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Cadmium uptake and sequestration in the pike (Esox lucius)
Camp. Biochem. Physiol. Vol. 95C, No. 2, pp. 217-221,
1990
0306~4492/90 $3.00 + 0.00
0 1990 Pergamon Press plc
Printed in Great Britain
CADMIUM
UPTAKE
AND SEQUESTRATION (ESOX
IN THE PIKE
LUCIUS)
C. G. NOREY,A. CRYERand J. KAY* Department
of Biochemistry,
University
of Wales College U.K.
(Received
21 October
of Cardiff,
P.O. Box 903, Cardiff
CFl
IST,
1989)
Abstract-l. Pike were exposed to cadmium dissolved in their aquarium water at separate concentrations of 60, 125 and 225pg/l and the distribution of the metal accumulated in the major body organs was determined. 2. The fractional retention coefficient ([pgCd/lOOg body wt]/[(pgCd/l) x weeks]) for cadmium was constant at each concentration and at different periods of exposure to the metal. The values measured were remarkably low. 3. When cadmium was introduced into the fish by intraperitoneal injection, the metal was found associated with metallothionein. 4. The amino acid composition and immunochemical cross-reactivity of pike metallothionein were comparable to those of other piscine metallothioneins. 5. The unexpected tolerance of pike to the toxic effects of cadmium when exposed to the metal in the aquatic environment is discussed.
INTRODUCTION
MATERIALSAND METHODS
Certain fish species, such as rainbow trout (Sulmo gairdneri), brown trout (Salmo trutta fario L.) and salmon (S&o s&r) are particularly sensitive to the toxic actions of cadmium when it is present in their aqueous environment. By contrast, coarse fish, including roach (Rut& rutilus L.) and stone loach (Noemacheilus barbatulus), can tolerate much higher concentrations of the dissolved metal (see for example, Brown et al., 1986). When exposed to sublethal concentrations of cadmium in their aquarium water, the tolerant species retain a significantly lower proportion of the metal in their tissues than do sensitive species such as rainbow trout (Brown et al., 1986). In previous reports (Kay et al., 1986; Brown et al., 1987) we have attempted to explain these differential sensitivities to cadmium and the observed differences in metal retention on a rational basis (Kay and Cryer, 1986; Kay et al., 1987). Such hypotheses however, must be tempered by the qualification that only data from salmonid species have been available to represent the cadmium-sensitive group of fish. Previous observations (EIFAC, 1977) have suggested that the pike (Esox lucius) is a non-salmonid species that may be considered sensitive to the toxic actions of dissolved cadmium. Thus, in order to broaden the basis of the hypothetical mechanisms and to ascertain whether these previous interpretations of the behaviour of resistant and sensitive fish species remain valid, the uptake, tissue distribution and sequestration of cadmium in the pike is reported herein.
The fish used in the present study were maintained in dechlorinated mains water (chlorine less than 0.1 ppm) at a total water hardness of approximately 120 ppm (90-100ppm as CaCO,). They we&housed in-tanks &ntaining 200 1 of water changed at a flow rate of 1.5-2 limin and were fed every 2-3 days. The pH of the water was neutral and the oxygen saturation was always greater than 80%. Pike (50-160 g body weight) were generously donated by Drs N. Giles and R. Wright (The ARC Wildfowl Centre, Milton Keynes, Bucks., U.K.). In the tanks, refuges for the fish were provided in the form of 45 cm lengths of 10 cm diameter opaque plastic pipe. Further cover for the fish was provided in the form of simulated floating reeds constructed from strips of black polythene sheet measuring approximately 4 cm x 40 cm. The fish were exposed to cadmium dissolved in the aquarium water at concentrations ranging from 60 to 225 pg/l for periods of between 2 and 11 weeks using a siphon doser (Shurben, 1978). Organs were removed from anaesthetized fish and samples were prepared for metal analysis as described previously (Roberts et al., 1979). Cd, Zn and Cu were determined by atomic absorption spectroscopy at 228.8, 213.9 and 324.8 nm respectively, using a Varian model AA 275 fitted with a background corrector (Varian Instruments, Walton-on-Thomas, Surrey, U.K.). In separate experiments, cadmium was administered to pike (200-450 g body weight) by the intraperitoneal injection of 0.2 mg/kg of the metal (as the sulphate salt) followed by a second injection (0.6 mg/kg) three days later. The fish were then sacrificed after a further two days and the main cadmium-containing protein was purified from liver as described by Brown et al. (1987). Samples of protein for amino acid analysis were hydrolysed in 6 M HCl in D~CUOat 105°C for 18 hr. Cysteine was determined as cysteic acid following oxidation of the protein samples with performic acid (Hirs, 1967) prior to
*Author for correspondence. 217
C. G. NOREYet al.
218
hydrolysis. Analysis was performed using a LKB 4150 alpha automated amino acid analyser, equipped with a recording integrator (Pharmacia LKB Biotechnology, Cambridge, U.K.). A competitive ELISA for the quantitation of fish MT was established as described previously (Norey et al., 1990), using a polyclonal antiserum to rainbow trout metallothionein raised in mice. Homogeneous rainbow trout MT was coated overnight onto the wells of Falcon “probind” microtitre plates from a solution of the protein (148 ng/ml) in carbonate-bicarbonate buffer, pH 9.6. Aliquots of the soluble competing antigen (either rainbow trout MT (in the range lo’-lo6 pg) in the standard curve or pike liver protein) were incubated overnight at 4°C with the mouse antimetallothionein antiserum at a final dilution of l/4000 prior to the incubation of these mixtures with the immobilized trout MT. The reaction between antibody and immobilized antigen was visualised using a rabbit-anti mouse IgG conjugated to horse radish peroxidase (Dako Ltd., High Wycombe, Bucks., U.K.) at a dilution of l/l000 and phenylenediamine as substrate. RESULTS AND DISCUSSION
Pike were exposed to cadmium in their aquarium water at concentrations of 60, 125 and 225 pg/l for up to 11 weeks. In common with our previously reported observations on other species (Brown et al., 1986, 1987), the liver, kidney and gills were the major organs in which cadmium accumulated in the pike. Similar patterns of accumulation were observed for pike kept at all three of the concentrations used. Therefore, for brevity, only the data collected from the experiment using 125 pg/l are presented in Fig. 1. In each of the three organs, the concentration of cadmium &g/g wet weight of tissue) increased progressively over the 11 week period of the study (Fig. la). This pattern was unaltered when the cadmium concentrations were expressed as pg/whole a /
organ/l00 g body weight. In each of the studies, heart, spleen, skeletal muscle and gonadal tissue were also analysed and found to contain no significant amounts of cadmium at any time. Thus, from the cadmium concentrations determined in the liver, kidney and gills, total body burdens of accumulated metal were calculated at each time point of analysis. The values obtained for pike kept at 125 pg of cadmium/l for 2, 5, 8 and 11 weeks were 0.55, 1.O, 1.2 and 2.2 pg (per 100 g body weight) respectively. The corresponding values for both sensitive and tolerant fish species reported previously (Brown et al., 1986) were, however, much higher. Indeed, the level of accumulation in pike kept at relatively high concentrations of the metal in their aquarium water, was exceptionally low, even in relation to the tolerant species studied previously. Despite this low overall level of accumulation in the pike, the distribution of the metal load among the liver, kidney and gills (expressed as a proportion of the total body burden accumulated; Fig. lb) was similar to that seen for other fish species (Thomas et al., 1983b; Brown et al., 1986, 1987). Initially, the major proportion of the body burden was carried by the gill but by 8 weeks of exposure, approximately equivalent loads were present in all three of the organs. Similar patterns were also observed with fish exposed at the lower (60 pg/l) and at the higher (225 pg/l) concentrations of cadmium when the data were expressed in similar terms (not shown). In order to permit comparisons of the accumulation of whole body cadmium by various species of fish, we have previously defined (Brown et al., 1986) a fractional retention coefficient for cadmium, as the quotient of the total body cadmium accumulation (pg/lOOg body weight) and the notional cadmium dose [(pg/l) x weeks]. Values of this fractional retention coefficient obtained in hard water for Cdtolerant species such as roach and stone loach were 7 x 10e3 and 14 x 10e3 respectively. These were considerably lower than the corresponding coefficient, (40 x 10m3) measured for Cd-sensitive species, e.g. rainbow trout. From the data presented above, the fractional retention coefficient for cadmium was calculated for pike on the same basis at each time point of analysis and at each concentration of cadmium. Table 1 shows that for pike, as reported for other species (Brown et al., 1986) the coefficient was unaffected either by the concentration of cadmium to which the fish were exposed or by the duration of exposure. The mean overall value for the coefficient in pike, Table 1. Comparison of the fractional retention of cadmium by pike at different cadmium concentrations
&05 TIME OF EXPOSURE [WEEM;’
Fig. 1. Uptake and distribution of cadmium in the tissues of pike exposed to the metal in their aquarium water (125 pg/l) for different lengths of time. Values are expressed as (a) tissue cadmium concentrations @g/g) and (b) distribution of the metal among the three major organs; gill (w----m), kidney (0-O) and liver (O----O).
Cadmium concentration (w3/0 60 125 225
Fractional retention coefficient (units x IO’) 1.8 + 1.0(6) 1.6CO.4(11) 1.4&0.6(10)
Fractional coefficients were calculated as units being ~;;;l~[ps Cd/100 g body weightl/[(lcgCd/t)
Values given are the means + SD, the number of observations are given in parentheses.
Cadmium in pike 1.5 + 0.7 x 10m3 units (n = 27) was, however, remarkably low when compared with those reported previously for rainbow trout, a cadmium-sensitive species. Indeed it was substantially below the values for the coefficient measured in the cadmium-tolerant species, roach and stone loach (Brown et al., 1986). Thus, contrary to previous indications (EIFAC, 1977; made with fish at a different stage of development than those used in the present study), the pike would appear to be a species that is relatively resistant to cadmium in the aquatic environment. This tolerance may be a reflection of the very low levels at which cadmium became sequestered in the body of the fish, as discussed previously (Kay and Cryer, 1986). Further investigations of this sequestration, by analysis of the tissues of fish exposed to cadmium in the environment, were precluded since so little of the metal accumulated over even quite protracted periods. Therefore, to ensure that sufficient cadmium accumulated in the tissues of fish for quantitative fractionation and analyses to be made, a different approach had to be adopted. Relatively large amounts of the metal were administered to pike by intraperitoneal injection, as described in the Materials and Methods section. The tissue distribution of cadmium administered in this fashion was measured and the nature of the proteins responsible for its sequestration was established. The distribution of the injected metal among the tissues studied is shown in Table 2. Approximately 80% of the cadmium retained in the bodies of the treated fish was accounted for collectively by the liver, kidney and gills. This pattern of distribution was similar to that seen not only with pike treated with environmental cadmium at the later times of exposure (Fig. lb) but also with other fish species into which cadmium had been introduced by intraperitoneal injection (Thomas et al., 1983b). In order to determine the nature of the protein(s) responsible for sequestration of cadmium, the liver was chosen for further study on the basis of (1) the amount of tissue available and (2) because previous observations (Thomas et al., 1983b, 1985; Brown et al., 1987) in other species of fish had revealed that the protein binding patterns exhibited by the other organs of accumulation (e.g. kidney and gill) are identical to those seen for the liver. Following the administration of cadmium to pike by intraperitoneal injection, the supernatant fraction from an homogenate of liver tissue was prepared as described previously (Thomas et al., 1983a, 1985). After concentration, the sample (39 ml) was subjected to gel filtration on a column of Sephadex G-75 (2.5 x 80cm). The cadmium was eluted as a single
219
symmetrical peak in a position (retention of 0.88 of the column volume) consistent with the metal being bound in a macromolecular form of approximately 10,000 daltons (data not shown for brevity). This pattern of elution was identical to those described previously with supernatants derived from other fish species to which cadmium had been administered by intraperitoneal injection (e.g. Overnell and Coombs, 1979; Thomas et al., 1983a). The average recovery of cadmium from this chromatographic step was 43%. As described previously (e.g. Brown et al., 1987), the cadmium-containing fractions were pooled, concentrated by ultrafiltration over a YM2 membrane and then buffer exchanged into 20 mM Tris-HCl buffer, pH 7.4, containing 2 mM mercaptoethanol. The concentrated material was subjected to ion exchange chromatography on a column of DEAE-cellulose (1.5 x 15 cm), as described by Brown et al., 1987. As found previously with material derived from stone loach and roach, the cadmium was eluted as a single peak which emerged at a concentration of 60 mM Tris in the eluting buffer (Fig. 2). This position of elution in the gradient is comparable to that observed with material from the stone loach (Brown et al., 1987) but somewhat later than that from roach. Further purification of this pike liver cadmium binding fraction, was carried out by concentration and desalting of the sample followed by resolution on an FPLC Mono-Q anion exchange column (Pharmacia, Uppsala, Sweden), equilibrated in 10 mM Tris-HCl buffer, pH 8.6. After washing, a linear gradient from 0 to 0.1 M NaCl in Tris-HCl buffer, pH 8.6, was applied. The pattern of AIs4 determined for the eluted fractions revealed one major cadmiumcontaining peak, emerging in the gradient at 0.028 M NaCl. The cadmium-binding protein thus isolated from the liver of pike was performic acid oxidized (see Material and Methods), prior to the determination of its amino acid composition (Table 3). This revealed
,I-/
L__
L
20
Table 2. Cadmium distribution among tissues and organs of pike exposed to the metal by intraperitoneal injection Cadmium Tissue/organ Liver Kidney Gill Heart Spleen
Gonad Skeletal muscle
recovered W) 31 33 15 3 9
9 0
40 FRACTION
60
20
NUMBER
Fig. 2. Ion exchange chromatography of the pooled fractions obtained after gel filtration on Sephadex G-75 of extracts of liver from pike exposed to cadmium by intraperitoneal injection. The cadmium-containing material was applied to a DEAE cellulose column (1.5 x 15cm), equilibrated in 20 mM Tris-HCI buffer, pH 7.4 containing 2 mM mercaptoethanol. After extensive washing, a linear gradient of 20-120 mM Tris-HCl buffer (200 ml each) containing 2mM mercaptoethanol was used for desorption. e-0 = cadmium.
C. G. NOREYet al.
220
,I
0
3
Loa MT (PP)
Fig. 3. Cross immunoreactivity of rainbow trout metallothionein (a---0) and the purified cadmium-containing protein from pike liver (O----O), with mouse antirainbow trout metallothionein serum using the competitive ELISA format. Concentrations for the two fish proteins were determined by amino acid analysis.
a high content of cysteine and a total absence of tyrosine, phenylalanine and arginine. This finding not only indicates the homogeneity of the cadmium binding protein but also confirms its identity as a metallothionein. As further substantiation of this, samples of the purified pike liver protein were compared with the previously well-characterized metallothionein from rainbow trout, using a mouse anti-rainbow trout metallothionein antiserum in the competitive ELISA system defined in the Materials and Methods section (Fig. 3). The pike protein displayed an identical degree of cross-reactivity to the rainbow trout metallothionein. The mid A/Ao values were at 4.0 and 3.2 ng of competing antigen for the pike and trout proteins respectively. This together with the amino acid composition shown in Table 3, indicates unequivocally that the cadmium binding protein isolated from the liver of pike under the present conditions is a metallothionein. Thus by analogy with other cadmium-tolerant species of fish (Brown et al., 1987) and in contrast to Table 3. Amino acid composition of the major cadmium-containing protein isolated from the liver of Dike Amino acid
Cyst&e Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine fsoleucine Leucine Histidine Lysine
16.9 6 4 8.8 6 3.4 9.1 5.1 2.5 2.1 2.1 0.7 5.9
Samples were hydrolysed for 18 hr in uacuo at IOS’C in 6 M HCI following performic acid oxidation. Values are given as residues per mole of protein assumpting an integral value for aspartic acid of 6.0.
the situation observed for the sensitive rainbow trout, it would not be unreasonable to conclude that the very low amounts of cadmium which did penetrate the body tissues of the pike upon administration of the metal in the aquarium water, were likely to have been sequestered by the cysteine-rich protein, metallothionein. In keeping with the tolerance of high concentrations of dissolved cadmium in the water, the calculated coefficient of retention for the pike was very low. As suggested previously (Kay and Cryer, 1986; Kay et al., 1987), this index is a valuable comparator to indicate the likely responses of fish to the toxic challenges posed by cadniium. The underlying mechanisms which account for the relative differences in the levels of the metal accumulated in the tissues of tolerant and sensitive fish species however remain unclear. Ongoing studies are attempting to establish whether the uptake of cadmium from the aquatic environment or the rates of its exchange among and excretion from the organs of the fish are the primary factors that distinguish cadmiumsensitive from tolerant species of freshwater fish. Acknowledgements-This
study was supported by a grant from NERC (GR3/6179A). It is a pleasure to acknowledge our colleagues Miss Wendy Lees and Mrs Barbara Darke, for their help, advice and constant encouragement. We are also grateful to Drs N. Giles and R. Wright, ARC Wildfowl Centre and Messrs T. Jones and C. Strange from the Welsh Water Authority for their invaluable assistance during this study.
REFERENCES Brown M. W., Thomas D. G., Shurben D., Solbe J. F. de L. G., Kay J. and Cryer A. (1986) A comparison of the differential accumulation of cadmium in the tissues of three species of fresh water fish, Salmo gairdneri, Rutilus rutilus and Noemacheilus barbatulus. Comp. Biochem. Physiol. 84C, 213-217.
Brown M. W., Shurben D., Solbe J. F. de L. G., Cryer A. and Kay J. (1987) Sequestration of environmental cadmium by metallothionein in the roach (Rutilus rutilus) and the stone loach (Noemacheilus barbatulus). Comp. Biochem. Physiol. 8’?C, 65-69.
EIFAC Working Party Report on Water Quality Criteria for European Freshwater Fish (1977) EIFAC Technical Papers 30, l-21. Hirs C. H. W. (1967) Performic acid oxidation. Meth. Enzymol. 11, 197-199. Kay J. and Cryer A. (1986) Environmental cadmium and metallothionein gene expression in freshwater fish. N.E.R.C. Journal, pp. 9-11. Kay J., Thomas D. G., Brown M. W., Cryer A., Shurben D., Solbe J. F. de L. G. and Garvey J. S. (1986) Cadmium accumulation and protein binding patterns in tissues of the rainbow trout, Salmo gairdneri. Env. Health Persp. 65, 133-139.
Kay J., Brown M. W., Cryer A., Solbe J. F. de L. G., Shurben D., Garvey J. S. and Thomas D. G. (1987) Cadmium poisoning in freshwater fish and metallothionein gene expression. In Metallothionein and Other Low Molecular Weight Metal Binding Proteins (Edited by Kagi J. H. R.), pp. 247-251. Birkhauser Verlag, Basle, Switzerland. Norey C. G., Lees W. E., Darke B. M., Stark J. M., Baker T. S., Cryer A. and Kay J. (1990) Immunological distinction between piscine and mammalian metallothioneins. Comp. Biochem. Physiol. %B, 597401.
Cadmium in pike Ovemell J. and Coombs T. L. (1979) Purification and properties of plaice metallothionein, a cadmium binding protein from the liver of the plaice (Pleuronectesplaressa). Biochem. J. 183, 277-283. Roberts K. S., Cryer A., Kay J., Solbe J. F. de L. G., Wharfe J. R. and Simpson W. R. (1979) The effects of exposure to sub-lethal concentrations of cadmium on enzyme activities and accumulation of the metal in tissues and organs of rainbow trout and brown trout (Salmo gairdneri and Salmo trutto Fario L.). Comp. Biochem. Physiol. 62C, 135-140.
Shurben siphon 111-112.
CBFC 9512-H
D. G. (1978) A self-breaking concentricdosing apparatus. Laboratory Practice 27,
221
Thomas D. G., Solbe J. F. de L. G., Kay J. and Cryer A. (1983a) Environmental cadmium is not sequestered by metallothionein in rainbow trout. Biochem. Biophys. Res. Commun. 110, 584-592. Thomas D. G., Cryer A., Solbe J. F. de L. G. and Kay J. (1983b) A comparison of the accumulation and protein binding of environmental cadmium in the gills, kidney and liver of the rainbow trout (Salmo gairdneri). Comp. Biochem. Physiol. 76C, 241-246.
Thomas D. G., Brown M. W., Shurben D., Solbe J. F. de L. G., Cryer A. and Kay J. (1985) The sequestration of cadmium and zinc in the tissues of rainbow trout following exposure to the metals either singly or in combination. Comp. Biochem. Physiol. 82C, 55-62.
1990
0306~4492/90 $3.00 + 0.00
0 1990 Pergamon Press plc
Printed in Great Britain
CADMIUM
UPTAKE
AND SEQUESTRATION (ESOX
IN THE PIKE
LUCIUS)
C. G. NOREY,A. CRYERand J. KAY* Department
of Biochemistry,
University
of Wales College U.K.
(Received
21 October
of Cardiff,
P.O. Box 903, Cardiff
CFl
IST,
1989)
Abstract-l. Pike were exposed to cadmium dissolved in their aquarium water at separate concentrations of 60, 125 and 225pg/l and the distribution of the metal accumulated in the major body organs was determined. 2. The fractional retention coefficient ([pgCd/lOOg body wt]/[(pgCd/l) x weeks]) for cadmium was constant at each concentration and at different periods of exposure to the metal. The values measured were remarkably low. 3. When cadmium was introduced into the fish by intraperitoneal injection, the metal was found associated with metallothionein. 4. The amino acid composition and immunochemical cross-reactivity of pike metallothionein were comparable to those of other piscine metallothioneins. 5. The unexpected tolerance of pike to the toxic effects of cadmium when exposed to the metal in the aquatic environment is discussed.
INTRODUCTION
MATERIALSAND METHODS
Certain fish species, such as rainbow trout (Sulmo gairdneri), brown trout (Salmo trutta fario L.) and salmon (S&o s&r) are particularly sensitive to the toxic actions of cadmium when it is present in their aqueous environment. By contrast, coarse fish, including roach (Rut& rutilus L.) and stone loach (Noemacheilus barbatulus), can tolerate much higher concentrations of the dissolved metal (see for example, Brown et al., 1986). When exposed to sublethal concentrations of cadmium in their aquarium water, the tolerant species retain a significantly lower proportion of the metal in their tissues than do sensitive species such as rainbow trout (Brown et al., 1986). In previous reports (Kay et al., 1986; Brown et al., 1987) we have attempted to explain these differential sensitivities to cadmium and the observed differences in metal retention on a rational basis (Kay and Cryer, 1986; Kay et al., 1987). Such hypotheses however, must be tempered by the qualification that only data from salmonid species have been available to represent the cadmium-sensitive group of fish. Previous observations (EIFAC, 1977) have suggested that the pike (Esox lucius) is a non-salmonid species that may be considered sensitive to the toxic actions of dissolved cadmium. Thus, in order to broaden the basis of the hypothetical mechanisms and to ascertain whether these previous interpretations of the behaviour of resistant and sensitive fish species remain valid, the uptake, tissue distribution and sequestration of cadmium in the pike is reported herein.
The fish used in the present study were maintained in dechlorinated mains water (chlorine less than 0.1 ppm) at a total water hardness of approximately 120 ppm (90-100ppm as CaCO,). They we&housed in-tanks &ntaining 200 1 of water changed at a flow rate of 1.5-2 limin and were fed every 2-3 days. The pH of the water was neutral and the oxygen saturation was always greater than 80%. Pike (50-160 g body weight) were generously donated by Drs N. Giles and R. Wright (The ARC Wildfowl Centre, Milton Keynes, Bucks., U.K.). In the tanks, refuges for the fish were provided in the form of 45 cm lengths of 10 cm diameter opaque plastic pipe. Further cover for the fish was provided in the form of simulated floating reeds constructed from strips of black polythene sheet measuring approximately 4 cm x 40 cm. The fish were exposed to cadmium dissolved in the aquarium water at concentrations ranging from 60 to 225 pg/l for periods of between 2 and 11 weeks using a siphon doser (Shurben, 1978). Organs were removed from anaesthetized fish and samples were prepared for metal analysis as described previously (Roberts et al., 1979). Cd, Zn and Cu were determined by atomic absorption spectroscopy at 228.8, 213.9 and 324.8 nm respectively, using a Varian model AA 275 fitted with a background corrector (Varian Instruments, Walton-on-Thomas, Surrey, U.K.). In separate experiments, cadmium was administered to pike (200-450 g body weight) by the intraperitoneal injection of 0.2 mg/kg of the metal (as the sulphate salt) followed by a second injection (0.6 mg/kg) three days later. The fish were then sacrificed after a further two days and the main cadmium-containing protein was purified from liver as described by Brown et al. (1987). Samples of protein for amino acid analysis were hydrolysed in 6 M HCl in D~CUOat 105°C for 18 hr. Cysteine was determined as cysteic acid following oxidation of the protein samples with performic acid (Hirs, 1967) prior to
*Author for correspondence. 217
C. G. NOREYet al.
218
hydrolysis. Analysis was performed using a LKB 4150 alpha automated amino acid analyser, equipped with a recording integrator (Pharmacia LKB Biotechnology, Cambridge, U.K.). A competitive ELISA for the quantitation of fish MT was established as described previously (Norey et al., 1990), using a polyclonal antiserum to rainbow trout metallothionein raised in mice. Homogeneous rainbow trout MT was coated overnight onto the wells of Falcon “probind” microtitre plates from a solution of the protein (148 ng/ml) in carbonate-bicarbonate buffer, pH 9.6. Aliquots of the soluble competing antigen (either rainbow trout MT (in the range lo’-lo6 pg) in the standard curve or pike liver protein) were incubated overnight at 4°C with the mouse antimetallothionein antiserum at a final dilution of l/4000 prior to the incubation of these mixtures with the immobilized trout MT. The reaction between antibody and immobilized antigen was visualised using a rabbit-anti mouse IgG conjugated to horse radish peroxidase (Dako Ltd., High Wycombe, Bucks., U.K.) at a dilution of l/l000 and phenylenediamine as substrate. RESULTS AND DISCUSSION
Pike were exposed to cadmium in their aquarium water at concentrations of 60, 125 and 225 pg/l for up to 11 weeks. In common with our previously reported observations on other species (Brown et al., 1986, 1987), the liver, kidney and gills were the major organs in which cadmium accumulated in the pike. Similar patterns of accumulation were observed for pike kept at all three of the concentrations used. Therefore, for brevity, only the data collected from the experiment using 125 pg/l are presented in Fig. 1. In each of the three organs, the concentration of cadmium &g/g wet weight of tissue) increased progressively over the 11 week period of the study (Fig. la). This pattern was unaltered when the cadmium concentrations were expressed as pg/whole a /
organ/l00 g body weight. In each of the studies, heart, spleen, skeletal muscle and gonadal tissue were also analysed and found to contain no significant amounts of cadmium at any time. Thus, from the cadmium concentrations determined in the liver, kidney and gills, total body burdens of accumulated metal were calculated at each time point of analysis. The values obtained for pike kept at 125 pg of cadmium/l for 2, 5, 8 and 11 weeks were 0.55, 1.O, 1.2 and 2.2 pg (per 100 g body weight) respectively. The corresponding values for both sensitive and tolerant fish species reported previously (Brown et al., 1986) were, however, much higher. Indeed, the level of accumulation in pike kept at relatively high concentrations of the metal in their aquarium water, was exceptionally low, even in relation to the tolerant species studied previously. Despite this low overall level of accumulation in the pike, the distribution of the metal load among the liver, kidney and gills (expressed as a proportion of the total body burden accumulated; Fig. lb) was similar to that seen for other fish species (Thomas et al., 1983b; Brown et al., 1986, 1987). Initially, the major proportion of the body burden was carried by the gill but by 8 weeks of exposure, approximately equivalent loads were present in all three of the organs. Similar patterns were also observed with fish exposed at the lower (60 pg/l) and at the higher (225 pg/l) concentrations of cadmium when the data were expressed in similar terms (not shown). In order to permit comparisons of the accumulation of whole body cadmium by various species of fish, we have previously defined (Brown et al., 1986) a fractional retention coefficient for cadmium, as the quotient of the total body cadmium accumulation (pg/lOOg body weight) and the notional cadmium dose [(pg/l) x weeks]. Values of this fractional retention coefficient obtained in hard water for Cdtolerant species such as roach and stone loach were 7 x 10e3 and 14 x 10e3 respectively. These were considerably lower than the corresponding coefficient, (40 x 10m3) measured for Cd-sensitive species, e.g. rainbow trout. From the data presented above, the fractional retention coefficient for cadmium was calculated for pike on the same basis at each time point of analysis and at each concentration of cadmium. Table 1 shows that for pike, as reported for other species (Brown et al., 1986) the coefficient was unaffected either by the concentration of cadmium to which the fish were exposed or by the duration of exposure. The mean overall value for the coefficient in pike, Table 1. Comparison of the fractional retention of cadmium by pike at different cadmium concentrations
&05 TIME OF EXPOSURE [WEEM;’
Fig. 1. Uptake and distribution of cadmium in the tissues of pike exposed to the metal in their aquarium water (125 pg/l) for different lengths of time. Values are expressed as (a) tissue cadmium concentrations @g/g) and (b) distribution of the metal among the three major organs; gill (w----m), kidney (0-O) and liver (O----O).
Cadmium concentration (w3/0 60 125 225
Fractional retention coefficient (units x IO’) 1.8 + 1.0(6) 1.6CO.4(11) 1.4&0.6(10)
Fractional coefficients were calculated as units being ~;;;l~[ps Cd/100 g body weightl/[(lcgCd/t)
Values given are the means + SD, the number of observations are given in parentheses.
Cadmium in pike 1.5 + 0.7 x 10m3 units (n = 27) was, however, remarkably low when compared with those reported previously for rainbow trout, a cadmium-sensitive species. Indeed it was substantially below the values for the coefficient measured in the cadmium-tolerant species, roach and stone loach (Brown et al., 1986). Thus, contrary to previous indications (EIFAC, 1977; made with fish at a different stage of development than those used in the present study), the pike would appear to be a species that is relatively resistant to cadmium in the aquatic environment. This tolerance may be a reflection of the very low levels at which cadmium became sequestered in the body of the fish, as discussed previously (Kay and Cryer, 1986). Further investigations of this sequestration, by analysis of the tissues of fish exposed to cadmium in the environment, were precluded since so little of the metal accumulated over even quite protracted periods. Therefore, to ensure that sufficient cadmium accumulated in the tissues of fish for quantitative fractionation and analyses to be made, a different approach had to be adopted. Relatively large amounts of the metal were administered to pike by intraperitoneal injection, as described in the Materials and Methods section. The tissue distribution of cadmium administered in this fashion was measured and the nature of the proteins responsible for its sequestration was established. The distribution of the injected metal among the tissues studied is shown in Table 2. Approximately 80% of the cadmium retained in the bodies of the treated fish was accounted for collectively by the liver, kidney and gills. This pattern of distribution was similar to that seen not only with pike treated with environmental cadmium at the later times of exposure (Fig. lb) but also with other fish species into which cadmium had been introduced by intraperitoneal injection (Thomas et al., 1983b). In order to determine the nature of the protein(s) responsible for sequestration of cadmium, the liver was chosen for further study on the basis of (1) the amount of tissue available and (2) because previous observations (Thomas et al., 1983b, 1985; Brown et al., 1987) in other species of fish had revealed that the protein binding patterns exhibited by the other organs of accumulation (e.g. kidney and gill) are identical to those seen for the liver. Following the administration of cadmium to pike by intraperitoneal injection, the supernatant fraction from an homogenate of liver tissue was prepared as described previously (Thomas et al., 1983a, 1985). After concentration, the sample (39 ml) was subjected to gel filtration on a column of Sephadex G-75 (2.5 x 80cm). The cadmium was eluted as a single
219
symmetrical peak in a position (retention of 0.88 of the column volume) consistent with the metal being bound in a macromolecular form of approximately 10,000 daltons (data not shown for brevity). This pattern of elution was identical to those described previously with supernatants derived from other fish species to which cadmium had been administered by intraperitoneal injection (e.g. Overnell and Coombs, 1979; Thomas et al., 1983a). The average recovery of cadmium from this chromatographic step was 43%. As described previously (e.g. Brown et al., 1987), the cadmium-containing fractions were pooled, concentrated by ultrafiltration over a YM2 membrane and then buffer exchanged into 20 mM Tris-HCl buffer, pH 7.4, containing 2 mM mercaptoethanol. The concentrated material was subjected to ion exchange chromatography on a column of DEAE-cellulose (1.5 x 15 cm), as described by Brown et al., 1987. As found previously with material derived from stone loach and roach, the cadmium was eluted as a single peak which emerged at a concentration of 60 mM Tris in the eluting buffer (Fig. 2). This position of elution in the gradient is comparable to that observed with material from the stone loach (Brown et al., 1987) but somewhat later than that from roach. Further purification of this pike liver cadmium binding fraction, was carried out by concentration and desalting of the sample followed by resolution on an FPLC Mono-Q anion exchange column (Pharmacia, Uppsala, Sweden), equilibrated in 10 mM Tris-HCl buffer, pH 8.6. After washing, a linear gradient from 0 to 0.1 M NaCl in Tris-HCl buffer, pH 8.6, was applied. The pattern of AIs4 determined for the eluted fractions revealed one major cadmiumcontaining peak, emerging in the gradient at 0.028 M NaCl. The cadmium-binding protein thus isolated from the liver of pike was performic acid oxidized (see Material and Methods), prior to the determination of its amino acid composition (Table 3). This revealed
,I-/
L__
L
20
Table 2. Cadmium distribution among tissues and organs of pike exposed to the metal by intraperitoneal injection Cadmium Tissue/organ Liver Kidney Gill Heart Spleen
Gonad Skeletal muscle
recovered W) 31 33 15 3 9
9 0
40 FRACTION
60
20
NUMBER
Fig. 2. Ion exchange chromatography of the pooled fractions obtained after gel filtration on Sephadex G-75 of extracts of liver from pike exposed to cadmium by intraperitoneal injection. The cadmium-containing material was applied to a DEAE cellulose column (1.5 x 15cm), equilibrated in 20 mM Tris-HCI buffer, pH 7.4 containing 2 mM mercaptoethanol. After extensive washing, a linear gradient of 20-120 mM Tris-HCl buffer (200 ml each) containing 2mM mercaptoethanol was used for desorption. e-0 = cadmium.
C. G. NOREYet al.
220
,I
0
3
Loa MT (PP)
Fig. 3. Cross immunoreactivity of rainbow trout metallothionein (a---0) and the purified cadmium-containing protein from pike liver (O----O), with mouse antirainbow trout metallothionein serum using the competitive ELISA format. Concentrations for the two fish proteins were determined by amino acid analysis.
a high content of cysteine and a total absence of tyrosine, phenylalanine and arginine. This finding not only indicates the homogeneity of the cadmium binding protein but also confirms its identity as a metallothionein. As further substantiation of this, samples of the purified pike liver protein were compared with the previously well-characterized metallothionein from rainbow trout, using a mouse anti-rainbow trout metallothionein antiserum in the competitive ELISA system defined in the Materials and Methods section (Fig. 3). The pike protein displayed an identical degree of cross-reactivity to the rainbow trout metallothionein. The mid A/Ao values were at 4.0 and 3.2 ng of competing antigen for the pike and trout proteins respectively. This together with the amino acid composition shown in Table 3, indicates unequivocally that the cadmium binding protein isolated from the liver of pike under the present conditions is a metallothionein. Thus by analogy with other cadmium-tolerant species of fish (Brown et al., 1987) and in contrast to Table 3. Amino acid composition of the major cadmium-containing protein isolated from the liver of Dike Amino acid
Cyst&e Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine fsoleucine Leucine Histidine Lysine
16.9 6 4 8.8 6 3.4 9.1 5.1 2.5 2.1 2.1 0.7 5.9
Samples were hydrolysed for 18 hr in uacuo at IOS’C in 6 M HCI following performic acid oxidation. Values are given as residues per mole of protein assumpting an integral value for aspartic acid of 6.0.
the situation observed for the sensitive rainbow trout, it would not be unreasonable to conclude that the very low amounts of cadmium which did penetrate the body tissues of the pike upon administration of the metal in the aquarium water, were likely to have been sequestered by the cysteine-rich protein, metallothionein. In keeping with the tolerance of high concentrations of dissolved cadmium in the water, the calculated coefficient of retention for the pike was very low. As suggested previously (Kay and Cryer, 1986; Kay et al., 1987), this index is a valuable comparator to indicate the likely responses of fish to the toxic challenges posed by cadniium. The underlying mechanisms which account for the relative differences in the levels of the metal accumulated in the tissues of tolerant and sensitive fish species however remain unclear. Ongoing studies are attempting to establish whether the uptake of cadmium from the aquatic environment or the rates of its exchange among and excretion from the organs of the fish are the primary factors that distinguish cadmiumsensitive from tolerant species of freshwater fish. Acknowledgements-This
study was supported by a grant from NERC (GR3/6179A). It is a pleasure to acknowledge our colleagues Miss Wendy Lees and Mrs Barbara Darke, for their help, advice and constant encouragement. We are also grateful to Drs N. Giles and R. Wright, ARC Wildfowl Centre and Messrs T. Jones and C. Strange from the Welsh Water Authority for their invaluable assistance during this study.
REFERENCES Brown M. W., Thomas D. G., Shurben D., Solbe J. F. de L. G., Kay J. and Cryer A. (1986) A comparison of the differential accumulation of cadmium in the tissues of three species of fresh water fish, Salmo gairdneri, Rutilus rutilus and Noemacheilus barbatulus. Comp. Biochem. Physiol. 84C, 213-217.
Brown M. W., Shurben D., Solbe J. F. de L. G., Cryer A. and Kay J. (1987) Sequestration of environmental cadmium by metallothionein in the roach (Rutilus rutilus) and the stone loach (Noemacheilus barbatulus). Comp. Biochem. Physiol. 8’?C, 65-69.
EIFAC Working Party Report on Water Quality Criteria for European Freshwater Fish (1977) EIFAC Technical Papers 30, l-21. Hirs C. H. W. (1967) Performic acid oxidation. Meth. Enzymol. 11, 197-199. Kay J. and Cryer A. (1986) Environmental cadmium and metallothionein gene expression in freshwater fish. N.E.R.C. Journal, pp. 9-11. Kay J., Thomas D. G., Brown M. W., Cryer A., Shurben D., Solbe J. F. de L. G. and Garvey J. S. (1986) Cadmium accumulation and protein binding patterns in tissues of the rainbow trout, Salmo gairdneri. Env. Health Persp. 65, 133-139.
Kay J., Brown M. W., Cryer A., Solbe J. F. de L. G., Shurben D., Garvey J. S. and Thomas D. G. (1987) Cadmium poisoning in freshwater fish and metallothionein gene expression. In Metallothionein and Other Low Molecular Weight Metal Binding Proteins (Edited by Kagi J. H. R.), pp. 247-251. Birkhauser Verlag, Basle, Switzerland. Norey C. G., Lees W. E., Darke B. M., Stark J. M., Baker T. S., Cryer A. and Kay J. (1990) Immunological distinction between piscine and mammalian metallothioneins. Comp. Biochem. Physiol. %B, 597401.
Cadmium in pike Ovemell J. and Coombs T. L. (1979) Purification and properties of plaice metallothionein, a cadmium binding protein from the liver of the plaice (Pleuronectesplaressa). Biochem. J. 183, 277-283. Roberts K. S., Cryer A., Kay J., Solbe J. F. de L. G., Wharfe J. R. and Simpson W. R. (1979) The effects of exposure to sub-lethal concentrations of cadmium on enzyme activities and accumulation of the metal in tissues and organs of rainbow trout and brown trout (Salmo gairdneri and Salmo trutto Fario L.). Comp. Biochem. Physiol. 62C, 135-140.
Shurben siphon 111-112.
CBFC 9512-H
D. G. (1978) A self-breaking concentricdosing apparatus. Laboratory Practice 27,
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Thomas D. G., Solbe J. F. de L. G., Kay J. and Cryer A. (1983a) Environmental cadmium is not sequestered by metallothionein in rainbow trout. Biochem. Biophys. Res. Commun. 110, 584-592. Thomas D. G., Cryer A., Solbe J. F. de L. G. and Kay J. (1983b) A comparison of the accumulation and protein binding of environmental cadmium in the gills, kidney and liver of the rainbow trout (Salmo gairdneri). Comp. Biochem. Physiol. 76C, 241-246.
Thomas D. G., Brown M. W., Shurben D., Solbe J. F. de L. G., Cryer A. and Kay J. (1985) The sequestration of cadmium and zinc in the tissues of rainbow trout following exposure to the metals either singly or in combination. Comp. Biochem. Physiol. 82C, 55-62.