV absorption spectra at neutral and acidic pHs in a spectrophotometer (Hitachi).
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The basal levels of MT-like metal-binding proteins in the liver and kidney of crucian carp maintained under mild air-pumping conditions were 29.39 5.7 and 18.69 4.3 mg MT equivalent/g tissue, respectively. When fish were cultured under violent air-pumping stress, the increases of MT-like metal-binding proteins were apparently observed in both the liver and kidney (Fig. 1). Both levels gradually increased with the period of exposure to stress, reaching the highest value of 100–110 mg MT equivalent/g tissue. However, there was a difference in the time-dependent induction profiles in these tissues. Hepatic MT-like proteins were rapidly and significantly induced within 12 h and the level had doubled 24 h after exposure. On the contrary, the renal MT-like proteins were hardly induced within 24 h, but reached a level similar to hepatic ones after around 4 days.
3.2. Purification of MT-like metal-binding proteins in crucian carp exposed to stress
2.6. Immunological analysis A major isoform of MT purified from the livers of cadmium-injected fish was polymerized with glutaraldehyde and subcutaneously injected into several sites on the backs of rabbits with Freund’s complete adjuvant. Boost injections of the protein with Freund’s incomplete adjuvant were repeated twice at an interval of 3 weeks. Blood was collected through the carotid artery after 1 week of the final injection and antisera were obtained. Immunological cross-reactivity of the antiserum containing the highest titer of antibody was examined by the enzyme-linked immunosorbent assay method. Various concentrations of each MT were adsorbed in 96-well immunoplates at 37°C for 2 h. After blocking the wells with 0.5% gelatin – 20 mM phosphate-buffered saline – 0.05% Tween 20, each well was treated with the antiserum diluted by 1:1000 at 37°C for 1 h, followed by incubation with peroxidase-conjugated goat anti-rabbit IgG antibody at 37°C for 1 h. The plates were finally incubated with an ABTS solution (pH 4.0) as a substrate and then monitored for absorbance at 415 nm in a plate reader (BioRad).
Hepatic extracts of crucian carp exposed to stress for 6 days were subjected to sequential chromatography on Sephadex G-75 and DEAE–Sepharose columns. Gel filtration gave a typical elution profile similar to those observed in the separation of mammalian MTs and a small UV absorption peak with an intermediate molecular weight (around 10 kDa) contained a high concentration of cadmium (data not shown). Ion-exchange chromatography of this fraction gave two peaks, which were detected by both absorbance at 254 nm and the cadmium concentration (Fig. 2).
2.7. Statistical analysis Data were analyzed by the Student’s t-test with significant probability levels of less than 0.05 or 0.01.
3. Results
3.1. Induction of MT-like metal-binding proteins in crucian carp by 6iolent air-pumping stress
Fig. 1. Time course of induction of MT-like metal-binding proteins in crucian carp by violent air-pumping stress. At the indicated periods of cultivation, fish were sacrificed and the contents of MT-like metal-binding proteins in the liver () and kidney () were determined by the cadmium saturation assay method. The contents were calculated as the amounts equivalent to MT. All data are means 9 SD from three to six fish at each point examined. Significantly different from the corresponding controls, *P B0.05, **PB 0.01.
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3.4. Induction of MT in crucian carp li6ers and kidneys by dexamethasone
Fig. 2. Detection of two MT-like metal-binding proteins in crucian carp liver by ion-exchange chromatography. Hepatic extracts of fish exposed to stress for 6 days were subjected to gel filtration on a Sephadex G-75 column after saturation with cadmium. The cadmium-rich intermediate-molecular weight fractions were then chromatographed using linear gradient Tris elution (——) on a DEAE – Sepharose column. The fractions were monitored for absorbance at 254 nm () and the cadmium concentration ().
Since glucocorticoid hormones physiologically participate in the homeostatic control following exposure to various kinds of stress in mammals, dexamethasone, a synthetic glucocorticoid, was evaluated for its potential to induce MT synthesis in crucian carp. A single injection of dexamethasone caused a dose-dependent rise in MT production in livers 24 h after its administration, resulting in 2- and 3-fold increases at doses of 4 and 40 mg kg − 1, respectively, compared with that of vehicleinjected fish (Fig. 5). The induction of renal MT was only weakly detected at a dose of 40 mg kg − 1, but its inducing effect was not detectable in the intestine. Similar induction was observed in both the liver and kidney 24 h after injection of the same doses of the water-soluble form, dexamethasone 21-phosphate, though their responses were a little lower than those in dexamethasone-treated fish (data not shown).
3.5. Comparison of induction profiles of MT isoforms by 6arious stimuli These two peaks (1 and 2) were homogeneous, as shown by native polyacrylamide gel electrophoresis and had the same mobilities as those of rabbit MT-I and MT-II, respectively (Fig. 3a). Both purified proteins showed no absorption at 280 nm and a weak absorption at around 240 – 250 nm at neutral pH, but such typical profiles diminished completely at acidic pH (Fig. 3b). These results indicate the presence of a metal –thiolate structure in their molecules. Judging from these biochemical characteristics, both proteins could be classified as MT and, therefore, they were designated ccMT-1 and ccMT-2, respectively. To identify the metal endogenously bound to these MTs in fish exposed to stress, the hepatic extracts were applied to gel filtration without cadmium saturation. An intermediate molecular weight peak containing MTs coincided with the peak of zinc concentration (data not shown). This indicates that these inducible MTs are mainly in zinc-bound forms.
A single intraperitoneal injection of cadmium chloride (1 mg kg − 1) or zinc chloride (10 mg kg − 1) induced a drastic rise (150–200 mg MT equivalent/g liver) in MT synthesis after 24 h. These levels were about 2 times higher than their maximum values in fish exposed to aeration stress and dexamethasone. The induction profile of MT isoforms in the livers of fish treated with these metal ions and dexamethasone (20 mg kg − 1) were compared with that by aeration stress. The soluble fractions of liver homogenates from crucian carp treated with these inducers were saturated with cadmium to stabilize MT molecules and then subjected to sequential chromatographies. Fig. 6 illustrates the elution profiles of major MT isoforms in ion-exchange chromatography. Dexamethasone was found to induce both MT isoforms effectively, while the metals induced to synthesize ccMT-2 predominantly. These results reveal that the induction profile of MT isoforms by violent air-pumping stress resembles that with dexamethasone.
3.3. Immunological characterization of crucian carp MT isoforms
4. Discussion
Immunological cross-reactivity of the antiserum raised against cadmium-containing ccMT-2 with crucian carp MTs, rabbit MT-I and rat MT-II was determined by the enzyme-linked immunosorbent assay method. The antiserum only exerted immunological specificity to the crucian carp MT isoforms and it was more sensitive to ccMT-2 than ccMT-1 (Fig. 4). The serum showed no reactivity with rat and rabbit MTs.
In this experiment, we first demonstrated the production of MT-like metal-binding proteins in the liver and kidney of crucian carp maintained under violent airpumping conditions for 6 days. These inducible proteins were purified from liver extracts by the same chromatographic procedure as mammalian MTs were. They possessed some typical biochemical similarities to mammalian MTs, such as a low molecular weight, a
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Fig. 3. Electrophoretic and spectroscopic characteristics of the purified MT-like metal-binding proteins of crucian carp. (a) Crucian carp and rabbit proteins (15 – 20 mg per lane) were electrophoresed on a 10% acrylamide gel under native conditions and stained with Coomassie brilliant blue. (b) Crucian carp proteins were dissolved at an appropriate concentration in 10 mM phosphate buffer (pH 7.0) and monitored for their UV spectra (——). The solutions were then acidified by adding 1/100 volume of 1 N HCl and their UV spectra were recorded (- - -).
high metal-binding capability, and a spectroscopic feature characteristic of a metal – thiolate complex. Consequently, these inducible proteins were concluded to be MT in crucian carp. Stress-induced synthesis of MTs was observed in a time-dependent manner in both the liver and kidney, but their induction kinetics were considerably different. Judging from their responses, the liver was found to be more sensitive to environmental variables than the kidney, resulting in the rapid de novo synthesis of MTs. Similar responses were observed in metal-injected fish, although the MT induction in livers was considerably high. These findings suggest that this protein has essential functions, especially in the liver, to maintain homeostasis. The culture
conditions used in this study are thought to continuously generate sound, vibration, and over-oxygenation in the tank, which may become physical stresses to induce the synthesis of some proteins. Recently, several papers have described the effect of physical stresses on the induction of MT synthesis in fish. Capture stress and handling stress increased the amount of MT-like proteins in mullet (Mugil cephalus L.) liver [1] and rainbow trout [34], respectively. Culture temperature affected MT synthesis especially in the liver of goldfish (Carassius auratus) [6]. In addition, restraint stress in largemouth bass (Micropterus salmoides) [35] and starvation stress in Atlantic cod (Gadus morhua) [23] also enhanced the levels of metal-binding proteins and MT
Fig. 4. Immunoreactivity of the antiserum raised to ccMT-2 with various kinds of MTs. Each MT isoform was adsorbed onto the wells of 96-well immunoplate at the indicated concentrations for 2 h at 37°C. After washing the plate, diluted antiserum raised to ccMT-2 was added to each well, followed by treatment with peroxidase-labeled second antibody. Bound enzyme activity was measured at 415 nm after incubation with the substrate.
Fig. 5. Induction of MT in crucian carp by dexamethasone. Fish were given an intraperitoneal injection of dexamethasone at the indicated doses. After 24 h, the liver, kidney and intestine were excised and the MT contents in these tissue homogenates were determined by the cadmium saturation assay method. All data are means 9SD from three fish. Significantly different from the corresponding controls, *P B0.05.
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Fig. 6. Chromatographic comparison of MT isoforms induced by various stimuli in crucian carp. Hepatic extracts from fish treated with dexamethasone (20 mg/kg) (a), cadmium chloride (1 mg/kg) (b) or zinc chloride (10 mg/kg) (c) were chromatographed according to the isolation procedure, and their ion-exchange chromatograms were demonstrated. Each fraction was monitored for absorbance at 254 nm () and the cadmium concentration ().
mRNA, respectively. This evidence indicates that the stress-induced MTs may play a role in self defense and/or adaptation to environmental changes. The induction of two isoforms of MT in crucian carp livers was confirmed by ion-exchange chromatography. This means that crucian carp have the potential to synthesize two major isoforms of MT. Among several fish species, rainbow trout [20,31] and gibel (Carassius auratus langsdorfi) [32] have been reported to synthesize two types of MT, while roach (Rutilus rutilus) and stone loach (Noemacheilus barbatulus) produce a single isoform of MT [4,19]. We also found that there was a marked difference in the induction profiles of crucian carp MTs by several inducers. The administration of dexamethasone resulted in the moderate induction of both isoforms, as did exposure to aeration stress, whereas the injection of cadmium or zinc induced synthesis of the MT-2 isoform more prominently than the
MT-1 isoform. This finding suggests that stress-induced MT synthesis may be partly mediated through the release of endogenous factors, such as glucocorticoid hormones. Although stress is known to release glucocorticoids in mammals, there are only a few reports dealing with their release and function on MT induction in fish. Tort et al. [34] have recently reported that handling stress increased the levels of liver MT and plasma cortisol in rainbow trout. Receptor-like binding of cortisol was demonstrated in hepatic cytosols of rainbow trout [30] and the elevation of serum cortisol levels in response to restraint stress was observed in largemouth bass [35]. In addition, in vitro studies have demonstrated the induction of MT synthesis in cultured fish cells by glucocorticoids [5,12,16,26]. However, opposite findings that stress (due to catching), dexamethasone, and cortisol failed to stimulate MT synthesis in plaice (Pleuronectes platessa) [29], and that dexamethasone did not induce an increase of MT mRNA in winter flounder (Pseudopleuronectes americanus) [8] have been reported. Thus, hormonal control of MT synthesis in different fish species has not been established. In order to clarify the induction mechanism of MT synthesis by glucocorticoids in crucian carp, it will be necessary to identify steroid regulatory elements in the 5%-flanking region of the MT-coding genes. Mammalian MT genes have been revealed to contain both a metal responsive element and a glucocorticoid responsive element within the 5%-flanking regions of the MT genes [33]. However, very little is known about regulatory elements flanking MT genes in non-mammalian vertebrates, such as fish. Only a limited analysis of cDNA clones which encode MTs for rainbow trout, pike, and stone loach demonstrated that the primary structures of the proteins were highly homologous among these three fish species [2,19,27]. Especially, in rainbow trout, sequences with similarity to glucocorticoid responsive element have been identified in the distal portion of the promoter sequence of MT-A gene, in addition to six metal responsive elements [27]. On the contrary, analyses of regulatory elements flanking the MT genes in pike and stone loach have shown that neither gene locus shows any homology with the glucocorticoid or interferon responsive elements [19]. We have observed MT induction in crucian carp by the administration of dexamethasone as well as metals. This study suggests the presence of glucocorticoid-dependent regulatory mechanism for MT synthesis in this fresh-water fish. Based on these facts, it is very important to clarify the dynamics of MT induction in response to various aquatic environmental variables. It has been shown that there is no immunological homology between fish and chicken or mammalian MTs, indicating apparent differences in their amino acid sequences [9,10,14,25]. Two antigenic determinant peptides which are distributed in the NH2-terminal
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regions (residues 1– 5 and residues 20 – 25) were identified in mammalian MT molecules [36]. However, fish (rainbow trout, pike and stone loach) MTs have been demonstrated to have distinct sequences in these two antigenic regions [20]. In this study, the antiserum raised against ccMT-2 exhibited preferential reactivity with ccMT-2 and moderate cross-reactivity with ccMT1, but absolutely no cross-reactivity with rat and rabbit MTs. This means that the antibody strictly recognizes NH2-terminal peptide characteristic of crucian carp MTs. Indeed, our preliminary study for analyzing the amino acid sequence of ccMT-2 suggests the deletion of asparagine at position 4 within the NH2-terminal region of mammalian MTs (unpublished results). In addition, their structural differences should be clarified to explain the difference between the two crucian carp MTs in their immunoreactivity with this antibody. Since the tissue levels of MTs in crucian carp reflect the extent and degree of physical and chemical stresses, the assay methods specific to each MT may be useful for assessment of the presence of environmental stressors in fish. Although a radioimmunoassay system has been established only for perch MT [14], it is necessary to develop a specific and sensitive immunoassay system applicable to crucian carp in the fresh-water environment. This approach may lead us to establish a reliable biological monitoring system for evaluating aquatic environmental contamination.
Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research (C) (No. 06680506) and Monbusho International Scientific Program for Joint Research (No. 07044145) from the Ministry of Education, Science, and Culture of Japan.
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